Translational control in mammalian cells.

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TRAl'JSLATIONAL CONTROL IN

Annu. Rev. Biochem. 1991.60:717-755. Downloaded from www.annualreviews.org Access provided by Yale University - Law Library on 01/01/15. For personal use only.

MAMMALIAN CELLS John W. B. Hershey Department of Biological Chemistry, School of Medicine, University of California, Davis, Calilfornia KEY WORDS:

95616

protein synthesis, phosphorylation, initiation factor, elongation factor, ribo

some.

CONTENTS PERSPECfIVES AND SUMMARy . . . . . . . . . . . . . . . . ............. . . . . . . . ..... . . . . . . . . . .. . . . . . . . .. .

717

PATHWAY AND MECHANISM OF PROTEIN SYNTHESIS ........ . . . . . . . ........ . . . . . .

��ftf�:�� .":!. .��':':.��.::::::::::::::::::::::::::::::::::::::: : ::::::::::::::: : : : : : ::: : :: : :

719 721 727 729

APPROACHES TO STUDYING TRANSLATIONAL CONTROL . . . . . . . ......... . . ... . . . .

730

Determinants of Protein Synthesis Rates .................................................... .

730 733 734

GLOBAL CONTROLS BY PHOSPHORYLATION..................... . . . . . . . . . .. . .. . . . .... .

738 738 741 743

Initiation .

. ....... . . . . ...................... ................................. . . . . . . . . . . . . . . . . . . . . . . . .

Measuring Protein Synthesis Rates ........................................................... . Targets 0./ Translational Control ............................................................. .

Stimulation of Protein Synthesi,' and Cell Gruwth ........................................ . Repression of Translation ...................................................................... . Other Phosphorylation Targets................................................................ .

OTHER CONTROL MECHANISMS ............ ... . . . . . . . ..... ............ . ...... . ... .......... . Polyadenylation of mRNAs ..................................................................... . Poliovirus-Infected Cells........................................................................ . Ferritin ..................................................................................... Frameshijiing and Readthrough............................................................... .

mRNA

Miscellaneous Control Mechanisms .......................................................... .

744 745 746 746 747 749

PERSPECTIVES AND SUMMARY Protein synthesis is an integral part of the pathway of gene expression and makes important contributions to modulation of the expression of specific genes. As a major consumer of energy in the cell, protein synthesis must be integrated into the overall metabolic activity of the cell by controlling the rates 717

0066-4154/9110701-0717$02.00


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of global protein synthesis . In order to understand how translation is regulated at the molecular level, knowledge of the mechanism of protein synthesis is required. In the five years since protein synthesis was reviewed in this series (1), a more detailed understanding of the mechanism of mRNA binding to ribosomes and the catalytic function of initiation factors has been gained. In particular, the ribosome scanning model for initiation has been well es tablished and an alternate mechanism involving ribosome binding directly into internal regions of mRNAs has been demonstrated. A plausible working model of the pathway of protein synthesis is postulated, but a detailed molecular description of how mRNA and aminoacyl-tRNAs bind and interact on ribosomes and how soluble factors promote these interactions is not yet possible. Nor is it certain that all of the components of the translational apparatus have been identified. The rate-limiting step of translation usually but not always occurs during the initiation phase. Methods are available to measure changes in rates of initiation and elongation for global or specific protein synthesis on poly somes, and to detect changes in the distribution of mRNAs between active polysomes and messenger ribonucleoprotein particles (mRNPs) . The intrinsic activity or strength of most mRNAs is determined first by accessibility of the capped 5' -terminus to initiation factors, then by the ability of the 40S ribosomal subunit to bind and scan the mRNA distally , and finally by the frequency of recognition of the initiator codon and surrounding context. The mobilization of mRNAs from repressed mRNPs into active polysomes is controlled separately from ribosome initiation on polysomes and is an impor tant regulator of the expression of specific mRNAs. The physical basis for mRNP repression is poorly understood in most cases, but insights into mRNP structure and activity are beginning to emerge . In particular, the repression of ferritin mRNA translation is explained by the binding of a heme-regulated repressor protein to a specific cis-acting RNA sequence in the 5' -untranslated region of the mRNA. The dominant mechanism of control of global protein synthesis is phosphorylation /dephosphorylation of translational components, primarily initiation and elongation factors . In the stimulation of protein synthesis, eIF-4F (the mRNA cap-binding protein complex) is the major phosphoryla tion target, although phosphorylation of other proteins such as elF-3 , eIF-4B , and ribosomal protein S6 may be important also. The fact that overexpression of the eDNA encoding the a-subunit of eIF-4F is oncogenic , whereas synthe sis of a mutant form that resists phosphorylation is not, serves to emphasize the importance of translation and its regulation through phosphorylation in growth control . Phosphorylation also causes repression of protein synthesis , primarily by modifying the a-subunit of initiation factor eIF-2 or the elonga-


TRANSLATIONAL CONTROL IN MAMMALS

719

tion factor eEF-2. Phosphorylation of eIF-2a appears to be a general mech anism for inhibiting initiation of translation, whereas eEF-2 phosphorylation affects elongation rates and may be used less frequently. The kinases thus far identified as responsible for the phosphorylation of translational components are usually highly specific, whereas the phosphatases exhibit a broad range of specificity. Their regulation is mostly obscure and represents one of the larger challenges for the future. A complex net of interactions, some redundant or overlapping in effect, may serve to tie translation to the overall metabolic state of tht� cell. A wide variety of control mechanisms operate on classes of mRNAs or specific mRNAs. The poly(A) tail at the 3 ' -terminus of most mRNAs in fluences both mRNA stability and translational efficiency. Trans-acting pro teins that biind to special cis-acting regions of an mRNA may be a general way to regulate the synthesis of specific proteins. Ribosomal frameshifting and translational readthrough of stop codons also contribute to the regulated expression of specific genes, especially the reverse transcripta se gene in retroviruses. In a growing number of examples, gene expression is regulated at the level of translation by unknown mechanisms. We can anticipate con fronting a great variety of new and exciting regulatory mechanisms whose elucidation presents a challenge for future studies. PATHWAY AND MECHANISM OF PROTEIN SYNTHESIS

Protein synthesis is divided into three phases: initiation; elongation; and termination. The reactions in each phase are promoted by soluble protein factors (see Table 1) that transiently interact with the ribosome, mRNA, and aminoacyl-ItRNAs. A striking feature of the pathway is the plethora of non covalent binding interactions among the protein and RNA components and the dearth of covalent bond-making and -breaking reactions. Thus, during the initiation phase, only the hydrolysis of pyrophosphate bonds in ATP and GTP occurs, whereas a great many macromolecular binding and dissociation reac tions occur. A thorough understanding of the pathway and mechanism re quires knowledge of the structures of the various intermediates and of the rules goveming protein and nucleic acid interactions. Neither aspect is known to a satisfactory degree, but a working model of the pathway may be proposed (Figure 1) to assist in our discussion of the reactions involved. The model is based primari ly on in vitro studies with purified components, where stable putative intermediate complexes are detected and then arranged in a logical order. Such a model should be used with caution and skepticism, since in vivo evidence and kinetic confirmation are largely lacking Most of the work .

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HERSHEY

Table 1

Mammalian soluble factors" Other

Factor

Functions

Factor

Subunits

mass (kOa)

mass (kDa)

stimulates formation of 40S

15

names

Cloned cDN spe cies (Ref

Initiation factors eIF-I

preinitiation complexes


eIF-IA

eIF-4C

ribosome dissociation; stimu-

lates 40S preinitiation

17.6

complexes e1F-2

GTP-dependent Met-tRNA

130

binding to 40S ribosome

a

36. 1

human, rat (23

f3

38.4

human (2 34)

a

26

f3

39

l' 55

eIF-2A

AUG-dependent Met-tR NA

65

binding to 40S ribosome eIF-2B

GEF

GTP:GDP exchange

272

l' 58 5 67

eIF-2C

Co-eIF-2A

eIF-3

stabilizes ternary complex stimulates formation of 40S

E

82

94 550

preinitiation complex

a

35

f3

36

E

47

'Y 40 (j 44

, 67 1/ 115

eIF- 3A

e IF-6

0170 ribosome dissociation; binds

25

60S ribosome eIF-4A

RNA-dependent ATPase;

eIF-4B

helicase; stimulates mRNA

helicase; stimulates mRNA binding

44.4

mouse (235)

80

human (236)

binding eIF-4F

CBP-II eIF-4E

cap recognition; helicase;

270

stimulates mRNA binding

a

25

f3

44.4

human (2 37) mouse (238)

eIF-4A p220 eIF-5 A

eIF-4D

l' 220

cleaved by poliovirus stimulates ribosome junction

eIF-5

stimulates 1 st peptide bond

150 16.7

human (2 39)

formation eEF-la

binds aa-tRNA; GTPase

51

human (240) mouse (241)

TRANSLATIONAL CONTROL IN MAMMALS Table 1

721

(Continued) Other

Factor

names

eEF-lfl

eEF-ly

Functions

GTP:GDP exchange eEF-la GTP:GDP exchange

Factor

Subunits

Cloned cDNA

mass (kDa)

mass (kDa)

species (Ref.)

on

23

Anemia (242)

with

49

Anemia (243)

cEF-lfl


eEF-2

100

stimulates translocation;

rat, hamster

GTPase Termination factor eRF

rabbit (245)

54.0

recognizes stop codons, stimulates peptidyl-tRNA cleavage and release

•

Information in the table was obtained in part from

(4),

where further references may be found.

leading to the model was conducted prior to 1985 and therefore has been reviewed in detail in this series (1). An extensive, up-to-date treatment of eukaryotic translation also is available (2) as well as descriptions of the structure, function, and genetics of ribosomes (3). In the following sections, I merely sketch the outlines of the various reactions and concentrate on aspects relevant to translational control.

Initiation During the initiation phase, two important tasks are achieved: (a) an mRNA among many available candidates is selected for translation by the initiating ribosome; and (b) the ribosome identifies the initiator codon and begins translation in the appropriate reading frame. The various binding reactions are promoted by at least 10 initiation factors (abbreviated elF), whose physical characteristics are listed in Table 1. These proteins have been highly purified, and a number of their cDNAs or genes have been cloned and sequenced. Throughout this review, I employ the revised nomenclature for soluble factors as specified by the Nomenclature Committee of the International Union of Biochemistry (4). Previous names are included in Table 1 to facilitate the reader's tr�lnsition to the new nomenclature. We identify four major steps in the initiation pathway:

1. ribosome dis

sociation illlt o 40S and 60S subunits; 2. Met-tRNAi binding to the 40S ribosomal subunit to form a 40S preinitiation complex; 3. mRNA binding to the 40S preinitiation complex to form a 40S initiation complex; and 4. junction of the 40S initiation complex with the 60S subunit to form an 80S initiation complex. I concentrate primarily on steps 2 and 3, and limit comments Ito descriptions of our current understanding of the pathway.

(244)

�

@� _____ 0

Met L eIF-2C

� EO --1� @ 3

r----

..l- AUG--An

Met

ATP

&

-r lS

An :t-@@ r- �@@@@ AUG--An

f4t)�LAUG ATP p. ADP I


�@ � Met

1

AUG--An

�r-

ATP ,f-- ADP Pi +

Met

��J--- An e1F-5

mRNA +

tRNAs

Figure

1

Model of protein synthesis pathway in mammalian cells



723

DISSOCIATION OF 80s RIBOSOMES At Mg2+ concentrations thought to be physiologi(;al (> 1 mM) , 80S ribosomes predominate in active equilibrium with dissociated subunits. Two initiation factors, eIF- l A and eIF-3, shift the equilibrium towards dissociation by binding to 40S subunits and preventing their association with 60S subunits (5). Another factor, eIF-3A, binds to 60S subunits and prevents subunit association in vitro (6), although the role of this protein in translation is not well established. The mechanism of ribosome dissociation has been reviewed in detail ( 1 ); little additional work has been reported since then. Since the levels of eIF-3 and possibly eIF-lA are nearly equal to those of ribosomes , one would expect extensive 80S dissociation in translationally repressed cells, yet mostly 80S ribosomes rather than pre dominantly subunits are observed . Either the in vitro studies do not reflect the process of dissociation in vivo accurately, or other components interact with 80S ribosomes to favor their association. METHIONYL-tRNA B INDING AND eIF- 2 FUNCTION The binding of the initia tor methionyl-tRNA (Met-tRNA;) to 40S ribosomal subunits is a step com mon to the translation of all mRNAs. eIF-2, in a binary complex with GTP, binds Met-tRNAj to form a ternary complex. The ternary complex is an obligatory intermediate that can be readily identified and isolated. Ternary complexes are formed in high yield with purified components at physiological concentrations of factor and tRNA (ca. 1 JLM) (7, 8). At lower concentrations «0. 1 JLM), stabilization by ancillary factors has been observed. One of these, eIf.-2C (formally called Co-eIF-2A8o; a 94 kDa protein) , acts stoichiometrically to stabilize the complex and prevent its disruption when naked mRNA is added (9). Another is eIF-3, which also stimulates ternary complex fOlrmation at low concentrations of e1F-2 and Met-tRNAj ( 10) . In the case of eIF-3, disruption by mRNA is not prevented, however. It is not clear if the interactions of eIF-2 C and e1F-3 with the ternary complex actually occur in vivo off the ribosome or whether they reflect subsequent interactions on the surface of the 40S ribosomal subunit. Since e1F-2 binds GDP 400-fold more tightly than GTP ( 1 1 ) , the proportion of e1F-2 in potentially active binary complexes with GTP will be sensitive to the energy charge of the cell (see also discussion of eTF-2 recycling beloW). The ternary complex binds to the 40S ribosomal subunit to form a 40S preinitiation complex that is sufficiently stable to be isolated by sucrose density gradient centrifugation (12). Such complexes are stabilized by e1F-3 and elF-l A , but do not require the presence of mRNA ( 1 3 , 1 4) . Furthermore, GTP hydrolysis does not occur and nonhydrolyzable GTP analogues can function in these steps. Since stable mRNA ·40S complexes are not detected in the absence of Met-tRNAj, the proposed pathway of initiation places Met-


724

HERSHEY

tRNAj binding prior to mRNA binding . An important caveat is that other pathways involving unstable intennediates are possible. Initiation complexes are most frequently analyzed by sucrose density gradient centrifugation, which usually requires hours of centrifugation to separate ribosome-bound and free components. Complexes are detected only if their dissociation rates are slower than the centrifugation times, yet many physiologically relevant complexes may dissociate in seconds rather than hours. Thus we cannot rule out a pathway that includes initial binding of mRNA to 40S ribosomal subunits in the absence of eIF-2 and Met-tRNAj (see section below). A kinetic analysis of the possible reactions is required to define the pathway more precisely. mRNA B INDING An initiating 40S ribosomal subunit interacts with mRNA in the fonn of either a free mRNP particle or a polysome with associated poly somal RNP proteins and translating ribosomes. It is generally believed that the 40S preinitiation complex with Met-tRNAj, eIF-2, eIF-3, and eIF- l A is the active form of the ribosomal subunit ( 14), but this view is not firmly established. Either of two modes of mRNA binding occur: the ribosome binds to the 5' -terminus of the mRNA, then scans linearly down the mRNA until it recognizes the initiator codon (the scanning model); or the ribosome binds to an internal region of the mRNA, either directly at the initiator codon, or somewhat upstream followed by a scanning process (internal initiation mod el) . In either case, formation of the 40S initiation complex is thought to be promoted by ATP hydrolysis and by a number of initiation factors . A tentative pathway for the scanning model is depicted in Figure 1 . The scanning model accounts for initiation of the large majority of cellular mRNAs ( 1 5). Important features of mRNA structure [reviewed in ( 1 5 , 1 6)] include the presence of a 7-methyl-guanylic acid "cap" at the 5' -tenninus, aspects of secondary and tertiary structurc, the initiator codon (nearly always AUG) and surrounding sequences (context), and the poly-adenylate lpoly(A)] tail at the 3' -terminus (discussed in greatcr dctail below) . The role of initiation factors in the scanning mechanism is only beginning to be understood [reviewed in ( 1 7 , 1 8) J. A working hypothesis is that eIF-4F binds to the cap through its a-subunit (eIF-4E) , which is known as the cap-binding protein because it binds and crosslinks to the cap structure. eIF-4A and eIF-4B may then join the complex. Highly purified eIF-4A or eIF-4F in combination with eIF-4B exhibits an ATP-dependent RNA helicase activity ( 19-2 1 ) . eIF-4A is an RNA-dependent ATPase (22) and is homologous to other "D-E-A-D box" proteins with helicase activity (23); eIF-4B contains a consensus RNA-binding domain present in a number of other RNA-binding proteins (236). It is not yet known if eIF-4A and eIF-4B continue their heliease activity until the entire 5' -untranslated region (5'-



725

UTR) of the mRNA is melted up to the initiator codon, with the 40S subunit passively following, or if the 40S ribosome joins these factors earlier, and is an essential contributor to the melting and movement towards the AUG. In either case,. ATP hydrolysis is essential for the unwinding process but may or may not be directly involved in 40S ribosomal subunit migration. It is possible that higher-order initiation factor complexes are involved in cap recognition . Both eIF-4F and eIF-4B bind to eIF-3, and evidence for such super-factor complexes has been reported (25 , 26). Since eIF-3 binds tightly to 40S ribosomal subunits and also to mRNA , it may be the link between the 40S ribosomal subunit and the mRNA-factor complexes. Alternatively, the a-subunit of eIF-4F alone may interact first with the cap , followed by the binding of the other factors and the ribosome, as suggested by Rhoads ( 1 6) . Clearly, additional biochemical studies are needed t o elucidate how the initiation factors contribute to the scanning mechanism and how the 40S ribosomal subunit slides distally from the cap along the mRNA. Following recognition of the cap structure by the initiation factors and the melting out of secondary structure near the 5' -terminus, the 40S preinitiation complex binds to the cap-proximal region of the mRNA. The 40S ribosomal subunit then begins the scanning process in search of the initiator codon. The scanning model predicts that the 5 ' -proximal initiator codon should be pre ferred, and this is true for >90% of cellular mRNAs (27). A modulating feature is the context in which the AUG occurs. Important elements in a strong context include purines at positions -3 and +4 (where the A of AUG is + 1) (28) . When a scanning ribosome encounters an AUG with a weak consensus sequence, it may pass over the AUG and continue to scan the mRNA. What recognizes the AUG and consensus sequences? The anticodon of Met-tRNAi surely interacts with the AUG, as has been shown conclusively in yeast (29). Genetic studies also implicate eIF-2 in the recognition process , where mutations in the lX- and l3-subunits of eIF-2 alter codon selection in yeast (30, 31). However, the macromolecules that recognize and interact with the surrounding consensus sequences remain a mystery . It is difficult to see how Met-tRNAi can play this role, nor has evidence been generated for rRNA involvement. Specific recognition of AUG-ribosome binding sites by eIF-2 (32) and eIF-4B (33) has been proposed, but needs further experimental verification. Whatever the mechanism, the 40S initiation complex presum ably pauses at AUGs with strong consensus sequences, thereby enabling the 60S junction reaction to occur. mRNA secondary structure properly placed downstream of the initiator AUG promotes initiation, presumably by enhanc ing pausing near the AUG and impeding further scanning (24). mRNA binding to ribosomes by the "internal initiation" mechanism has been demonstrated for poliovirus (34) and encephalomyocarditis (EMC) virus RNAs (35 ) . The long 5'-UTRs and the presence of numerous upstream AUGs


726

HERSHEY

between the uncapped 5 ' -terminus and initiator codon strain the credibility of the scanning mechanism for these RNAs. Proof for an internal initiation mechanism has been accomplished by constructing di-cistronic genes with the ribosome entry region of the poliovirus or EMC virus 5' -UTRs inserted between the cistrons (34, 35) . Enhanced in vivo expression of the downstream cistron in transfected cells, even when translation of the upstream cistron is inhibited, indicates ribosomal entry between the cistrons. The possibility that translation of the second cistron is due to partial RNA cleavage has been ruled out by Northern blot analyses of polysomes actively translating only the second cistron. Whereas most evidence for internal initiation has been obtained with viral RNAs, there is a report of a cellular mRNA encoding the glucose-regulated protein 78/immunoglobulin heavy chain binding protein (GRP78/BiP) that utilizes a cap-independent process and may initiate by the internal binding mechanism (36). Cellular mRNAs with long 5 ' -UTRs that contain numerous AUG's [e.g. human c-abl mRNA (37)] , also are good candidates for translation by the cap-independent mechanism. Whereas the internal initiation mechanism likely is a minor contributor to initiation in mammalian cells, it may play an important role in enabling the translation of a small class of mRNAs when the cap-dependent scanning mechanism is repressed. A detailed molecular mechanism of internal initiation is not yet available, nor have essential initiation factors been identified. It appears likely that the same initiation factors are involved in both mechanisms, except that internal initiation does not involve cap recognition and therefore may not require a functional eIF-4F. An attractive hypothesis is that eIF-4F is non-essential and that eIF-4A and eIF-4B bind directly to the internal region and begin their helicase activity . An added complexity may be the binding of mRNA-specific proteins to specific structures in these 5 ' -UTRs . Poliovirus-specific proteins of 52 kDa (38) and 50 kDa (39) and a 57 kDa EMC virus-specific protein (40) have been identified that may assist in directing ribosome binding. In the case of poliovirus , ribosome entry appears to occur upstream of the initiator AUG, since secondary structure introduced between the ribosome entry site and the initiator AUG inhibits translation (34) . In contrast, with EMC viral RNA, the ribosome appears to bind directly at the lith AUG, the authentic initiator codon (41). 80s INITIATION COMPLEX FORMATION AND INITIATION FACTOR RECY CLING The 60S ribosomal subunit binds to the 40S subunit carrying mRNA and Met-tRNAj positioned at the initiator codon. The reaction requires the function of e1F-5 and the hydrolysis of the GTP molecule bound to eIF-2. The GTPase reaction is promoted by eIF-5 in the absence of 60S subunits and results in the ejection of eIF-2·GDP and other bound factors such as eIF- l A



727

and eIF-3 (14, 42, 43) . Subsequent binding of the 60S subunit may be rapid, since the stability of bound Met-tRNAj to 40S ribosomes in the absence of e1F-2 is low. The physical nature of e1F-5 remains controversial. Initial purification and characterization of the factor identified a protein of 1 50-1 70 kDa [reviewed in ( 1)] , whereas a 62 kDa protein with qualitatively compara ble activity has been isolated (44) and shown not to be a proteolytic fragment of the larger form (45). The eIF-2·GDP binary complex must exchange the GDP for GTP in order for e1F-2 to catalyze another round of initiation . The rate of guanine nucleo tide exchange is slow at Mg2+ concentrations in the physiological range and the reaction thus requires catalysis. eIF-2B , frequently called GEF (for guanine nucleotide exchange factor) , interacts with eIF-2'GDP to enhance the exchange rate by a mechanism that remains controversial ( 1 1 , 46). A separate issue is the thermodynamics of the reaction: e1F-2 binds GDP ca. 400-fold more avidLy than GTP. The high GTP/GDP ratio (energy charge) and the removal oJ the eIF-2'GTP product of the equilibrium reaction by ternary complex formation contribute to a reaction flux towards the eIF-2'GTP complex . Following 80S initiation complex formation , the elongation phase of pro tein synthesis commences. However, formation of the first peptide bond has unique features that distinguish it from subsequent peptide bond formation reactions: the Met-tRNAj in the ribosomal P site (donor site) has a charged a-amino group, whereas in subsequent rounds of the elongation cycle this aminoacyl.. tRNA derivative is acylated as peptidyl-tRNA. The initiation fac tor eIF-5A appears to promote formation of the first peptide bond, usually measured by methionyl-puromycin synthesis (47) . The factor is uniquely modified on Lys50 by the polyamine, spermidine, and subsequently is hy droxylated to form a hypusine residue [NC(2-hydroxy-4-aminobutyl)lysine] (48). The hypusine (or deoxyhypusine) modification is essential for elF-SA activity in the in vitro methionyl-puromycin synthesis assay (49) and is required for elF-SA function in vivo in yeast (J. Schnier, H . G. Schwelberger, Z . Smit-McBride, H . Kang, J . W. B . Hershey, manuscript submitted). How eIF-5A and its hypusine residue function in the reaction is not known . Elongation and Termination

The elongation phase of protein synthesis is a cyclic process that adds one amino acid residue to the C-terminal end of the nascent polypeptide chain per turn of thl:: cycle. It is promoted by four proteins, called elongation factors (abbreviated eEF) , which have been purified and whose cDNAs have been cloned and sequenced (Table 1 ) . The pathway (Figure 1 ) involves four major steps: 1 . b:inding of aminoacyl-tRNA to the A site of the ribosome, catalyzed by eEF- lo; 2. GTP hydrolysis and ejection of eEF- l a·GDP; 3. formation of


728

HERSHEY

the peptide bond, catalyzed by the peptidyl transferase center in the 60S ribosomal subunit; and 4. translocation, promoted by eEF-2 . Considerable energy is expended during each cycle: at least one high-energy pyrophosphate bond in GTP is cleaved at both the binding and translocation steps; and two additional high-energy bonds are required to synthesize the aminoacyl-tRNA that is consumed. The process is rapid, a ribosome incorporating up to six amino acids per second, although not so rapid as in bacteria ( 1 5-1 8 residues/ sec). Key features are high fidelity, i.e. the ability to match properly the aminoacyl-tRNA and the codon in the mRNA template, and processivity, i.e. the ability to synthesize long polypeptides without premature dissociation of the peptidyl-tRNA. The termination phase, in contrast, is relatively simple. When a termination codon is positioned in the A site of the ribosome, a release factor (eRF) (Table 1 ) promotes cleavage of the completed peptidyl tRNA, releasing the protein . PATHWAY AND MECHANISM Our knowledge of the molecular events of elongation and termination in mammalian cells is primarily by analogy to mechanisms elucidated in bacteria, where some important advances have been made recently. The components and pathway have been reviewed by Moldave in this series ( 1 ), and more currently by Slobin (50) . Aminoacyl tRNA forms a ternary complex with eEF- l a and GTP prior to binding to the A site of the ribosome . Following ribosome binding but before peptide bond formation can occur, the GTP is hydrolyzed by the factor, and eEF- I a dissociates as a binary complex with GDP. The ejection of eEF- l a-GDP may be the rate-limiting step of elongation, as it appears to bc in bacteria (5 1 ), and is thought to provide an additional opportunity for incorrectly bound ami noacyl-tRNA to dissociate from the ribosome (proofreading). In order for eEF- l a to promote another binding reaction, the GDP must be exchanged for GTP. Reminiscent of the situation with eIF-2, the release of GDP from eEF- l a is slow [ 1 4 x 10-3 s-lfor the Artemia protein (52)], and requires catalysis by a factor complex called eEF- II3y. eEF- l a is a highly abundant protein in cells, and is posttranslationally modified by methylation of a number of lysine residues (53, 54) . It also is uniquely modified on two glutamic acid residues by formation of an amide linkage to glycerylphos phorylethanolamine (55, 56) . However, the role of these modifications in vivo is unclear; they do not appear to be required for factor activity in vitro CW. C. Merrick, personal communication). Little is known about the catalytic entities on the 60S ribosomal subunit that promote peptide bond formation. In contrast, significant advances in our understanding of the translocation reaction have been made for the Es cherichia coli system. Using RNA modification methods and refined models of the bacterial ribosome, Noller and coworkers were able to distinguish at the



729

structural level between bound peptidyl-tRNA before and after translocation (57). In the pretranslocation state , the anticodon end of the peptidyl-tRNA is bound in the A site, but the peptidyl end already is located in the P site. The translocation reaction actually occurs prior to GTP hydrolysis (i.e. with the GMP-PNP analogue) (58). It therefore is likely that eEF-2·GTP binding alone promotes the movement of the mRNA and the anticodon portion of the peptidyl-tRNA into the P-site. How eEF-2 promotes the movement of mRNA and peptidyl-tRNA on the ribosomal surface is unclear. eEF-2 also is uniquely modified by conversion of a specific histidine residue to diphthamide. Diph thamide in tum is the target of toxins from either Corynebacterium diphtheri ae or Pseudomonas aeruginosa, which ADP-ribosylate the factor and inhibit its activity (59 60). Transfection of mammalian cells with mutant forms of the cDNA encoding eEF-2, where the target His is altered to Lys or Arg, does not result in toxin resistance (6 1 ) . It is possible that the mutant factor lacking diphthamide does not function in protein synthesis, or that endogenous eEF-2 when ADP-ribosylated inhibits dominantly. However, yeast mutants lacking enzymes in the biosynthetic pathway for diphthamide are viable, indicating that the His residue fully modified to diphthamide is not essential for protein synthesis (.59). Since the diphthamide modification reactions are reversible, a regulatory role for this modification remains a possibility. ,

Reinitiation

Nearly all cellular mRNAs are monocistronic, i.e. only a single translational start and stop signal are recognized and utilized. This is in contrast to protein synthesis :In bacteria, where polycistronic mRNAs are common and are translated either independently or by coupling translation of the downstream cistron to that of the upstream cistron. Recent work suggests that eukaryotie ribosomes also are capable of termination at one open reading frame (ORF) and initiating at another downstream ORF, a process we call reinitiation. The following observations indicate that reinitiation occurs at low frequency in mammalian cells. An mRNA with an upstream AUG and strong consensus sequence inserted in the 5' -UTR translates the major protein coding sequence poorly, but does so more efficiently when a termination codon is introduced in-frame and distal to the upstream AUG (62). Translated upstream ORFs are usually short (63), although low-level reinitiation after a major ORF has been reported for the xanthine-guanine phosphoribosyl transferase (64) and di hydrofolat'� reductase (65) coding regions . Placement of the upstream "mini cistron" affects the efficiency of reinitiation; increased efficiency is seen as the distance separating the two ORFs becomes greater (66). The mechanism of reinitiation remains unclear: after termination , either 80S ribosomes or 40S ribosomal subunits resume scanning in a downstream or upstream direction.


730

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Translation of the small upstream ORF may result in incomplete dissociation of soluble factors following termination, so that reinitiation is facilitated. Alternatively, sufficient distance separating the ORFs may provide enough time for Met-tRNAj and initiation factors to bind to the scanning ribosome. Reinitiation plays a key role in regulating the translational efficiency of the yeast GCN4 mRNA, where four upstream minicistrons are found [reviewed in (67 , 68)] . Elucidation of this complicated control mechanism with the assis tance of yeast genetics is providing insight into the mechanisms of initiation, termination, and reinitiation . APPROACHES TO STUDYING TRANSLATIONAL CONTROL Determinants of Protein Synthesis Rates

In order to change the rate of protein synthesis, a cell must alter the kinetic parameters of the step that is overall rate limiting. Changing the rates of other steps will not lead to translational regulation so long as the rate-limiting step remains constant. Therefore it is pertinent to ask: What limits the rate of protein synthesis in cells? The answer varies, depending on the cell type and its physiological state . A priori , rates depend on the level of the translational machinery and its specific activity. For specific protein synthesis, the limiting components are the particular mRNA's cytoplasmic concentration and specif ic activity, the latter determined by the mRNA's intrinsic activity (i.e. "strength") and by trans-acting elements . For global protein synthesis, the limiting parameters are more difficult to identify. We examine here those features of the translational machinery that contribute to determining the rates of specific and global protein synthesis. The level of an mRNA is determined by its rate of synthesis (transcription), the efficiency of posttranscriptional pro cessing and transport out of the nucleus, and its degradation rate in the cytoplasm . These issues lie outside the scope of this review and are not considered further. We note only that in terms of global protein synthesis , mRNAs seem not to be limiting. In cells such as hepatocytes following feeding , or cultured cells in exponential growth, where at least 90% of the ribosomes are active in polysomes , there remains a sizeable portion of cytoplasmic mRNA in the non-active mRNP compartment. The intrinsic efficiency whereby a mRNA is translated depends on the structure of the specific RNA [reviewed in detail by Kozak ( 1 5) and by Rhoads ( 1 6)] . Some mRNAs are translated efficiently, whereas others are translated inefficiently. What determines such efficiency? Four features appear to be important for mRNAs translated by the scanning mechanism. mRNA LEVELS AND STRUCTURE



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1. The presence and accessibility of a 5'_m 7a cap structure enhances mRNA translation, as demonstrated in vitro. All mammalian cellular mRNAs are thought to be capped, but uncapped mRNAs are found in some virus infected cells. The accessibility of the 5 I -cap structure to eIF-4F correlates with mRNA strength; higher-order structures that bury the cap reduce trans lational efficiency (69, 70). 2. Secondary and tertiary structures of mRNAs play an important role, depending on their stability and position. Secondary structure in the 5 I -UTR may affect cap accessibility and may impede the scanning 40S ribosomal subunit if sufficiently stable (>50 kcallmol) ( 1 5 , 7 1 , 72) . However, when placed just downstream from the initiator codon, such structures cause the scanning 40S ribosomal subunit to pause at the AUG and thereby enhance initiation (28). If the cap is accessible, the translational machinery can melt out moderately stable structures during scanning. This is in contrast to bacterial initiation, where efficiency is greatly influenced by secondary struc ture near the initiator AUG (73, 74) . Secondary structure in the coding region of mammalian mRNAs, even when very stable , does not block or greatly impede the elongating ribosome (75) , nor do long cDNAs annealed to this region of the mRNA (76). This likely is due to partial opening (breathing) of such structures, allowing the ribosome to advance a codon at a time, thereby eventually melting out the entire structure, regardless of its overall stability. A problem in evaluating the role of secondary and tertiary structures in trans lational efficiency is that these structures are difficult to determine ex perimentally or to predict with confidence from the mRNA sequence. S e quences in the coding or 3 I -untranslated region can bind to the 5 I -UTR by l ong-range interactions , thereby affecting initiation. A further complication is that mRNAs are presented to ribosomes , not as naked RNAs, but as mRNP particles. Very little is known about how the proteins in mRNPs influence higher-order structures and thus the process of translation (see below). 3 . The placement and context of the AUG initiator codon affect translation al efficiency as discussed earlier. The use of non-AUG initiator codons, while rare, reduces initiation strength (24). 4. Finally, the presence and length of poly(A) tails at the 3 I -terminus contribute to translational efficiency , as described in a later section of this review . The poly(A) tail length for a mRNA may vary with time, thereby affecting its translation. In summary , mRNA strength is a measure of the frequency of binding of a 40S ribosomal subunit to the AUG initiator codon and initiation of translation there. Cap accessibility is critical for opening up the 5 '-terminus of mRNAs to allow 40S preinitiation complexes to bind. Bound 40S subunits must migrate along the mRNA towards the initiator AUG, during which secondary structure and/or premature dissociation from the mRNA may influence the

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frequency of reaching the AUG. The initiator codon and its context then i nfluence the efficiency of productive initiation events. It is obvious that one cannot predict the innate strength of an mRNA on the basis of its primary structure or initiator codon context alone. LEVEL OF TRANSLATIONAL MACHINERY Ribosome levels appear to define the overall capacity of a cell to synthesize proteins. Tissues relatively active i n protein synthesis, such a s liver and brain, contain large amounts o f ribosomes and their associated soluble protein factors, whereas the opposite is true for many other cell types. Shortly after feeding , essentially all liver ribosomes are engaged in translation, and are functioning at their maximum elongation rate (77). Thus in this situation , ribosome levels limit protein synthesis. However, some hours later, only a fraction of available ribosomes are functioning; some other component becomes rate limiting. It is generally believed that the rate-limiting step of protein synthesis under most physiological conditions is the initiation phase (78). This view is based in part on analyses of polysome size, where ribosomes are usually spaced along the mRNA at intervals of 80-1 00 nucleotide residues. Were initiation rates fast relative to elongation, ribosomes could bind as close as one every 30 nucleotides, as is observed when ribosomes stack up behind pause sites in the mRNA (75). Another method to distinguish initiation vs elongation as the rate-limiting step is to treat cells with low concentrations of the elongation inhibitor, cycloheximide (79 , 80); mRNAs whose translation is elongation rate-limited are highly sensitive to the inhibitor, whereas mRNAs with relatively weak initiation rates are more resistant. Kinetic studies of translation indicate that both the 40S preinitiation com plex (8 1 ) and the i nitiation factor eIF-4F (82-84) contribute to limiting the overall rate of translation. However, polysome profiles show the presence of "half-mers," where a 40S ribosomal subunit is bound to a polysome, implying that the 60S junction step also may be relatively slow and near-limiting. It i s likely that a number o f different steps may b e close to rate-limiting, so that an i ncrease in the level of a single limiting component may cause little overall effect. The cellular concentrations of the soluble factors are likely coordinate ly regulated with ribosome levels, as has been shown for some of the initiation factors in lymphosarcoma cells down-regulated by treatment with glucocorti coids (85) . On the other hand, the ratio of eIF-2B to eIF-2 varies sub stantially, depending on the cell type; the ratio for rabbit reticulocytes is ca. 0. 1 5 whereas that for Ehrlich ascites cells is ca. 0.5 (86). Similarly, the relative and absolute levels of the two forms of eIF-4A, as deduced from mRNA concentrations, vary appreciably in different tissues (87) . Such vari ability raises the possibility that a component limiting in one cell type or physiological state may not be limiting in another. Unfortunately, a com prehensive analysis of the levels of translational components is l acking.



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SPECIFIC ACTIVITY OF TRANSLATIONAL MACHINERY Translation rates may be regulated by the activity of mRNAs, ribosomes, and their associatcd soluble factors. Regulation of specific activities rather than levels allows the cell to alter protein synthesis rates rapidly, and to reverse the inhibition or activation equally rapidly. The translational control of ferritin synthesis de scribed below is an example of the cell's need to respond quickly to an environmental change, namely the presence of iron . Posttranslational cova lent modification of protein components of the translational machinery, such as phosphorylation or methylation, is known to occur. Phosphorylation! dephosphorylation appears to be the most prevalent mechanism of translation al control, especially at the global level. The synthesis or regulation of trans-acting factors specific for one or a small class of mRNAs also may occur. How the activity of the translational machinery is regulated is the major focus of the remainder of this review.

Measuring Protein Synthesis Rates

The rate of protein synthesis is most frequently determined by measuring the incorporation of radioactive amino acid precursors into protein. Its application to estimating global protein synthesis rates is obvious; the major caveat is that intracellular amino acid pools may vary, thereby altering the specific radioactivities of the precursors. It appears that in most cases the specific radioactivity of amino acids in the intracellular pool equilibrates within one to two minutes with that of the extracellular pool (88, 89), but exceptions have been reported (90). Measurement of rates of synthesis of specific proteins requires their separation from the bulk of proteins, usually accomplished by immunoprecipitation or fractionation by two-dimensional isoelectric focus sing-SDS polyacrylamide gel electrophoresis . The methods provide a mea sure of the number of amino acids incorporated per unit time, from which the number of protein molecules synthesized per unit time can be calculated if the specific or average size and amino acid composition of the protein(s) are known. An alternative method for estimating protein synthesis rates measures the number of ribosomes actively engaged in protein synthesis together with the average rate of amino acid incorporation by the ribosomes (91). For bulk protein synthesis, the number of active ribosomes is determined by sucrose density gradient centrifugation, which separates active ribosomes (poly somes) from nontranslating 80S particles and ribosomal subunits. For specific proteins, the average number of ribosomes per mRNA and the number of specific mRNAs in polysomes may be determined from gradient analyses by hybridization techniques, yielding the total number of ribosomes engaged in protein synthesis on the specific mRNA. The rate of amino acid incorporation by active ribosomes (elongation rate) is determined by measuring the ribo-

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some transit time, the time required for a ribosome to translate a mRNA . Ribosome transit times are measured for bulk or specific protein synthesis by in vivo labeling with amino acid precursors followed by separation of released proteins from ribosome-bound (nascent) protein by centrifugation (89, 92). The method is not sensitive to variations in amino acid pools or specific activities, but requires that the synthesis of similar-sized products be com pared. The elongation rate is equal to the number of amino acid residues encoded by the mRNA divided by the ribosome transit time. Thus: protein synthesis rate

=

[# active ribosomes]

x

[# amino acid residues]

'"-------------=-

--------

ribosome transit time

Values obtained usually fall in the range of 3 to 6 amino acids incorporated per ribosome per second at 37°C. A priori, a third method is to measure the absolute rate of initiation of specific or bulk proteins. Since the rate of protein synthesis can be expressed as the number of proteins synthesized per unit time, this value under steady state conditions is equal to the number of initiation events irrespective of the elongation rate (assuming that each initiation event results in a completed protein product, a condition that holds approximately) . No general method exists for measuring initiation events in vivo directly, so this approach is not usefu l . An absolute initiation rate, however, can be calculated from polysome size and elongation rate measurements (91):

# init iation events/min

=

[ # ribosomes in polysome] ribosome transit time (min)

An efficiently translated mRNA at 37°C initiates protein synthesis once every 5 to 6 sec. Targets of Translational Control

Regulation of protein synthesis requires altering the rates of one or more of the phases of protein synthesis . To increase clarity, the initiation phase needs to be further divided and defined . We restrict the use of the term initiation to the process of ribosome binding to a preexisting polysome . The binding of the first ribosome to an mRNP to begin the formation of a polysome is quite distinct (see below) and is called mobilization. When a ribosome terminates translation on one cistron and then initiates translation on a distal cistron of the same mRNA, the process is called reinitiation (as defined earlier) . MOBILIZATION OF mRNPs INTO POLY SOMES Cytoplasmic mRNAs are found in two distinctly different functional states: those actively being trans-



735

lated by Iibosomes, in polysomes; and those that are not active, in mRNP particles. Regulation of the proportion of active to non-active mRNAs con stitutes a type of translational control that is readily distinguished from changes in the initiation rate on polysomes. A substantial amount of total cytoplasmic mRNA is found as free mRNPs in a wide variety of cells. For example, 30% of mRNAs are in mRNP particles in active muscle cells (93). An example of a shift of mRNA from polysomes into mRNPs, with little change in polysome size, is the inhibition of translation of ribosomal protein mRNAs upon treatment of mouse lymphosarcoma cells with dexamethasone (94). The amount of mRNA decreases in polysomes and increases in mRNPs, but the number of ribosomes present per mRNA changes very little. A shift of mRNA from mRNPs into polysomes is a dominant event in early develop ment, especially in sea urchins and in the maturation of Xenopus oocytes (95). Many specific mRNAs are present simultaneously in free mRNPs and in large polysomes. For example, the distribution of ferritin mRNA is bimodal in iron-depleted cells (96). Insulin mRNA is found primarily in mRNPs in islet cells treakd with low glucose, but is mobilized into polysomes in the presence of high gllucose (97). The fact that mRNPs (which have zero bound ribo somes) fall well outside the Poisson distribution for the number of ribosomes bound to a specific mRNA means that the binding of the first ribosome to an mRNP is kinetically different from the initiation rates that help maintain a polysome size. One plausible explanation is that mRNPs are repressed or masked by a trans-acting element, blocking initiation. Another is that initia tion by a ribosome on an mRNP may differ mechanistically from initiation on a polysome. What regulates the inability of mRNPs to be translated (i.e. their apparent repression)? mRNPs comprise about 75% protein and 25% RNA, with up to 30 different protein species ranging in mass from 25 to 150 kDa. Few of these proteins have been well characterized except the 72 kDa poly(A) binding protein (PABP) (98 , 99). Most of the proteins appear to be present in the RNPs of a wide range of rnRNA species , whereas it is expected (but not yet widely demonstrated) that some proteins may be restricted to a given RNA or class of RNAs. Lacking are determinations of where the proteins bind (except for PABP), their stoichiometry in the mRNP particle, and their precise functions. Some of the proteins may remain bound as the mRNA is mobilized into polysomes , since polysomal mRNA stripped of ribosomes still is com plexed with proteins. The most abundant of the polysomal RNP proteins are the PABP and a 50 kDa protcin. Recent work begins to provide some insight into how mRNPs are repressed and activated. The rate of recruitment of ribosomes into polysomes was studied in a nuclease-treated reticulocyte lysate with exogenous globin mRNA ( 100). The first ribosome bound to the mRNA rapidly, but subsequent


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ribosome binding was slower, requiring about 1 5 minutes to reach full-sized polysomes. This result is unexpected, since in vivo analyses suggest that binding of the first ribosome is a slow step. A possible explanation is that once a ribosome translates an mRNA and then terminates, it preferentially and efficiently reinitiates protein synthesis on the same mRNA. The introduction of new ribosomes from the free pool of ribosomes would appear to be a relatively slow process in this study. In another study, the ability of mRNAs to be translated in vitro, either as naked RNA or in RNP complexes, was compared in rabbit reticulocyte and wheat germ lysates ( 1 0 1 ) . In the reticulo cyte lysate, high concentrations of naked mRNA but not mRNPs inhibit protein synthesis, perhaps explained by the fact that naked RNA binds and sequesters initiation and elongation factors essential for translation ( 1 02) . The wheat germ lysate does not translate the mRNPs well, indicating that it lacks the ability to unmask the particles. Treatment of mRNPs with high salt or with the ribosomal high salt wash from reticulocyte lysates activates the mRNPs for translation in the wheat germ system, apparently by removing a masking factor ( 1 03). A 50 kDa protein , purified from mRNPs, inhibits translation of naked mRNAs in wheat germ lysates (04) and may be the putative masking factor. Standart and coworkers ( l 05) have developed a "competitive unmask ing" assay with clam oocyte mRNPs and a rabbit reticulocyte lysate. Transla tion is stimulated by addition of antisense RNAs complementary to relatively short regions in the 3 ' -UTR of the mRNAs. This suggests that the small RNAs interfere with the binding to the 3 ' -UTR of a repressor protein(s) that is responsible for masking the activity of the mRNA. The latter two studies point towards a role for trans-acting factors that specifically repress one or a small class of mRNAs. The repression of ferritin mRNA described in detail below is another example of masking by a trans-acting protein . Additional studies of the mechanism of mRNP mobilization are required to understand better how this process is controlled in cells. ALTERATION OF INITIAnON RATES Changes in initiation rates are reflected in polysome size, which is proportional to the initiation rate (and coding length of the mRNA) and inversely proportional to the elongation rate . Since the elongation rate can be determined from ribosome transit time measure ments, a change in the relative rate of initiation can be calculated (as dis cussed above) (9 1 ) . At constant elongation rate, an increased rate of initiation results in larger polysomes , whereas a decreased rate results in smaller polysomes. Severe inhibition of initiation results in essentially complete run-off of ribosomes from mRNAs and the disappearance of polysomes. A change in the rate of global initiation can be detected by shifts in the polysome profile measured by absorption at 260 nm of sucrose density gradient frac tions. Examples are the mild inhibition of protein synthesis observed during


737


mitosis in HeLa cells (92), or more severe inhibitions seen upon heat shock ( 1 06) or Sl!rum deprivation ( 107, 1 08). A change in the rate of initiation of a specific protein is most readily detected by measuring polysome sizc by hybridization with specific DNA or RNA probes . MODULATION O F ELONGATION/TERMINATION RATES W e are concerned here with temporal modulations of elongation and termination rates, as op posed to intrinsic differences in elongation efficiencies caused by codon usage or mRNA secondary structure . A priori , translational control of the elongation or termination phases seems unlikely for a number of reasons: 1 . the initiation phase generally is rate-limiting, so modest changes in the elongation rate should have little or no effect on the overall rate of protein synthesis; 2 . the elongation and termination phases occur by the same basic mechanism for all mRNAs and are thought to be rather insensitive to mRNA sequence or structure , so regulation of specific mRNAs is difficult to envision; 3. stimula tion of elongation rates can only occur to a limited extent since most cells are functioning at near-maximal rates (ca. 5 amino acids/ribosome/sec); and 4. inhibition of elongation of specific mRNAs would result in an inefficient use of ribosomes, since they would be unavailable for the translation of other mRNAs . Nevertheless, small changes in elongation rate are seen and often accompany changes in initiation rates. For example, HeLa cells deprived of serum for 24 h exhibit an elongation rate that is half that of serum-fed cells (89) . Severe inhibition of elongation has been reported for Drosophila mRNAs following heat shock ( 1 09) [although the postulated block at elonga tion is disputed ( 1 10)] and for mRNAs in HeLa cells stressed by amino acid analogues ( I l l) . The viral S l mRNA in reovirus-infected cells also appears to be regulated at the level of elongation ( I 12). Perhaps the best documented example of such regulation concerns translation of the HSP 70 mRNA in chicken reticulocytes ( 1 1 3) . In this case, the mRNA in nonstressed cells is present in polysomes but is translated very slowly, whereas following heat stress , the elongation block is removed and the efficiency of HSP 70 synthesis increases 10-fold. During elongation, ribosomes may shift the reading frame , or upon reach ing a termination codon, may read through the stop signal. These events are usually regarded as translational errors and they occur at low frequencies. It is now appa:rent, however, that the frequency of such errors can be increased substantially by cis-acting sequences in the mRNA, and can be utilized as a means to control gene expression. Regulation through frameshifting and readthrough is developed in more detail below. The yidd of protein product from an mRNA is usually thought to be directly proportional to the number of translational initiation events . This is not strictly true , however, since not all ribosomes that initiate actually com-


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plete synthesis of the protein. As has been shown in prokaryotic cells, there is a measurable frequency whereby ribosomes abort synthesis by falling off the mRNA before reaching the termination codon. This rate, approximately 2 X 10-4 abortive reactions per codon translated ( 1 14), is substantial but may be less in mammalian cells given that extremely large proteins (>500 kDa) are known to be synthesized. The ability of a ribosome to retain the peptidyl tRNA at each step depends on the affinity of peptidyl-tRNA binding. Howev er, high-affinity binding is expected to result in slow rates of tRNA release at steps such as translocation. Thus processivity and rate of protein synthesis are diametrically opposed. It is tempting to speculate that the need to synthesize very long proteins is the cause for the threefold slower overall rate of elongation in mammalian cells compared to prokaryotes. Little or nothing is known about ribosome processivity in mammalian cells, or about if this aspect of the elongation phase is regulated under special circumstances . GLOBAL CONTROLS B Y PHOSPHORYLATION Phosphorylation/dephosphorylation of numerous protein components of the translational apparatus appears to play an important role in controlling the overall rate of protein synthesis in mammalian cells [reviewed in ( 1 1 5)] . A large number of phosphoproteins have been identified: at least 1 3 initiation factor subunits; 3 of the 4 proteins comprising the elongation factors; 3 ribosomal proteins; and a number of aminoacyl-tRNA synthetases . Phosphorylation of a subset of the components correlates with inhibition of translation, whereas phosphorylation of others may cause stimulation. Cur rent research in this area seeks to identify the precise sites of in vivo phosphorylation and the protein kinases and phosphatases involved, to demonstrate in vitro a change in activity of the protein due to phosphoryla tion, to elucidate how the extent of phosphorylation is controlled, and to establish that phosphorylation actually causes modulation of translation rates in vivo. S timulation of Protein Synthesis and Cell Growth

The phosphorylation of a number of initiation factors , namely eIF-2B , eIF-3 , eIF-4B , and eIF-4F, as well as ribosomal protein S6, correlates with activa tion of protein synthesis in vivo and/or with increased factor activity in vitro ( 1 15). We discuss those factors implicated in mRNA binding here, and postpone consideration of eIF-2B to the following section on repression of translation. The best-studied initiation factor in this group is the relatively low abundance cap-binding protein, eIF-4Fa (eIF-4E) , which is phosphorylated

eIF-4F



739

in vivo primarily at Ser53 ( 1 1 6) . The same site is phosphorylated in vitro by protein kinase C ( 1 17 , 1 1 8) , where the preferred substrate is the eIF-4F complex rather than the free e1F-4Fa subunit ( 1 1 9) . Another kinase specific for elF-4lFa has been identified ( 1 20) , but is not yet well characterized. Phosphorylation of eIF-4Fa does not appear to alter its affinity for m7G cap structures ; however, numerous correlative data link phosphorylation and the active state of the factor. The factor is dephosphorylated in eells during mitosis ( 1 2 1 ) or when treated at high temperatures (25), conditions that inhibit protein synthesis, and is phosphorylated by treatment of quiescent cells with serum, insulin, tumor necrosis factor a, or the mitogen , phorbol ester (TPA) ( 1 1 8 , 1 22, 1 23). These results suggest that phosphorylation of e1F-4Fa activates the factor. In vitro studies with radiolabeled protein expressed from e1F-4Fa cDNAs demonstrate that the protein binds to 40S initiation com plexes, where it is mainly in the phosphorylated state ( 1 24) . In contrast, a mutant form, where Ala is substituted for Ser53 thereby preventing phosphorylation at that site, does not bind to the complexes . A startling finding is that the wild-type e1F-4Fa eDNA, when overexpressed in murine 3T3 cells or in rat 2 fibroblasts, causes the cells to undergo malignant transformation and become tumorigenic ( 1 25). Furthermore , microinjection of eIF-4F or e1F-4Fa into serum-starved NIH 3T3 cells results in the stimula tion of DNA synthesis and a transient morphological transformation ( 1 26) . A role for phosphorylation in these events is implied by the fact that overexpres sion or microinjection of the Ser53-Ala mutant form of eIF-4Fa is not oncogenic . The results above implicate the factor as a key target in the phosphor l ation cascades that lead to control of cell growth through signal transduction mechanisms and protein kinase C. The Y-8ubunit of eIF-4F, p220, also is phosphorylated in 3T3-Ll cells treated by insulin and phorbol esters ( 1 27) . Furthermore, protein kinase C and protease-activated kinase I and II phosphorylate e1F-4Fy in vitro ( 1 1 9) . When eIF-4F is treated with protein kinase C, there is a fivefold increase in the factor's al�tivity in a cell-free globin synthesis assay ( 1 28). Since the a subunit also is phosphorylated by protein kinase C, it is not yet clear if phosphorylation of either or both the a- and y-subunits causes enhanced activity. llhe site(s) of phosphorylation of e1F-4Fy have not yet been de tennined, nor is it certain that protein kinase C is responsible for the protein's phosphorylation in vivo. y

S6 is phosphorylated in vivo on up to five serine RIBOSOMAL PROTEIN s 6 residues near the C-terminus of the protein [reviewed in ( 129)] . Phosphoryla tion corre1ates with activation of protein synthesis caused by mitogens and growth factors , but no compelling evidence has been generated to show that S6 phosphorylation enhances the activity of 40S ribosomal subunits in trans-


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lation. Protein kinases relatively specific for S6 have been purified from a variety of cell types ( 1 30-1 32) , and a cDNA encoding a mouse S6 kinase has been cloned recently ( 1 33). The mammalian S6 kinase itself is stimulated upon phosphorylation on serine/threonine by an unidentified kinase ( 1 34) , although MAP-2 kinase has been implicated in the Xenopus system ( 1 35 ) . Protein kinase C also i s implicated i n the pathway , since treatment o f cells with phorbol esters increases S6 phosphorylation ( 1 36) . Phosphoprotein phos phatase type- l dephosphorylates S6 and is differentially regulated by insulin and epidermal growth factor ( 1 37). Thus, a complex net of kinases and phosphatases appears to be involved in establishing the phosphorylation level of S6. A role for S6 in the regulation of protein synthesis may be better established now that its kinase has been cloned. OTHER INITIATION FACTORS eIF-4B , like S6, is phosphorylated at multiple sites, namely on eight or more serine residues. The hyperphosphorylation of eIF-4B in vivo correlates with activation of protein synthesis, and partial dephosphorylation occurs upon repression of translation caused by heat shock and serum deprivation ( l 06, 1 38). eIF-4B is phosphorylated in vivo when cells are treated with serum or phorbol esters ( 1 27) and in vitro by the mammalian or Xenopus S6 kinases (R. Duncan , unpublished results) as well as by protein kinase C and a variety of other kinases ( 1 1 7) , but it remains to be shown whether or not the sites are identical to those phosphorylated in vivo . There is as yet no evidence that the phosphorylation state of eIF-4B affects the factor' s activities in vitro or in vivo. The difficulty in demonstrating activity changes due to phosphorylation stems from the low activities of highly fractionated assays , and to rapid changes in the phosphorylation states in more active, crude assay systems. Another problem may be that simultaneous phosphorylation of a number of different translational components is required to effect a measurable change in protein synthesis. eIF-3 is another initiation factor whose phosphorylation on the TJ-subunit ( 1 1 5- 1 20 kDa) is enhanced by insulin and phorbol esters ( 1 27). eIF-3 com prises at least eight different polypeptide subunits ( 1 39), and evidence for in vivo phosphorylation on the 8 - , E - , g- , 1)- , and 8-subunits exists ( 1 39, 140). No reports implicate phosphorylation of these subunits in translational con trol, however. The simultaneous stimulation of the phosphorylation on eIF-4F, eIF-4B , eIF-3, and ribosomal protein S6 by phorbol esters and growth factors suggests that there is coordinate regulation of these translational components. Too little is known about the kinases involved in modifying these proteins to speculate how the apparent coordinatio:l is achieved , but it seems safe to postulate that protein kinase C is a part of at least one of the pathways. eIF-4F seems to be a particularly important target for control of cell growth and translation rates . It


74 1

remains to be established whether or not phosphorylation of the other proteins is necessary for, adds only marginally to, or is entirely gratuitous with respect to change5: in rates of protein synthesis.


Repression of Translation Repression of global protein synthesis is seen in cells deprived of serum, starved for amino acids, and subjected to stress by heat, among other things. In each of these cases , the extent of phosphorylation of elF-4Fa and eIF-4B is decreased ( 106, 1 3 8 , 1 4 1 ) and may contribute to reduced protein synthesis activity, as described above. Thus increased phosphatase action or decreased kinase activity on any of the proteins whose phosphorylation is implicated in stimulation of protein synthesis is expected to contribute to translational repression .. In addition to this kind of control, inhibition of protein synthesis may be c2lUsed by enhanced phosphorylation of two other soluble factors:

cIF-2 and eEF-2. eIF-2 AND eIF-2B

The phosphorylation of the a-subunit of eIF-2 was first detected ( 142) in rabbit reticulocyte lysates deprived of hemin [the reader is referred to Jackson's recent review ( 143) of the extensive literature on this subject] . The absence of hemin results in an activation of a highly specific protein kinase, called the hemin regulated inhibitor (HRI, or HeR). The phosphorylation of eIF-2a in many other cell types also correlates with inhibition of the initiation rate, suggesting that this is a common mechanism for controlling protein synthesis ( 1 1 5). At least one other highly specific eIF-2a kinase, an interferon-induced protein called the double-stranded RNA activated inhibitor (DAI, or dsI), phosphorylates the same site, namely Ser5 1 ( 144, 145). The phosphate on Ser5 1 turns over rapidly , and broad-specificity phospho-protein phosphatases of type- l and type-2A are implicated. Phosphorylation of only 25-30% of the factor is sufficient to cause a strong inhibition of the initiation phase in reticulocyte lysates. Phosphorylation does not directly inhibit formation of the ternary complex or ribosomal initiation complexes, as these reactions proceed efficiently in vitro with completely phosphorylated factor ( 1 46). Rather, it impedes the guanine nucleotide ex change reaction that enables the factor to recycle and promote multiple rounds of initiation (Figure 1 ) [reviewed in ( 147)] . Phosphorylated eIF-2·GDP binds more tightly to elF-2B , but does not exchange GDP for GTP ( 1 1 , 46) . Since the cellula:r level of eIF-2B is thought to be lower than that of eIF-2 , only a portion of dF-2 need be phosphorylated in order to inhibit eIF-2B activity. In effect, eIF-2B is sequestered, thereby preventing its catalysis of GTP ex change on nonphosphorylated eIF-2. Although this mechanism appears to explain the inhibition caused by HRI or DAI, a number of issues remain


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unresolved. Evidence suggests that the guanine exchange reaction may occur on the surface of the ribosome, and that phosphorylation may result in a failure of eIF-2 ·GDP to be ejected, thereby inhibiting 80S initiation com plex formation ( 1 48-1 50) . Elucidation of the molecular mechanisms that control HRI and DAI is incomplete, and still other eIF-2a kinases may he involved. Although the phosphorylation of eIF-2a correlates with inhibition of pro tein synthesis and results in loss of factor activity in vitro, these findings do not prove that eIF-2a phosphorylation actually causes translational repression in vivo. To address this point, mutant forms of cDNA encoding eIF-2a altered at the site of phosphorylation were transfected and expressed in mammalian cells. Expression of eIF-2a with the Ser5 1 residue substituted with Asp, which resembles phosphoserine , results in severe inhibition of global protein synthesis following accumulation of small amounts of the protein ( 1 5 1 ) . The mutant form of eIF-2a exchanges into the endogenous eIF-2 complex (S . -Y . Choi, unpublished results) and presumably mimics phosphorylated eIF-2 . Phosphorylation therefore appears to be sufficient to cause inhibition in cells under the growth conditions used in this study . The mutant factor, eIF-2a(Ser5 1 -Ala), stimulates the translation of both plasmid derived mRNAs in transiently transfected COS - l cells ( 1 5 1 ) and viral mRNAs in human 293 cells infected with a mutant form of adenovirus lacking the VA-/ gene ( 1 52). In these cells, DAI kinase is activated ( 1 53, 154) but the repressive effects of the kinase are prevented by the mutant eIF-2a protein, which is not phosphorylated . The results implicate the importance of eIF-2a phosphorylation by the DAI kinase and indicate that eIF-2a phosphorylation is the cause of translational repression in intact cells. eIF-2 phosphorylation is perhaps the best-characterized translational con trol mechanism in mammalian cells. A large body of evidence points to its involvement in many instances of translational repression, although not every kind of inhibition involves this mechanism ( I SS). A complication in the interpretation of the above results is the observation that inhibition of protein synthesis does not always occur when elF-2a is phosphorylated. For ex ample, 2-aminopurine prevents host mRNA shut-off following adenovirus infection, although DAI remains active and eIF-2a is phosphorylated to a relatively high extent ( 1 56) . A possible explanation is that eIF-2B may be modified to suppress the effects of eIF-2a phosphorylation. Dholakia & Wahba ( 157) have shown that eIF-2B is phosphorylated in vitro on the e-subunit (82 kDa) by casein kinase II, resulting in a fivefold stimulation of its specific activity in the guanine nucleotide exchange reaction. They also argue that NADPH binding to the protein alters its function ( 1 58). It is therefore possible, but not yet demonstrated, that repression by eIF-2a phosphorylation may be modulated by modifications of eIF-2B structure and/or activity .



743

eEF-2 A :;econd phosphorylation pathway for inhibition of protein synthesis involves the elongation factor, eEF-2. Evidence for the phosphorylation of this factor comes from analyses of reticulocyte lysates incubated with [y32p]ATP ( 1 59) and of mammalian fibroblasts treated with serum or growth factors ( 1 60) . A highly specific kinase , called the Ca2+ Icalmodulin dependent protein kinase III or eEF-2 kinase , has been identified and shown to phosphorylate primarily Thr56 ( 1 61 , 1 62), but Thr53 and Thr58 are also labeled on prolonged treatment. These Thr residues reside in a region that may be involved in eEF-2 binding to ribosomes ( 1 63). Phosphorylated eEF-2 binds to 80S ribosomes but does not promote in vitro the translocation reaction with GTP ( 1 64) . Thus phosphorylation inactivates eEF-2 and inhibits the elongation phase of protein synthesis. This is consistent with the findings that cAMP stimulation of reticulocyte lysates correlates with dephosphoryla tion of eEF-2 ( 1 65), whereas okadaic acid inhibition correlates with its phosphorylation ( 1 66) . What is the physiological role of eEF-2 phosphoryla tion? Celis and coworkers ( 1 67) reported that in transformed human amnion cells, eEF-2 becomes phosphorylated during mitosis, when a transient in crease in Ca2+ concentration occurs. The view that elongation is inhibited during mitosis may not apply generally, however, as there is contradictory evidence for a decreased initiation rate with no change in elongation rate in mitotic HeLa cells (92). It is perplexing that eEF-2 is transiently phosphory lated in fibroblasts treated with serum or growth factors ( 1 60), circumstances that cause a stimulation of protein synthesis. Since treatment of cells with cycloheximide causes ribosomes to shift into polysomes (79) , a transient inhibition of the elongation rate by phosphorylation of eEF-2 also may result in the mobilization of ribosomes and mRNPs. The widespread occurrence of eEF-2 phosphorylation and its proven effects on protein synthesis in vitro likely will attract considerable interest in elucidating the physiological roles played by this phosphorylation mechanism . Other Phosphorylation Targets The phosphorylation of a variety of other protein components of the trans lational apparatus has been described but not yet studied extensively. Although no clear role in translational control is indicated at this time, some of these modifications may turn out to be especially significant. The f3-subunit of eIF-2 is phosphorylated in vivo, but there are only minor changes in its phosphorylation status when protein synthesis is either inhibited or stimulated ( 106, 1 68). eIF-2f3 is phosphorylated on Ser2 by casein kinase II and on Ser 1 3 b y protein kinase C ( 1 69 , 1 70) . eIF-5 i s phosphorylated at multiple sites in vivo and by protein kinase II in vitro ( 140), but changes in the extent of phosphorylation have not been determined. Phosphorylated eEF- l a has been reported ( 17 1 ) but not characterized. Potentially interesting is eEF- l f3y,


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proteins involved in the guanine nucleotide exchange reaction with eEF- I a, which are substrates for P34cdc32 protein kinase ( 1 72) . Here, phosphorylation may contribute to inhibition of protein synthesis during mitosis, together with the dephosphorylation of eIF-4Fa ( 1 2 1 ) and the phosphorylation of eEF-2 ( 1 67). Artemia eEF- l f3 is phosphorylated on SerS9 in vitro, resulting in a decrease in its GTP exchange activity ( 173). Besides ribosomal protein S6, the large subunit acidic proteins PI and P2 are phosphoproteins ( 1 74) , and the small subunit proteins S2 and S 1 3 are phosphorylated following infection of cells by vaccinia virus ( 1 75 ) . Whether or not the phosphorylation status of any of these ribosomal proteins affects translation rates remains to be determined, although phosphorylation of P I -P2 is required for their in vitro assembly into 60S particles ( 1 76) . Finally, up to eight different aminoacyl-tRNA syn thetases are phosphorylated ( 177) , although there appears to be no change in their capacities to charge tRNAs [see the recent discussion by Clemens ( 1 7S)] . In summary, there exists a large number of phosphorylation targets among the proteins in the translational apparatus . It is possible that many of these modifications are gratuitous , without functional effects. Yet is also is likely that regulation by phosphorylation proceeds by mUltiple, redundant mech anisms, thereby ensuring proper control of this crucial pathway. OTHER CONTROL MECHANISMS Control of global rates of protein synthesis is important in coordinating translation with the overall metabolism of the cell, whereas regulation of a single mRNA or a class of mRNAs may be employed to provide temporal control of specific gene expression. Specific translational controls are known to operate in bacteria, where the synthesis of ribosomal proteins ( 1 79) and a variety of other proteins ( 1 80) are under autogenous control . That is, the gene product, upon reaching a given critical concentration in the cell, binds to its mRNA and inhibits its own synthesis . Likewise, bacteria and their phages regulate the synthesis of specific or groups of proteins with translational repressors ( I SO) . It is therefore likely that mammalian cells would employ translational control mechanisms to regulate the synthesis of specific proteins. Such regulation is expected also because control at the level of transcription and RNA processing involves a time delay of 1 5 minutes or more in the response, whereas translational control can be much more rapid. A number of cases involving regulation of specific mRNAs have been described and a detailed mechanism has been elucidated for ferritin mRNA (see below) . We focus on a few examples of control mechanisms involving whole classes or individual mRNAs with the intent to provide some insight into the kinds of regulation that can occur.


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Polyadenylation of mRNAs Most mammalian mRNAs are polyadenylated soon after transcription in the nucleus, and carry 200-250 adenylate residues at their 3 ' -termini as they are transported into the cytoplasm. The poly(A) tails are further metabolized in the cytoplasm, where in general they are gradually shortened to about 50-70 residues, but also can be lengthened by cytoplasmic enzymes. Besides possi ble functions in nuclear processing and transport, the poly(A) tails are thought to affect both mRNA stability and translational efficiency. The role of poly(A) taitls in mRNA degradation and translation has been reviewed recent ly ( 1 8 1 ) . We shall limit our discussion to their influence on the efficiency of translation. A role for poly(A) is indicated by in vitro studies of rabbit reticulocyte lysates, where mRNAs carrying poly(A) tails are translated more efficiently than their deadenylated counterparts ( 1 82) . The requirement of a poly(A) tail is not absolute, as considerable translation occurs with poly(A)- mRNAs. Further in vitro evidence comes from the observation that addition of oligo(A) molecules inhibits protein synthesis ( 1 82, 1 83). Reversal of inhibition by the poly(A) binding protein (PABP) suggests that the oligo(A) molecules com pete for the PABP and implies that the PABP and poly(A) tail together are positive effectors of translation. A detailed investigation showed that poly(A)- mRNAs are recruited less efficiently into polysomes compared to poly(A)+ InRNAs, possibly through a defect in the junction step of the 60S subunit with the 40S initiation complex ( 184). A compelling case for poly(A) stimulation in vivo comes from studies of the tissue plasminogen activator (TPA) mRNA ( 1 85). Its poly(A) tail is lengthened to 400--600 adenylate residues during mouse oocyte maturation, resulting in a strong activation of translation . Polyadenylation and translational activation also occur upon microinjection of an in vitro chimeric transcript carrying the 3 ' -UTR of the TPA mRNA inserted next to a reporter gene. Experiments involving microinjection of mammalian mRNAs into Xenopus oocytes provide evidence in support of the view that polyadenylation affects translational efficiency. mRNAs with poly(A) tails are translated much more efficiently than those lacking poly(A) , a result that is not attributable to differences in mRNA stability ( 1 86, 1 87). In a recent study of the mobiliza tion of maternal mRNAs into polysomes , Richter, Dworkin, and coworkers found that the act of cytoplasmic polyadenylation, not the size of the poly(A) tail per se , is required for activation of the mRNA ( I 88). However, it is uncertain whether or not results from Xenopus oocytes can be extrapolated to mammalian systems. Finally , genetic studies in yeast provide some insight into the role of poly(A) and the PABP in translation. Deletion of the PABP gene is lethal and

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second site revertants have been obtained, one of which is a 60S ribosomal protein ( 1 89) . Careful examination of the polysome profiles from PABP deficient cells indicates that the amount of RNA in polysomes is decreased, but not the number of ribosomes per mRNA. This suggests that poly(A) and PABP are involved in establishing the distribution of mRNA between mRNPs and polysomes rather than in the initiation rate on polysomes.


Poliovirus-Infected Cells When poliovirus infects a mammalian cell, host mRNA translation is strongly inhibited, whereas poliovirus mRNA is translated vigorously [for a com prehensive review, see ( 1 90)] . The mechanism for selectively inhibiting the cell' s mRNA translation involves proteolytic cleavage of the y-subunit of eIF-4F ( 1 9 1 ) , thereby inactivating the factor and reducing translation of capped mRNAs by the scanning mechanism. Poliovirus mRNAs are not capped and initiate by the internal initiation mechanism, thereby evading the effects of eIF-4Fy cleavage. Apparently the infection also activates an eIF-2a kinase, leading to the phosphorylation of eIF-2 ( 1 92) . It is proposed that cleavage of eIF-4FI' results in only a partial inhibition of capped mRNA translation, and that eIF-2a phosphorylation further inhibits these mRNAs. It is unclear how the internal initiation mechanism would be less sensitive to repression by eIF-2 phosphorylation . The poliovirus genome encodes two proteases, named 2A and 3C, which are involved in the processing of the polyprotein translation product. Protease 2A is implicated in eIF-4Fy cleavage, yet appears not to cleave the initiation factor directly, but to activate a cellular protease. It has been shown recently that both protease 2A and eIF-3 are required to generate the eIF-4Fy protease activity ( 1 93). Possibly eIF-3 itself carries the catalytic site for eIF-4Fy cleavage, or eIF-3 alters the substrate or protease conformation to promote cleavage. It is interesting to speculate that there may be cellular mechanisms not involving poliovirus protease 2A that activate cIF-4Fy cleavage and cause inhibition of protein synthesis. Indeed, specific cleavage of eIF-4Fy compara ble to that seen in poliovirus-infected cells, is occasionally observed. In this regard, it has been shown recently that eIF-4Fy is a substrate for multiple Ca2+ -dependent enzymes ( 1 94) . It remains to be shown whether or not such enzymes play a role in regulating translation by cleavage of eIF-4Fy. Ferritin mRNA Ferritins are ubiquitous intracellular proteins that bind and store iron mole cules, thereby protecting cells from the toxic effects of free iron. Expression of the two highly homologous genes, H and L, is dramatically regulated at the translational level by the extracellular iron supply [for a review, see ( 1 95)] . Ferritin mRNA is repressed in iron-deficient cells, but the rate of ferritin



747

synthesis is stimulated 1 0--40-fold when cells are shifted to an iron-containing medium. Whereas a part of the induction may be attributed to enhanced transcription in some cells ( 1 96) , most is due to an increase in translational efficiency of preexisting mRNAs. Analysis of cis-acting mRNA sequences identified a conserved 28-nucleotide region in the 5 ' -UTR of all ferritin mRNAs that is necessary for iron regulation ( 1 97 , 1 98) and that confers such regulation when inserted into other mRNAs. The sequence, called the iron responsive element (IRE), may fold into a stem-loop structure . Mutations affecting the strength of the putative stem-loop structure alter the regulatory properties of the IRE ( 1 99). It is interesting to note that transferrin receptor mRNA contains five similar IREs , but in contrast to ferritin mRNA, they are situated in the 3 ' -UTR. The transferrin receptor IREs play a role in protecting the mRNA from degradation when iron levels are low. A trans-acting ferritin repressor protein (FRP) has been purified to homogeneity from mammalian cells or tissue on the basis of its binding to RNA sequences containing the IRE (200--202). The protein inhibits ferritin mRNA translation in a wheat germ cell-free system (203), leading to the conclusion that the FRP is a repressor of ferritin mRNA translation in the absence of iron . Thach and coworkers have shown that hemin, not iron itself, reverses the inhibition of in vitro ferritin synthesis by FRP (204). They argue that hemin may be a physiological inducer in mammalian cells (205). An alternative" though not necessarily a mutually exclusive, view is that the redox state of the FRP is critical. When the FRP is in an oxidized state, its affinity for the IRE is low (Kd 5 nM); upon reduction, a high-affinity (Kd �20 pM) ]FRP form is generated (206). Klausner and coworkers propose a disulfide-sulfhydryl switch regulated by iron (207). The position of the IRE in the 5 ' -UTR is critical: no inhibition by the FRP occurs when the IRE lies more than 67 nucleotides distal from the cap (208). This suggests that the IRE-FRP complex inhibits cap recognition or the initial 40S ribosomal sub unit binding at the 5 ' -terminus rather than the scanning process itself. The recent cloning of the cDNA encoding the FRP (209) should provide a tool for better evaluating the mechanism of mRNA binding and derepression. =

=

Frameshifting and Readthrough A translating ribosome normally advances along the mRNA by precisely three nucleotides; at each tum of the elongation cycle, then terminates synthesis when a nonsense codon is encountered in the A site. However, at a frequency estimated to be less than 1 x 10-3, a mistake (other than incorporation of a wrong amino acid at a sense codon) is made and the ribosome either shifts its mRNA reading frame or binds an aminoacyl-tRNA at the nonsense codon and continues translation. In both prokaryotic and eukaryotic cells, the structures


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of some specific mRNAs have evolved so as to increase the tendency to frameshift or read through termination codons. By increasing the frequency of such errors, the ribosome is able to translate a more distal part of the mRNA at low but significant levels . In addition, bacterial ribosomes can "hop" over a significant number of nucleotides, leaving an internal section of the mRNA untranslated, as seen in the bacteriophage T4 DNA topoisomerase gene 60 mRNA (2 1 0, 2 1 1 ) . No evidencc for hopping in mammalian cells has been found, however. We examine here a number of examples of how frameshift ing and readthrough are employed to express downstream regions in mamma lian mRNAs [reviewed recently in (21 2)] . FRAMESHIFfING Frameshifting is usually caused by slippage of a tRNA derivative from one codon to an overlapping codon in the - l or + 1 reading frame . For example, tRNALeu, which binds initially to CUU in the sequence CUU UGA of the RF-2 mRNA of E. coli, slips to UUU, thereby establishing a new + 1 reading frame (2 1 3). Runs of homopolymers facilitate frameshift ing and thereby create potential shift sites. In mammalian systems , frameshifting is frequently encountered in the gag-pol region of retroviruses, where a - 1 shift is invariably seen. The frameshift enables the virus to express at low levels the pol gene, which lies in a different translational reading frame from the upstream gag gene. In many cases, two adjacent tRNAs shift in tandem, for example at the sequences A AAA AAG in mouse mammary tumor virus (MMTV) (214), and at U UUA AAC in the coronavi rus IBY ( 2 1 5 ) . The frequency of the frameshift event is increased by other elements in the mRNA sequence, particularly those that cause pausing of the ribosome over the shifty sequence. Examples of stimulators of frameshifting are: secondary structure elements, particularly pseudoknots, located about seven nucleotides downstream from the shift site, seen in Rous sarcoma virus (2 1 6) and coronavirus !BY (2 17); an adjacent stop codon , as in the E. coli RF-2 mRNA (2 1 3); and (perhaps only in bacteria) the upstream Shine/ Oalgamo region of the RF-2 mRNA that interacts with 16S rRNA (2 1 8) . Frameshift frequencies range from a few percent to a s high a s 2 3 % (for MMTY) , quite sufficient to synthesize adequate amounts of the pol gene product. READTHROUGH

Readthrough enables the ribosome to continue to translate a downstream, in-frame region of an mRNA that is distal to a stop codon . The readthrough phenomenon is seen in the murine leukemia virus, where the gag and pol genes are separated by a UAG stop codon that is read at 10% frequency by tRNAG1n to generate a fusion protein (2 1 9) . Other examples are readthrough of UGA in Sindbis virus (220) and UAG in feline leukemia virus (22 1 ) . As in frameshifting, secondary structure elements may enhance the



749

frequency of such readthrough events (222, 223). The insertion of selenocys teine into glutathione oxidase in mammalian cells is coded by a UGA codon (224) , and therefore represents a special class of the readthrough mechanism . Selenocysteinyl-tRNA is synthesized from a minor seryl-tRNA that recog nizes the UGA codon. The mechanism of mammalian cell recognition of the specific UGA codons where selenocysteine is incorporated is not known. However, in the synthesis of formate dehydrogenase in E. coli, nucleotides 3 ' to the UGA are critical (225) and a specific EF-Tu-like elongation factor is involved (226). It will be interesting to elucidate further the molecular mechanisms that lead to specific incorporation of selenocysteine in both prokaryotic and eukaryotic proteins. Miscellaneous Control Mechanisms A variety of additional translational controls are not addressed due to limita tions of space . Global controls by intracellular pH and energy charge have been observed, and Ca2+ levels are reported to affect protein synthesis [for a recent review , see (227)] . Cells subjected to nutrient deprivation , heat shock, or heavy metals are translationally repressed [reviewed in (228)] , likely by mechanisms involving the phosphorylation/dephosphorylation of translational components . In contrast, hypertonic shock of cells causes an inhibition of the initiation phase (229) by an unknown mechanism not involving changes in eIF-2 or eIF-4B phosphorylation ( 1 38). A sizeable body of evidence points to an involvement of the redox potential in the cell, and sugar phosphates or other smaU metabolite molecules may affect the translational machinery [see discussion by Jackson ( 143)]. Hormones influence protein synthesis, likely in large part through the phosphorylation mechanisms discussed earlier, but other pathways of control may occur as well. Infection of mammalian cells by viruses frequently leads to inhibition of host protein synthesis, and a number of different mechanisms are involved [reviewed in ( 1 54)] . Small cytoplasmic RNAs may specifically inhibit translation, in some cases through com plementary interactions (230). Polysomal mRNAs appear to be attached in some way to the cytoskeleton (23 1 ) , yet little is known about the nature of the attachment or its importance in translation . Finally , degradation of mRNAs is sometimes coupled to their translation. The best-studied example is the tubulin system, where translation of tubulin mRNA is required for mRNA degradation by a mechanism that recognizes the nascent polypeptide product (232). The main topics of this review and the above examples do not exhaust the list of translational control possibilities , but indicate the richness and variety of ways to regulate gene expression at thc translational level. As our knowledge of ribosome structure and the protein synthesis pathway is refined, it should be possible to describe these translational control mechanisms in detailed molecular terms .

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ACKNOWLEDGMENTS


I thank Drs. E. Ehrenfeld, H . Ernst, R . J. Kaufman, W. C . Merrick , N . Sonenberg, and R. E . Thach together with members of m y laboratory group for helpful comments on the text, and Drs. J . Bag , M . Clemens, R . Jackson, R. Rhoads, and L. Slobin for sharing manuscripts prior to publication . I am especially indebted to Drs . R. F. Duncan and R. J. Jackson for numerous stimulating discussions and insightful criticisms. The work from the author's laboratory was supported by USPHS grant GM22 1 35 from the N . I . H . Literature Cited 1 . Moldave, 2. 3.

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Translational control of ribosomal protein production in mammalian cells.

Stress-mediated translational control in cancer cells.

Optogenetic control of signaling in mammalian cells.

Automatic Control of Gene Expression in Mammalian Cells.

Orthogonal optogenetic triple-gene control in Mammalian cells.

Translational control in embryonic muscle.

Ribosome profiling reveals translational regulation of mammalian cells in response to hypoxic stress.

Ire1-mediated decay in mammalian cells relies on mRNA sequence, structure, and translational status.

[Synthetic RNA technologies to control functions of mammalian cells].

Translational Control during Calicivirus Infection.

Reporters transiently transfected into mammalian cells are highly sensitive to translational repression induced by dsRNA expression.

Translational control during early development.

Translational control in germline stem cell development.

The proteome under translational control.

Translational control in the mealworm, Tenebrio molitor.

Oligopyrimidine tract at the 5' end of mammalian ribosomal protein mRNAs is required for their translational control.

Positive mRNA Translational Control in Germ Cells by Initiation Factor Selectivity.

Glutamate-dependent translational control through ribosomal protein S6 phosphorylation in cultured bergmann glial cells.

Hormone-mediated translational control of protein synthesis in cultured cells of Glycine max.

Understanding translational control mechanisms of the mTOR pathway in CHO cells by polysome profiling.

Post-translational modification: Sweetening protein quality control.

Functional Integration of mRNA Translational Control Programs.

Translational control by oncogenic signaling pathways.

Cap-Independent Translational Control of Carcinogenesis.