Volume 14

Number 7 July 1975

Pages 445 - 506

International tditron in English Biosynthesis of Ribonucleic Acids By John P. Richardson[*] Biosynthesis of ribonucleic acids involves the polymerization of ribonucleotides on deoxyribonucleic acid templates and the conversion of these primary gene transcripts to mature RNA molecules. The present report reviews briefly our current understanding of these processes.

1. Introduction RNA is a significant and essential cellular component that can comprise as much as a quarter of the dry weight of a cell[’*21. Most RNA is involved in protein synthesis, but a small amount is needed in the replication of DUA and some RNA may also have a regulatory function. About half the RNA that is synthesized at any given moment goes into molecules that are stable. These are the ribosomal RNAs (rRNAs), which are structural components of ribosomes, and the transfer RNAs (tRNAs), which serve to adapt the amino acids to the genetic code during protein synthesis. The other halfgoes into RNA molecules with very low metabolic stability. These are the messenger RNAs (mRNAs), which are the templates for protein synthesis; many of the nuclear RNA molecules, which are precursors to the stable RNAs and mRNA; and the RNA intermediates involved in DNA replication. The lifetime of these RNAs is often less than one-tenth the cell generation time. Because such a large fraction of the RNA is unstable, the cell composition under-represents the fraction of the synthetic capacity devoted to RNA; hence, on a weight basis, RNA synthesis accounts for of all macromolecular synthesis. Chemically, RNA is a linear polymer of ribonucleotides linked together by phosphodiester bonds between the 5’ carbon of one ribonucleotide and the 3’ carbon of an adjacent ribonucleotide. The four common ribonucleotides in RNA have the bases adenine, guanine, cytosine, and uracil; but the stable [*] Prof. Dr. J. P. Richardson Department of Chcmistry Indiana University Bloomington. Indiana 47401 (USA)

RNAs, especially the tRNAs, also have small but significant fractions of other ribonucleotides that are formed by modification of the four common ones. In many of its chemical properties, RNA is similar to DNA. The major chemical differences are in the sugar moieties, where RNA has ribose instead of 2’-deoxyribose found in DNA, and in the presence of uracil as a common base instead of the thymine (5-methyluracil). Also, RNA molecules are usually smaller than DNA molecules, and they are generally single-stranded while DNA is usually double-stranded. Nonetheless, there are enough similarities for RNA to form stable complexes with DNA‘”. The structure of these complexes is a hybrid regular double-helix with the RNA strand antiparallel to a complementary DNA strandf4].The ability

DNA

RNA

1

1621

Fig. I . Scheme for RNA biosynthesis. When RNA polymerase (2x,B,B’,u) is incubated with DNA in the presence of subslrales (NTPs) (step 1 ) RNA molecules are synthesized from complexes of RNA polymerase and DNA. The D factor dissociales from the enzyme shoriiy after initiation of the RNA chain. When the enzyme reaches a transcription termination signal. it dissociates from the template and liberates the free RNA molecule (step 71. The synthesis of the RNA is completed in many cases by the action of nucleases that cleave segments of the initial transcript and of modification enzymes that alter specific nucleotides (step 3).

to form these structures is significant because i t is a clue to a possible mechanism for directing the polymerization of RNA. The biosynthesis of RNA molecules involves two major processes: polymerization of ribonucleotides and post-transcriptional maturation. These are shown schematically in Figure I.

2. Polymerization of Ribonucleotides 2.1. RNA Polymerase Nearly all RNA is polymerized directly from DNA templates by a process called transcription. This is inferred from the facts that nearly all RNA is homologous to sequences of DNA from the same organisms'']; that most RNA synthesis activity is found in DNA-dependent and that all RNA synthesis can be blocked by certain drugs, such as actinomycin D, known to bind very tightly to DNA"]. An obvious exception to this rule is that some viral RNAs are replicated directly from RNA templates. In addition, ribonucleotides can be added to the 3' ends of some RNAs without a template in reactions catalyzed by nucleotidyl transferases. Although it had long been considered possible that cellular RNAs might be duplicated on RNA templates there was no evidence to support this hypothesis until it was recently found that rabbit reticulocytes contain an RNA-dependent RNA polymerase[']. Since these cells no longer have DNA, their remaining mRNA, principally globin mRNA, might be replicated by such an enzyme. However, this has yet to be established. The enzyme responsible for RNA synthesis from DNA templates is called a DNA-dependent RNA polymerase. In bacteria one basic form, or core structure, of this enzyme has been identified'"]. It is sensitive to inhibition by rifampicin, and can be modified either by the binding of other protein factors or by chemical alterations. Another form has been postulated to be responsible for the RNA synthesis involved in chromosomal replication"01 since this synthesis is apparently not inhibited by rifampicin" ' I . However, the KNA polymerase responsible for this activity has not been isolated. In eukaryotic cells, four separate forms of RNA polymerases have been clearly distinguished'' 'I. One is from the mitochondria, and the other three are nuclear enzymes of which one comes from the nucleolus, the site of synthesis of ribosomal RNA precursors. The more abundant of the other two nuclear enzymes is inhibited by low levels of the mushroom toxin r-amanitin. This enzyme is thought to be responsible for the synthesis of precursors to mRNA. The other nuclear enzyme appears to be responsible for the synthesis of 5s rRNA and tRNA[''l. Most RNA polymerases are complicated, apparently asymmetric proteins, containing several subunits. With E. c d i RNA polymerase the smallest unit that has any RNA polymerase activity, known as the core, has a molecular weight of 390000["."1. I t consists offour subunits and contains a tightly bound zinc One polypeptide, the r subunit, is present in two copies. The other two, called p and p', are very large polypeptides with molecular weights > 150000 and although they are similar in size, they are distinctly different1"'. Highly purified enzyme also usually contains two other polypeptides. 446

One of these, called o, is a factor required for initiation of transcription from certain DNA templates and although it binds tightly enough to the free enzyme to be considered a subunit, it is released when the enzyme starts polymerization of an RNA chain. RNA polymerase with c is called the holoenzyme. The other polypeptide, called a), has a molecular weight of about loo00 and may actually be a contaminant even though enzyme preparations with a) generally have slightly higher specific activity than preparations without it. There is no evidence that nucleic acids are an important permanent component of RNA polymerase. However, covalently bound nucleotides and phosphate are known to be present under certain conditions and these substituents could play an important role in modulating enzyme activity"'! Core RNA polymerase isolated from exponentially growing E. coli appears to have less than 0.2 mole of phosphorous per mole of enzyme, so these substituents do not seem to be required for activity during normal cell growth. On the other hand, it has been reported that phosphorylation of (J factor by a protein kinase from rabbit muscle can activate RNA polymerase extensively[' 'I. The significance of this is not clear but it could be related to the presence of inactive polypeptides the same size as D factor in some enzyme preparations['"! The nucleolar and major nucleoplasmic RNA polymerase isolated from both calf thymus and rat liver nuclei are even larger proteins than the E. c d i enzyme'"'. They are also made up of several subunits including some peptides that are even larger than E. c d i p$' subunits. I t is not known yet whether any of these subunits are initiation factors analogous to 0. In contrast, RNA polymerase isolated from the mitochondria of Neurospora crassa consists of an aggregate of a single polypeptide with molecular weight of 64000[2"1, and the enzymes coded by bacteriophages T 3 and T7 have single polypeptides with molecular weight of about I07000[''~2 2 1 . Thus a complex protein structure is not a prerequisite for an RNA polymerase. However, these enzymes act on simpler genomes and the bacteriophage enzymes have rigid specificities. The more complicated structures of the bacterial and nuclear enzymes, therefore, may be characteristic of enzymes that recognize several kinds of genetic regions under strict regulation.

2.2. The Biosynthetic Reaction All RNA is synthesized by the polymerization of ribonucleoside triphosphates (ATP, GTP, CTP, and UTP) in a reaction that requires a divalent cation, usually Mg" ions. For each nucleotide added to the growing chain, a molecule of pyrophosphate is liberated. The postdated mechanism for the polymerization reaction is shown in Figure 2. The 3' hydroxyl group of the 3' terminal ribonucleotide of the nascent RNA chain is activated to perform a nucleophilic attack on the cx ( i . 6 ' . the first) phosphorus of a correctly aligned nucleoside triphosphate. The resulting phosphodiester bond is formed at the expense of the anhydride link between the cx and p phosphorus atoms, producing pyrophosphate as the leaving group. The correct alignment of the incoming nucleotide presumably dependson its binding to theenzymeand to the DNA template. Although the net change in free energy for the addition of monomer is not large (AGO'- -0.5 kcal/m01)[~~], the reac-

ternplate I DNA i

Fig. 2. Scheme for addition of a nucleotide to the 3' end of a nascent RNA chain.

tion proceeds well as long as the concentration of pyrophosphate is low. Physiologically, this is achieved by hydrolysis of the pyrophosphate to orthophosphate, which is a highly favorable reaction (A Go'= - 7.0 kcal/mol)catalyzed by ubiquitous pyrophosphatases. There is no indication that any further breakdown of nucleoside triphosphates is needed to drive the polymerization. The pure enzyme does not appear to catalyze any nucleotide hydrolysis reaction and the rate of RNA chain polymerization in citro is essentially identical to that in uiuo when physiological levels of substrates are used[24.''I. In E. coli growing at 3 7 T , this rate is 50 nucleotides per second for all RNAs of different types and size[". "I. It thus takes about one minute to synthesize an RNA molecule the size of the larger ribosomal RNA, which has a molecular weight of 1.1 x loh. This compares with an overall rate of nearly 900 nucleotides per second per nascent chain needed to replicate the DNA in that cell. The direction of RNA chain growth for the reaction catalyzed by E. coli RNA polymerase is as shown in Figure 2; each incoming nucleotide is added to the end with the free 3' hydroxyl Chain synthesis can be initiated by the same reaction used for elongation, except that the acceptor "RNA" is just another nucleoside triphosphate instead of the 3' end of a nascent RNA chain. This first nucleotide is the only one incorporated into the chain as a triphosphate; that is, without loss of its 0 and y phosphorus atoms. In general, this initiating nucleotide IS always either ATP or GTP[271. Although it is certainly possible that this specificity is a characteristic of all the different kinds of initiation sites recognized, the interaction of the purine nucleotides with the enzyme could also affect the positioning of the enzyme in an initiation site. ATP and GTP both bind to two sites on the free enzyme whereas UTP and CTP bind rather weakly to only one of these[291.The extra binding site for the purine nucleotides may be involved in the initiation reactions including the proper positioning of the enzyme with respect to the DNA template.

enzymes are double-helical DNAs, but single-stranded DNAand even RNA under some conditions--can substitute for double-helical DNA, but with much lower activity; with saturating amounts of DNA, E. co/i RNA polymerase is less than one-tenth as active with a single-stranded DNA than with a good double-helical DNAL6'. Analysis of the regions transcribed from a denatured DNA indicate that the enzyme is less discriminating when the template is in its single-stranded evidently RNA polymerase can initiate synthesis from many more regions on singlestranded DNA than on double-helical DNA and these include regions that are apparently not transcribed in the cell. The lower rate of RNA synthesis is not because of a lack of initiation sites but rather because RNA chain growth is slower, termination is more frequent, and possibly initiation is slower[28.3 1 1 . The overall transcription reaction on a double-helical DNA is fully conservative with respect to the structure of the DNA[321;no permanent alteration of the DNA occurs during the course of RNA synthesis. However, while it is used as a template, the DNA is firmly complexed with the enzyme and the nascent RNA In some cases the enzyme can be denatured and the RNA will remain attached to the DNA[34],but usually removal of the enzyme will result in a spontaneous dissociation of the RNA[331.Furthermore, most of the nascent RNA is susceptible to ribonuclease digestion. These facts suggest that the RNA is involved in a direct interaction with the DNA but that the site of this interaction is probably small and dependent on the enzyme for stability. Two basic models have been proposed as mechanisms for how a double-helical DNA can be used as a template for the assembly of a free single-stranded RNA without permanently altering the DNA structure. In one[35.361. a short single-stranded segment of the DNA, exposed by local unwinding, acts as a template by a mechanism similar to the process proposed by Watson and Crick for the duplication of DNA; a particular base in the DNA strand would only allow binding in the enzyme of the nucleotide that forms the correct WatsonCrick base pair (Fig. 3a). Presumably in this case, the RNA and the free template DNA strand would form a short hybrid

bi

5

5

2.3. The Role of DNA '162 3

With the DNA-dependent RNA polymerases, DNA acts as the template for the assembly of RNA. Without the template and without all the nucleoside triphosphates specified by the template, no RNA is synthesized. The best templates for most

3'

5

Fig. 3. Modelsof nascent RNA moleculeb forming interaction with nucleotides on only one DNA template strand (a) or on both DNA template strands (b). The DNA in ( a ) is in the "A" form and the DNA in (b) is i n the "B" form.

441

helix which, however, would be displaced by reformation of the more stable DNA-DNA helix during polymerization. In the alternative the two DNA strands do not separate completely and the RNA is assembled in the major groove of DNA by binding of the monomer ribonucleotides to both bases of each DNA pair. The specific interactions required for this binding have been demonstrated by model building[381(Fig. 3b). The major distinction between the two models is that a single DNA strand acts as the template in one whereas both strands are required in the other. The strongest evidence in favor of the former model is that single-stranded DNA can be used as a template, which suggests that the enzyme can use Watson-Crick base pairing for the interaction[351.However, the slower chain growth and frequent termination that occurs on the single-stranded DNA suggests that the second DNA strand must play some important role in stabilizing the interaction. Since both models require a distortion and partial unwinding of the helix, the fact that RNA polymerase does cause DNA to unwind about 180" per enzyme[3y1is not sufficient evidence to support either model. Instead, it is necessary to demonstrate the involvement of either one or both strands as template, and this has yet to be accomplished.

The model for recognition of the initiation site on DNA by RNA polymerase favored currently''"' postulates that the enzyme rapidly associates and dissociates from random sites on DNA molecules until it recognizes a special site. where it can form a tight binary complex (Fig. 4). This complex in turn is in equilibrium with another one in which the DNA strands unwind to expose the DNA bases. I t is enzyme in this latter complex that is able to initiate an RNA cham. Many elements of this model have been supported by studying the binding of RNA polymerase to T 7 DNA in the absence of nucleoside triphosphates and the conditions necessary for rapid chain initiation from complexes between RNA polymerase and DNA. The schema does not take into account the effect that the nucleoside triphosphates might have on the binding and recognition steps, but it is believed that the RNA polymerase can bind very close to the initiation site without the involvement of nucleoside triphosphates. closed complex

3. Selectivity of Transcription 3.1. Initiation

The synthesis of specific RNA molecules is a result of the initiation and termination of transcription at specific points on the DNA template[401.The E. coli DNA-dependent RNA polymerase has the ability to select the site on the DNA to be used for initiation; initiation does not occur at random, and as long as o factor is present these initiation sites can be regular double-helical sections of the DNA. The core enzyme also has a limited capacity to initiate specific RNA synthesis; it can use certain single-stranded segments and may be able to initiate slowly from double-helical segments. The main functions of the o factor are to stabilize the interaction between the enzyme and the initiation site, and to destabilize the nonspecific interaction that RNA polymerase can make with almost any region of DNA. It was originally suggested that o factor might direct the recognition of a particular DNA regionr411,but no new D factors that confer obviously different specificities on the activity of E . coli core RNA polymerase have been purified. Thus, strong support of this model has yet to be found. A second sigma factor from E. roli has been Isolated as a component of a minor RNA polymerase fraction. This new protein called G' has a molecular weight of 56000 and binds more tightly to the core enzyme than D [ ~ It~ ~apparently . performs the same functions, and D does not further activate enzyme saturated with 0'. No differences have been distinguished yet in their specificity except that o' more readily catalyzes the de novo synthesis of ribohomopolymers and the DNA-dependent synthesis of poly In its size, o' from E. coli is similar to the o isolated with B. subtilis RNA polymerase. Since E. coli o will substitute for the B. subtilis o factor, it appears that a basic interaction between factor and core has been conserved in different bacterial species[44! 448

,152 I

non-specific complex

Fig. 4. Model for recognition and activation of the initiation site on D N A by RNA polymerase (circle). N T P = a n y nucleoside triphosphate (adapted from ref. [40]).

The genetic element that is thought to govern initiation of RNA chain is called the promoter[45];mutants in promoter regions affect the rate of transcription from the genetic unit governed by that promoter. Two such promoter mutants that govern the transcription of the N gene in h, have been mapped at about 33 nucleotides from the DNA sequence that specifies the 5' terminal end of the RNA transcript for the h N 481. Even though the promoter site is neither coincident with nor immediately adjacent to the DNA sequences for the transcript it controls, it is within the 40 base pairs that presumably can be spanned by a single RNA polymerase molecule. It is thus close enough to influence directly the ability of the enzyme to use the template sequences for the start of the RNA molecule. The segment of DNA between the start of the N gene transcript and the site for its promoter mutations has been and the sequence contains several two-fold rotational symmetries (see Fig. 5). Certain of these symmetries are probably recognized by various L regulator proteins including the h repressor, and one of them may be important for RNA polymerase recognition. If this is the case then there is likely also to be a symmetry in the structure of RNA polymerase. This would have to involve the 2 subunit because it is the only one that is present in duplicate. A site in this LDNA sequence recognized by the Hind R I1 restriction nuclease is also indicated. When RNA polymerase is bound to hDNA the restriction nuclease no longer cleaves at that

ite el^'*^'! Other sites on hDNA and on several other DNA molecules recognized by this restriction enzyme are also protected from its nuclease activity when RNA polymerase is bound[491.However, most of the sites recognized by this nuclease on these DNA molecules are not protected by RNA polymerase. Thus, the sequence recognized by the nuclease is not sufficient to define the complete recognition site for RNA polymerase binding, but it may be a necessary sequence for at least one kind of polymerase binding site. Hind

1162 5

Fig. 5. Nuclcotide sequence of a h operator-promotor region. Sequence taken from ref. [48]. The overlined segment (GAT) indicates the start of the N gene transcript. Several two-fold rotational symmetries occur in this sequsnce. The axis of symmetry for the enclosed sequences is located by the vertical arrow above the sequence. The axes for two other symmetries are located by the two vertical arrow below the sequence. The site of cleavage by the Hind restriction nuclease is also indicated. R = purine nucleotide; Y = pyrimidine nucleotide.

Although no o factors with proven differences in specificity have been purified, there are many proteins that act in conjunction with the holoenzyme that affect the selectivity of transcription. The best characterized proteins of this sort are the repressors for the lactose[501and galactose[”] operons and for the early transcription units on bacteriophage hDNAlsZ1.These repressors specifically inhibit transcription of the genetic regions they control. With a bacteriophage DNA carrying sequences of the lactose operon, the lactose repressor only inhibits transcription of those sequences and not the sequences of the bacteriophage genes[”]. Similarly, h repressor specifically inhibits transcription from the early region of h DNA but does not inhibit transcription of bacteriophage 434 DNA, a closely related DNA that is immune to the action of the h repressor‘”! The repressor molecules are known to bind very tightly and specifically to the operator region of the appropriate DNAs. Since these operator regions are near or overlap with the regions recognized by RNA polymerase for the initiation of the synthesis of the genetic unit controlled by the repressor, it is quite possible that the repressor binding physically prevents interaction with RNA polymerase. This has been demonstrated with the h repressor[54! Alternatively, the repressor could just prevent the enzyme from moving through or away from the DNA regions blocked by it; there is also some evidence to support this mode of action as The h DNA sequences given in Figure 5 include a site recognized by the h repressor. In this case transcription appears to start just outside of the region recognized by the repressor. However, the transcript of a lactose operator containing an especially active promotor mutation includes the sequences for the lactose operator[56.571. Thus there may be several ways in which repressor-operator interactions can affect transcription of the genetic unit regulated. Two factors have been isolated that specifically activate transcription of particular genetic regions. One of these is the C gene product of the arabinose operon of E. coli. This protein in the presence of L-arabinose stimulates the transcription of the DNA region coding for the enzymes of L-arabinose metabolism[58,591. Its mechanism of action still remains to be elucidated. The other factor, which is known by several

names including catabolite gene activator protein and cyclicAMP receptor protein, is required along with its allosteric effector 3’-5‘ cyclic AMP for transcription, among others, of sequences coding for the proteins for lactose, galactose and arabinose utilization[“’. This factor binds to DNA and requires the presence of 3’-5‘ cyclic AMP to functionI6’]. It has been suggested that it may act to lower this activation temperature for unwinding DNA at the initiation sites for the affected transcription units[401. Several other protein factors have also been demonstrated to activate transcription of various DNA templates under certain conditions, but the role of many of these factors remains unclear140! On the other hand, certain proteins known to be required for the activation of certain genetic regions on T 4 DNA have been found as subunits of RNA polymerase isolated from TCinfected E. c.o/i[62, h31. Again, it is not known how these proteins effect this activation.

3.2. Termination Specific termination of transcription is required on bacterial and the larger bacteriophage genomes to prevent continued transcription from one genetic unit into an adjacent unit that should be controlled independently. Certain sequences in DNA are evidently recognized by E . coli RNA polymerase as termination signals because many of the transcripts synthesized in virro are discrete molecular species that correspond to particular genetic units on the The specificity of this termination recognition is also reflected in the characteristic sequence of -U-U-U-U-U-U that has been found at or near the 3’ ends of several RNAs synthesized in vitro; in most cases this sequence is P ~ r i n e - ( U ) , A ~(”I., ~ ~ ~ ~

m1

2

13

L

Fig. 6. Model for p function. P.T. and P I. indicate genetic positions for protein synthesis termination and initiation signals, respectively. State I shows nascent R N A with ribosomes nearing a termination site. State 2 shows a ribosome being released at the protein synthesis termination signal ( U A A ) on nascent RNA, and it shows a C-rich R N A stretch emerging from the R N A polymerase (large black circle). State 3 shows the R N A and RNA polymerase that have been released by action of p that recognized the C-rich region on the R N A (it also shows a p molecule still attached to the C-rich R N A region). State4 showsa nascent R N A whosesynthcsis wasnot terminated by p action. If the termination signal (P.T.) should arise as the result of a mutation t o a nonsense codon within a cistron (structural gene). p might have a chance to act at C-rich regions on the nascent R N A that would normally be obscured or blocked by the presence of a nbosome translating the RNA. This model could account for the strong polar effects caused by nonsense codons.

At least one other type of termination signal also exists. This is the signal recognized when an added protein, called p factor, is also present1661.However, in this case there is evidence that a nucleotide sequence is recognized on the RNA, not the DNA[671.Some recent experiments suggest that p interacts with specific sequences on nascent RNA molecules 449

to activate a nucleoside triphosphate phosphohydrolase (or phosphotransferase) activity that is essential for the p-induced termination ofRNA synthesis[h8."1. This sequence is probably near rather than at the 3' end of the terminated RNA chain because it is unlikely that p could fit into the polymerization site of RNA polymerase. From studies with the effects of p on transcribing the DNA region for the gal and lac operons. it has been suggested that p function may be to decrease the expression of genes at the end of a transcription unit compared to the genes near the beginning of that transcription unit['"]. A model for p function is presented in Figure 6.

endo-and eronucleolytic cleavages, modification

4. Post-transcriptional Maturation Reactions A627

The synthesis of many RNA molecules requires several biochemical steps in addition to the transcription of the nucleotide sequences for the RNA from the DNA template. These steps include cleavage of extra nucleotides from the transcripts, modification of some nucleotides and, in some cases, the addition of nucleotides at the 3' end of the RNA by a synthetic reaction that does not depend on a DNA template.

4.1. Removal of Extra Nucleotides The clearest examples of cleavage of sequences has come from studies of the synthesis of ribosomal RNAs in eukaryotic cells'711.In HeLa cells, for example, the 18S, 28S, and 7 s rRNAs are all derived from a single precursor molecule that is nearly twice as large a s the combined size of the rRNA products. This precursor has large stretches of RNA nucleotides that are degraded in the maturation process. In bacteria the sequences for the 16s and 23s rRNAs are also in single transcription units["', but in this case a full length RNA containing both 16s and 23 S rRNA sequences is rarely detected because the cleavage of the portion with the 16s rRNA sequence occurs before the synthesis of the 23s portion is finished. However, significant amounts of this full length RNA molecule arc found in a mutant that contains reduced levels of ribonuclease 111, an endonuclease specific for double-stranded RNA[731.This suggests that RNase Ill is responsible for the cleavage of the portion containing the 16s sequences from the portion containing the 23s sequences. Further steps in the maturation of the ribosomal RNAs in E. c d i involve the removal of a few extra nucleotides from the 5' and 3' ends of the cleaved precursors. In all, about 22'%, of the nucleotides from the initial transcript are Thus a greater fraction of the ribosomal RNA transcript is conserved in E. co/i than in HeLa cells. The synthesis of tRNA molecules also appears to depend on a similar pattern of cleavage and trimming. Several genes for tRNA molecules are found clustered on the chromosomes of E. c0/i17S1 and bacteriophage T4[''. "I. When a short labeling time is used it is also possible to detect labeled RNA molecules that contain the sequences for two or more tRNA molecules. Since segments containing sequences for other tRNAs could also have been cleaved during RNA polymerization the transcription units for the tRNA may contain sequences for several species plus spacer regions that are degraded when the primary transcriptsare converted t o mature tRNA molecules. An outline for these steps is given in Figure 7. Isolation of a precursor for the tyrosine tRNA (ptRNAT)') from E. c,o/i has made it possible to assay some of the

450

U

Lmononucleotldes

t

U

Fig. 7 . Scheme for processing of a transcript containing scqiienccs for two inaturc t R N A niolectiles. t K N A , and t R N A ? . The circlesat the 5' end indicate a triphosphate group.

enzymes involved in the removal of extra nucleotides from the 5' and 3' ends of the precursor. A ribonuclease called RNase P (for precursor) has been identified with this assay1781 and mutants with a temperature-sensitive form of this enzyme have been isolatedI"! When incubated a t the restrictive temperature, these mutant strains accumulated precursors containing as many as four tRNA sequences in a single RNA. Cleavage reactions are also believed to be very important in converting gene transcripts found in the nuclei of eukaryotic cells into the messenger RNA molecules found in the cytoplasm of these cells['"I. Most of the nuclear RNA molecules are much larger than the messenger RNAs in eukaryotic cells; molecules with molecular weights u p to 2 x lo7 have been found for the former, whereas even the largest mRNA molecules are less than one-tenth that size. The nuclear RNA molecules are also metabolically very labile. Over 90':(, of the larger RNA molecules synthesized in the nucleus are broken down there and only a small fraction is transferred to the cytoplasm. This fraction appears to correspond to the lo'%, of the RNA at the 3' end of the nuclear transcripts and is believed to be the source of mRNA. Although definite evidence for specific cleavage of precursor to a bacterial messenger RNA is lacking, many bacteriophage mRNA molecules are synthesized as long transcription units in citro. The T 7 RNA synthesized in ritro with E. c d i RNA polymerase has a molecular weight of 2.5 x 106[8'1, yet the early T 7 species found after infection in normal cells are all considerably smaller. However, when T 7 infects a strain of E. c d i that has a defective ribonuclease Ill, the T 7 RNA is as large as the in rifro products["! Thus ribonuclease 111 may be involved in processing of mRNA as well as rRNA. 4.2. Modification of Nucleotides

Maturation ofmany RNA molecules also involves modification of selected nucleotides in the initial transcripts. Examples of some modified nucleotides are given in Figure 8. These modifications are most extensive in the synthesis of tRNA molecules but are also important for the synthesis of ribosomal RNA molecules and may be important for the synthesis of some mRNA molecules[83- 8 S 1 . The most common modification reaction is the addition ofa methyl group to a nucleotide. The donor of the methyl group is S-adenosylmethionine and the reactions are catalyzed by specific methyl transferase enzymes that recognize the nucleotide to be modified in a

specific location. The products are primarily base methyl derivatives, but some 2’-O-methylribose derivatives and some dimethyl derivatives are also made. 0

X

0

\

\

\

111. x - 0 121. x = s

131

141

H, H,

N

,b las281

bH 151

,

I

O\ OH 161

Fig. 8. Structures of uridine ( 1 ), adenosine ( 5 ) , and their modified derivatives 4-thiouridine ( 2 ) . pseudouridine (5-P-D-ribofuranosyluracil) ( 3 ) , 5,6-dihydrouridine (41, and Nh-(2-isopentenyl)adenosinef6).

Another important modification is the isomerization of certain uridylate residues to pseudouridylate (5-ribosyluracil nucleotide). Almost every tRNA molecule has at least one pseudouridylate residue and many have two or more. This isomer has also been found in ribosomal RNAs of both eukaryotes and prokaryotes. An enzyme system that catalyzes the isomerization reaction for uridylate residue in tRNA molecules has been isolated from E. c ~ l i [ ~ and ~ ]mutants , of Salmonella typhimurian have been found that are lacking one of these enzymes; these mutant strains are defective in the regulation of certain amino acid biosynthetic pathwaysrX6l. Other modification reactions appear to occur exclusively in tRNA molecules. These include thiolation of uridylate residues, reduction of certain uracil bases to dihydrouracil, and the addition of isopentenyl group to 6-amino group of adenosine residues near certain anticodons. Many of these modifications are essential for function while others have roles that have not been identified. For both tRNA and rRNA some of the modification reactions can occur on the larger precursors. However, the unmodified tRNATy‘from E. coli that has been trimmed to its final size is a better substrate for modification than its larger precurs0r[873and some of the modifications of rRNA occur only after certain ribcisomal proteins have boundI7*I. Thus, nucleotide modifications can occur on the initial transcript but the completion of all the reactions may be one of the final steps in maturation of tRNA and rRNA molecules. 4.3. Addition of Residues

Finally, an important but not well understood maturation step for some RNA molecules involves the addition of nucleotides to the 3‘ end of the RNA in a reaction catalyzed by terminal nucleotide transferases that do not depend on a DNA template[801.Many of the large nuclear RNAs and mRNAs in eukaryotic cells have sequences of poly(A) at their 3’ end that are from 150 to 300 nucleotides long. The evidence that these adenylate residues are added by a post-transcripAngew. Chem. internat. Edit. J Val. 14 ( 1 9 7 5 ) / N o .7

tional reaction is that the appropriate template sequences for a poly(A) as long as 300 nucleotides have not been found in the DNA; that enzymes isolated from both nuclei and cytoplasm of many types of cells catalyze the attachment of adenylate residues to RNA; and that certain drugs selectively inhibit the synthesis of the poly(A) sequences. The exact function of these poly(A) sequences is not yet clear, but their addition appears to be a necessary step in the maturation of some mRNA species in higher organisms. The fact that they are found at the 3’ end of both the large nuclear RNA molecules and mRNA molecules is a further indication that the 3’ end of the nuclear RNA is a precursor to the mRNA. Although an enzyme capable of catalyzing the addition of adenylate residues to RNA has been found in bacteria, no evidence has been presented that such sequences are present in any bacterial RNA. Bacteria and other organisms also have enzymes that can add cytidylate and adenylate residues to tRNA molecules lacking the -pCpCpAoH sequence found at the 3’ terminal of every mature tRNArS8]. The one precursor tRNA that has been sequenced already, the ptRNATYrof E. coli contains internally the -pCpCpA- sequence of mature tRNAcx9].Thus the function of the enzymes that catalyze the addition may only be to replace this end if it is removed during the trimming process. However, the pCpCpAoHsequence may not be present in the primary gene products for all the tRNAs. In such cases, the post-transcriptional addition reaction catalyzed by the enzyme would be an important maturation step for the synthesis of these tRNA molecules. I thank Dr. 7: Blumenthal for a critical reading of this review. I am supported by a U.S. National Institutes of Health Career Development Award (GM-70,422). Received: October 30, 1974 [A 62 IE] German version: Angew. Chem. 87, 497 (1975)

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Asymmetric Membranes : Preparation and Applications By H.-D. Saier and H.

Strathmann[*l

The separation of molecular mixtures by semipermeable membranes under the driving force of hydrostatic pressure has assumed major importance in recent years. Among other factors the development of membranes with asymmetric structure which, while having the same separating properties, afford a filtration output many times greater than that of the previously known symmetrical membranes, was decisive for this method. In the present progress report the structures of asymmetric membranes are discussed, as well as their preparation from various polymers and their application to the separation of molecular mixtures.

1. Introduction Selective mass transport through membranes has been the subject of numerous scientific investigations for more than a century. In this work attention was first focused on biologi-

[*I

Dr. H.-D. Saier [ +] and Dr. H. Strathmann Forschungsinstitut Berghof GmbH 74 Tubingen-Lustnau, Berghof (Germany) [+] To whom correspondence should be addressed.

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cal membranes, the importance of which for metabolic processes in living organisms was recognized very early, but more recently synthetic membranes in particular and their application to separation problems in chemistry and industry have acquired economic significance. For example, membrane filtration is nowadays used to extract drinking water from the sea, for fractionating, concentrating, and purifying macromolecular solutions, for the treatment of industrial waste waters, and for the recovery of valuable constituents. Membrane filtraAngeir. Chem. internat. Edit. I Vol. 14 (197.5)

No. 7