Biochimie (1991) 73, 739-755 © Soci6t6 franqaise de biochimie et biologie moJ6culaire/ Elsevier, Paris

739

The assembly of prokaryotic ribosomes KH Nierhaus Max-Pianck-institut fiir Molekulare Genetik, Abt Wittmann, lhnestr 73, 1000 Berlin-Dahlem 33, Germany (Received 17 October 1990; accepted 12 November 1990)

Summary - - The targets of in vivo studies of the ribosomal assembly process are mainly the events of rRNA processing, whereas in vitro studies (total reconstitution) focus on principles of the assembly process such as assembly-initiation proteins, rate-limiting steps and a detailed sequence of assembly reactions (assembly map). The success of in vitro analyses is particularly remarkable in view of ionic and temperature requirements of the total reconstitution which differ significantly from the in vivo conditions. Features of the in vivo assembly are surveyed, however, the focal point is a description of experimental strategies and results concerning the in vitro assembly of ribosomes. ribosomal assembly / ribosomal reconstitution / assembly gradient

Introduction

The fact that a ribosome consists of a large number of different components (eg, E coil: 53 r-proteins and 3 rRNAs [1]) has two important consequences for the ribosomal biogenesis: both the synthesis and the assembly of the ribosomal components must occur in a highly coordinated fashion. The requirement for a highly coordinated synthesis is particularly demanding in those cases in which ribosomes contribute significantly to the dry mass of the cell. In bacteria the ribosomes can amount to more than 30% of the dry mass [2], whereas in eukaryotes they represent no more than 5% [3]. In fact, cells of Escherichia coli appear as sacs filled with ribosomes in images of transmission electron microscopy. Correspondingly, up to 40% of the total energy production is consumed by ribosomal biogenesis. It follows that a coordinated synthesis is not only a prerequisite for a successful and effective assembly, but is also a necessity for an economic consumption of the cell energy. We therefore find an intricate network of regulatory mechanisms for the synthesis of ribosomal components in proka~-Totes which does not exist in this perfection in eukaryotes (eg, the stringent response, see pertinent reviews [4, 5]). A reaction of a third order practically does not exist since the probability that three substrates react simultaneously is negligibly low. Therefore, the assembly of more than twenty components (in the case of the small ribosomal subunit) to a defined and relatively

compact particle is a series of reactions. This assembly is a self-assembly process, ie the total information for the assembly pathway as well as the quaternary structure of the active ribosomes resides completely in the primary sequences of the ribosomal proteins and rRNAs. This is indicated by the fact that fully active ribosomes can be reconstituted from the isolated components with the remarkably high efficiency of 50 to 100% of the input material. The self-assembly character in vitro does not exclude that some additional factors might occur in vivo, for example, to reduce activation energies of distinct, otherwise rate-limiting reactions thus facilitating and accelerating the whole assembly process. One of these factors is probably the 'assembly gradient' which marks the coupling of rRNA synthesis and ribosomal assembly in pro- and eukaryotes (Proteins essential f o r the early assembly: the assembly gradient). It is further possible that proteins exist which maintain an unfolded state of the de novo synthesized ribosomal proteins thus favouring the integration into ribosomal particles ('chaperonins', see [6]). Circumstantial evidence for a corresponding activity for the rRNA ('helicase'), possibly facilitating the attainment of distinct RNA conformations which favour the assembly process, has been already reported

[7].

In this review our knowledge on the assembly of ribosomes is surveyed. A detailed description of the various reconstitution techniques can be found elsewhere [8].

740

KH Nierhaus

Processing of rRNAs The processing of rRNAs has two possibly related aspects: 1) trimming of the rRNAs to the mature molecules found in native, active ribosomes (for review see [9]); and 2) modifications (mostly methylations) of the rRNAs. The common order of rRNA genes in the seven rRNA operons in E coli is 16S-spacer (containing either tRNA Ala and tRNAne or tRNAGlu)-23S-spacer5S. The complete, intact transcript, the '30S precursor rRNA', is practically not found in wild-type cells (I-2% of rRNA) but only in RNase Ill-deficient mutants. RNase III cleaves in spacer sequences bordering 16S and 23S rRNA; the spacer sequences can form impressive secondary structures flanking both 16S and 23S rRNA [10]. However, intramolecular hybridizations are not a prerequisite for RNase III activity since RNase III can cleave at the 5' end before the 3' e~.~,tof that molecule is transcribed [10]. A general fe~iiure is that processing begins before transcription of an rrn operon is finished [ 11 ]. This sequential processing in the 5' ---> 3' direction is compatible with the hypothesis that at least some processing steps are coupled to the ribosomal assembly, Other steps are certainly not coupled to the assembly process since a mutated 23S rRNA defective in assembly initiation and not appearing in precursor or mature 50S particles was processed at least to some extent [12]. Furthermore, '30S pre-rRNA' was cleaved with purified RNase III in vitro to produce pre-16S and pre-23S slightly larger than the corresponding mature species ([9] for review). The final maturation steps of pre-16S, pre-23S and pre-5S occur even after ribosomes are formed probably during early steps of protein biosynthesis [9]. RNase III cleavage yields precursor species of rRNA. The pre-16S species retains stretches of 115 and 33 nucleotides at 5' and 3' end, respectively, the pre-23S only stretches of 7 and 7-9 nucleotides, respectively. RNase III cleavages are not essential processing steps since mutants lacking RNase III are viable. In the absence of RNase III 50S with pre-23S are found whereas mature 16S rRNA molecules are formed at the same rate as in the wild-type strain [9]. Interestingly, 30S subunits containing pre-16S with a probable hybridization of the stretches flanking the mature 16S rRNA seem to be inactive (ie, mutants deficient in 16S maturation are not viable [13]) in contrast to 50S subunits containing pre-23S [14]. Note that in mature 30S subunits 5' and 3' ends are far apart from each other, whereas in 50S 5' and 3' ends are base paired ([15] and references herein). This has the important consequence that the maturation from pre-16S to mature 16S within 30S particles (removing the secondary structure flanking the 16S) triggers the activation of 30S subunits. When

the 16S rRNA processing is coupled to and depends on a correct 30S assembly, then this final processing step warrants that only active 30S subunits can initiate protein synthesis. Enzymes involved in this final processing step have not yet been identified; the spacer tRNA seems to be important for a correct maturation of the 3'-end [16]. As mentioned above, 50S subunits containing pre-23S rRNA can enter protein synthesis and are active, the mature 5'-termini can be only formed in polysomes [ 17]. Processing of 5S rRNA requires RNase E. RNase E deficient mutants accumulate a 9S species [18], which is not found in wild-type cells. RNase E form pre-5S with three extra nucleotides at both its 5' and 3' ends. The final processing of 5S rRNA might also occur during protein synthesis since pre-5S was found in polysomes [ 19]. 10 and 14 nucleosides are modified in 16S and 23S rRNA, respectively (table I; for review see [20]). Most of the modifications are methylations, three are pseudouridines and two are yet unidentified uridine derivatives. The methylations are not required for the trimming processes described above [21]. A few methyl groups are found at the 2'-ribose position and might protect sensitive rRNA regions against RNase attack. Most of the methyl groups are base modifications exposed at the ribosomal surface and clustered at functionally active sites of the ribosome [22]. Therefore, they are probably modifying and finetuning the ribosomal functions. The two adjacent m6A's at positions 1517/1518 are generated at a late assembly step of the 30S subunit, they improve the Table I. Modified nucleosides in E coli rRNA. For review and references see [20]. U+ and U++ are unidentified uridine derivatives. 16S rRNA

Location (nucleotide)

23S rRNA

Location (nucleotide)

m7G m2G msC m2G

526 965 966 1206

mIG W m5U m6A

745 746 747 1618

m4Cm msC

1401 1406

~ U+

1911 1915

Gm m2G m6A m6A Total

1496 1515 1517 1518 1541

~ msU m6A m7G Gm

1917 1939 2030 2069 2251 2449 2498 2552 2904

U ++

Cm Um Total

The assembly of ribosomes formation of initiation complexes, in particular the binding of IF-3, which has an anti-association activity. The absence of the four methyl groups confers resistante to the drug kasugamycin (for review and references see [23]). Precursor mediates

particles

and

reconstitution

inter-

Usually the assembly process is described from the point of view of the largest component, ie the sequence of additions to the 16S- or 23S-type of rRNA is considered. This sequence of additions represents a concatenation of reactions which differ in their respective activation energies. The highest activation energies function as energy barriers prior to which more or less stable precursor particles can accumulate. In fact, precursor particles have been found in the course of the in vivo assembly. The assembly of the small subunit (30S, E coli) is characterized by two particles, pt30S and p230S (p for precursor; [24, 25]). The pt30S particle sediments with 21S, its protein pattern [26] is listed in table II. The p230S particle Table II. Protein content of precursor particles and reconstitution intermediates of the 30S subunit from E coli ribosomes. Data are taken from [26] (pm30S) and [29] (RI30).

Protein

pt30S

R13o

Sl S2 $3 $4 S5 S6 $7 $8 $9

+ + + -

+ (+) + +

+

+

-

SI0

-

+ + + + + +. + (+) ÷ -

Sll S12 S13 S14 S15 S16 S17 S18 S19 S20 S21

+ + + + + +

741

contains a full complement of S-proteins (S for proteins from the small subunit), but still an immature '17S' rRNA, which is longer at both 5'- and 3'-ends (see Processing of rRNAs). Only one reconstitution intermediate, RI30, is found during the in vitro assembly of the 30S subunit. The total reconstitution ('total' marks a reconstitution from completely separated ribosomal proteins and rRNA) is a one-step procedure according to the formula [27]: 16S rRNA + TP30 - - [ 2 0 mM Mg2+,40oc, 20 min] ) 30S (l) TP30 stands for total proteins derived from 30S subunits. An incubation of both 16S rRNA and TP30 at 0°C leads to the RIa0 particle. This particle has to undergo a conformational change ('activation') according to: 40°C, 20 min RI3o ~ R130* (2) Only the RI30* particle can bind at 0°C the lacking complement of S-proteins thus forming active 30S particles [28]. Interestingly, the protein content of the RI3o particle [29] is very similar to the p~30S precursor (table II). Equation (2) describes the rate-limiting step of the 30S assembly, the activation energy of which has been measured to 63 kcal/mol [28]. The in vivo assembly of the large subunit (50S, E coli) occurs via three precursor particles pl50S, p250S and p350S sedimenting with 34S, 43S and 'near 50S', respectively [25, 30, 31]. The last precursor (p350S) contains again a full complement of Lproteins (L for proteins derived from the large subunit), the protein pattern corresponding to the pm50S and p250S particles are listed in table III. The p250S particles could be converted to active 50S subunits in the presence or TP50 and under methylating conditions (S-adenosyl-methionine, SI00 enzymes), whereas the pm50S particles could not [26]. A two-step procedure is required for the total reconstitution of active 50S subunits [32, 33]. (23s+5s) rRNA+TP50 - - [ 4 mM Mg2+,44°C, 20 min] (3) m[20 mM Mg2+,5O°C,90 mini ----~50S The two-step procedure is a consequence of the fact that the rate-limiting steps of the early and late assembly are conformational changes which differ in their ionic optima (see below). The two-step procedure is therefore a convenient way in which to separate early and late assembly events, and indeed allows a detailed analysis of possible reconstitution intermediates. Figure I A outlines the experimental strategy: a first-step incubation (see equation (3)) was performed, and at various times an aliquot was withdrawn and analysed for particles formed in a sucrosegradient centrifugation, a sister aliquot was subjected to a standard second-step incubation (20 mM Mg2+, 50°C, 90 min) and tested for active 50S subunits in a

742

KH Nierhaus

Table III. Protein content of precursor particles and reconstitution intermediates of the 50S subunit from E coli ribosomes. Data from [26] (pl50S and p250S) and from [34] (Rlso(I)). L8 is the pentamer LI0(L7/LI2)4; L26 is the 30S protein $20; L34, L35 and L36 have not yet been assigned.

Prowin L! L2 L3 L4 L5 L6 L7/LI2 L9 LI0 LII LI3 LI4 LI5 LI6 LI7 LI8 LI9 L20 L21 L22 L23 L24 L25 L27 L28 L29 L30 L31 L32 L33

pt50S

p250S

Rlso(1)

+ + + + + + + :t: + + + + + + + + + + + + -

+ + + + + + + + + + + + + + + + + + + + + + + +

+ (+) + + + + + + + (+) + + (+) + + + + + + (+)

poly(U) dependent poly(Phe) synthesis. Figure I B shows the kinetics of the particles formed. A distinct particle is present already at 0°C, which disappears during incubation at the cost of a second distinct particle which again slowly disappears whilst a third particle appears at the same rate. Obviously, three reconstitution particles are subsequently formed. When their relative amounts are drawn together with the activity monitored in the poly(U) system (fig I C) it becomes evident that the formation of the second particle is paralleled by the appearance of activity after the two-step incubation. It follows that the second particle is the essential product of the first-step incubation, this product can hardly be by-passed in the second step.

A protein analysis revealed that the first and the second particle contained the same complement of rRNAs and L-proteins in spite of the drastic difference in their respective S-values (33S and 41S, respectively, see table IV), whereas the third particle contained all the components of the active 50S subunit but was totally inactive. Accordingly, we termed the three reconstitution intermediates RIso(1), RIso*(1) and RI5o(2) [33]. It appears that the rate-limiting step of the first incubation is the conformational change RIs0(1) ---> RIs0*(l), and that of the second incubation the conformational change RI50(2) ---> 50S (table IV). The corresponding activation energies have been determined to be 70 and 54 kcal/mol, respectively [36]. The first value of 70 kcal/mol was found to be significantly smaller (43 _+ 4 kcal/mol [37]) when an improved technique for preparing TP50 was used [38]. The activation energy of the rate-limiting reaction of the first step is sensitive to the preparation methods of both rRNA [36] and TP50 [37] in contrast to that of the second step. It is striking that the precursor particles and the corresponding reconstitution intermediates have similar protein patterns (tables II and III) as well as similar S-values (fig 2) indicating that the in vivo assembly procedes via rate-limiting steps which are very similar if not identical to the corresponding ones of the in vitro assembly.

Assembly.initiator proteins Ribosomal proteins (r-proteins) which bind in vitro specifically to naked rRNA are called 'RNA-binding proteins'. About two-thirds of all r-proteins are RNAbinding proteins (see figs 11 and 12). The intriguing

question was whether all these RNA-binding proteins (about 20 L-proteins in the large subunit) do bind directly to rRNAs also in vivo without help of other r-proteins (binding without cooperativity), ie whether

these proteins can form assembly nuclei as independent assembly-initiation events. There were already indications that this was not the case, ie that only a small number of r-proteins were able to initiate the assembly process. Under unfavorable growth conditions, when the doubling time was about 10 h and thus 30 times

longer than the doubling time at optimal growth, the finely tuned balance of synthesis of rRNA on one hand and r-proteins on the other collapses and rRNA is produced in a three molar excess over r-proteins [39]. If all the 20 L-proteins, which are RNA-binding proteins, would be able to initiate the assembly process independently and thus would be distributed independently over the excess of rRNA, the outcome of active particles with a full complement of rproteins would be negligibly small. Since the E coli cells produce notable amounts of active ribosomes

The assembly of ribosomes

Intermediate Particles sucrose gradientonatysis f

I

I

1.step, 1` 1' 1' 1' T 1' 1'~ time I

$

I

standard 2.step~

8¢= .o

poty(Phe) synthesis

0rain Rlsoll)

2 min .

S rain

10min Rlso12)

20min

30rain

JO ,1¢

J 0.

Direction of sedimentation

743

even under unfavorable growth conditions, the number of assembly-initiator proteins must be significantly smaller than that of the RNA-binding proteins. An assembly-initiator protein is defined as an rprotein which binds without cooperativity to an rRNA molecule and is essential for the formation of an active ribosomal subunit. Only those rRNA molecules with a complete set of initiator proteins are able to perform a correct assembly thus forming active particles. The dependence of the output of active particles on both the number of assembly-initiator proteins and the excess of rRNA over r-proteins is illustrated in figure 3A. Assume that there would be only one assemblyinitiator protein (n -- 1). Then all the r-proteins (which are thought to be present in stoichiometric amounts) would assemble on only that rRNA molecule which carries the assembly-initiator protein. The output of active particles would be independent of the excess of rRNA, and 100% of the r-proteins would be found in active ribosomal particles (fraction A of TP50 appearing in active ribosomal particles = 1). With two assembly-initiator proteins (n = 2) and a 2:1 stoichiometric ratio of rRNA to r-proteins only 50% of the r-proteins will appear in active particles (A = 0.5) since only the complete initiator complexes with both assembly-initiator proteins will go through the whole assembly pathway, rRNA with only one of the two initiator proteins will bind some r-proteins thus producing assembly-dead ends. With three assembly-initiator proteins (n = 3) and a stoichiometry of 2:1 (rRNA: r-proteins) the formation of active particles reduces to one-fourth relative to the input of r-proteins (A = 0.25). The dependence of the amount of active particles on the number of assembly-initiator proteins and excess of rRNA is governed by the formula [40]: A = E I-,' (4) A is the fraction of total proteins which appears in active particles (A for activity), E is the molar ratio of

¢; 100

-~100 . . - :~

¢/t

I~ "e..

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

I

~'='=

-50

0

!

-0

!

0 ½ 5

10 20 Time (rain)

30

~ ~ , e.N

Fig 1. Reconstitution intermediates during the assembly of the large (50S) ribosomal subunit. A. Outline of the experimental strategy, rRNAs and TP50 were incubated under the conditions of the first step. At various time points, aliquots were withdrawn and analyzed for reconstitution intermediates by sucrose-gradient centrifugation. At the same time points, sister aliquots were taken, subjected to a second-step incubation and tested for activity in a poly(Phe) system after complementing with native 30S subunits. B. The pattern of the sucrose-gradient runs; 0 rain, the 50S peak is caused by native 50S subunits added for marking the 50S position. C. The relative amounts of the particles formed in (B) were drawn together with the obtained activity (- -) o, Rlso(l); A, Rico*(1); O, R15o(2); ®, poly(Phe)synthesis activity. B and C were taken from [33].

744

KH Nierhaus

Table IV. Sequential addition of proteins in the course of total reconstitution. Data of the small subunit were obtained from [28], those of the large subunit from [33]; proteins in bold indicate proteins essential and sufficient for the Rl*-formation, from [29] and [35], respectively. Subunit

Step

Small subunit

rRNA (RI)

1

16S

2

R13o

3

R13o*

I

23S+5S

Large subunit

2

Rls0(1)

3

Rlso*(l)

4

33S

p~50S ~ 325

4,1S

OoC

37°C

+

>

RI3o

21-22S

>

RI3o*

25-26S

>

30S

L1, L2, L3, L4, L5, L7/L12, L9, L10, L11, L13, L15, L17, LI8, L20, L21, L22, L23, L24, L26, L29, L33, L34

0°C > (4)

L6, LI4, LI6, L19, L25, L27, L28, L30, L31, L32

44°C, (4)

(20)

Rlso (2) ~

50S

48S

pa50S ~

50S

50S

Fig 2. Comparison of the precursor particles of the in vivo and in vitro assembly of the 50S subunit.

>

50°C, (20)

50°C

,~

Sediment coefficient

0°C

(4) +

Reconst interm (RI)

S I, $2, $3, SI0, S14, $21

44°C

415

p~50S ~

Temp (mM Mg 2+)

S4, $5, $6, $7, $8, $9, SI1, S12, S13, S15, S16, S17, S18, S19, $20

+

RIs0(2)

in vitro : 3 intermediates Rlso (1) - - ~ Rl*so (1) ~

in vivo : 3 precursors

Proteins

>

>

RIs0(1)

33S

Rlso*(1)

41S

Rlso(2)

48S

50S

distinct rRNA molecule is E-t, whereas for n initiator proteins the probability is E-n (independent events). Since the probability is the same for each rRNA molecule, the total probability of obtaining a complete initiation complex (ie a complex of rRNA and all n initiator proteins) is E x (E-n) = E~-n. This is identical with the fraction of TP (A), which appears in active particles, since only complete initiation complexes will form active particles. Hence, A = E~-n and lnA =(l-n)

rRNA to r-proteins (E for rRN,~ excess), and n represents the number of assembly-initiator proteins. Let us again assume that there is only one initiator protein. If the molar ratio rRNA:TP is E, then the probability of finding the initiator protein on one

lnE

(5)

Equation (5) gives us direct access to the experimental strategy for the elucidation of the number of initiator proteins. One keeps the level of TP50 constant and increases the input E of (23S+5$)rRNA. The reconsti-

The assembly of ribosomes

RNAexcess IE. motorratio rRNA:TPSO) 1,,

--=4

.m 1 o

"a "~:

2

-

2,,

3,,

-e--

A=I

,41.1---

~

: A:I -

Z.=.._

A:I

A=I ~

A:I/z

A:lh

+

"6 I=

--

A=I

A:tA

--e-

~-

~

A:lh A

100%

~

gl-n

~

< _= 10%

n-2

-3 n:10 1%

i_.__L. J 2 36 tnE

745

also given in figure 3B. The observed slope o f - l indicates the existence of only two initiator proteins. The two assembly-initiator proteins were identified according to the strategy outlined in figure 4A. Selected r-proteins were incubated with stoichiometric amounts of rRNA under the first-step condition (see equation (3)). The rRNA excess was then increased, eg 6-fold, TPS0 was added, and the incubation at conditions as for the first-step, was repeated before a standard second-step incubation was performed. Only if the selection of proteins contained both initiator proteins, stoichiometric amounts of complete initiator complexes were formed before establishing the rRNA excess thus leading to a high yield of active particles. In contrast, if the selection contains only one or none of the initiator proteins, complete initiation complexes can only be generated after establishing the rRNA excess leading to a low output of active particles. L3 and L24 were identified as the two assembly-initiator proteins of the large subunit with this strategy [40]. This kind of analysis implies the assumption that the many highly purified r-proteins used in the reconstitution experiment are of comparable activity, an assumption that cannot easily be verified. Therefore, an additional and independent assessment of the initiator proteins is desirable. A second strategy is sketched in figure 4B and is based on the non-cooperative character of the binding of the initiator proteins. (23S+5S) rRNAs are incubated with substoichiometric amounts of [14C]TPS0 (eg, rRNA:TPS0 = 1:0.5), then at various

, 10 Initiafor proteins

Fig 3. The dependence of tile fraction of active particles (,4) on the excess of rRNA (E) over ribosomal proteins and the number oi initiator proteins (n). A is measured as fraction of the input proteins appearing in active particles. A. Illustration of the dependence of (A) on both the rRNA excess (E) and the number of initiator proteins (n). Only those initiator proteins will show up in active particles which are present in complete assembly-initiation complexes containing all initiator proteins. B. Active fraction (A) predicted by the formula (equation (4), straight lines), and the corresponding experiment (points). For details see text. B is taken from [40].

A RNA excess experiment 1.step

I............................ rRNAil,;,se[.prot

L B

•

orRNA {6,I+TP50

lOm,~incub. , .. - ?> mm complex.

2.step

d 10minincub. >

pol.y (Phe) synlhesis

pulse chase experiment 1.step rRNA÷II4C]TPSO lO.S,,! ~ - - - ~ + []H]TP50 (ts.I I

tution is then performed and the output of active particles, A, determined. Figure 3B shows a double logarithmic plot I n A vs In E, where the theoretical output of active particles A (in percent of the input of TPS0) was drawn assuming various values for n between 1 and 10. The results of t h e c o r r e s p o n d i n g e x p e r i m e n t k e e p i n g c o n s t a n t TPSO a n d i n c r e a s i n g t h e i n p u t o f ( 2 3 S + 5 S ) r R N A [ 4 0 ] is

~

)

2"step -

'

iso[ation of ===:> ~ > SOSparticles

= = ~ ,6f'/3H

Fig 4. Strategies for the identification of the assemblyinitiator proteins. A. Test for complete initiation complexes; (1 x) and (6 x), the molar ratio rRNA:proteins is I:l and 6:1, respectively. B. Chasing strategy; (0.5 x) and (!.5 x), the molar ratio of TPS0:rRNA was 0.5: l and 1.5: l, respectively. Sel proteins, selected proteins. For further explanations, see text.

746

KH Nierhaus

times of the first-step incubation an excess of [3H]TP50 is added. The idea is that the earlier a protein is incorporated during the assembly process the less it should be susceptible to exchange, and the initiator proteins should be those proteins which can be chased least of all. After the two-step reconstitution the particles are isolated and their proteins separated on a two-dimensional gel electrophoresis. The protein spots are cut out and the corresponding ~4C/3H ratios determined. The largest 14CRH ratios indicate the lowest 'chasing efficiencies'. Figure 5 demonstrates the results. L24 was indeed the protein with the lowest 'chasing efficiency' followed by L3, confirming the data from the reconstitution analysis with individual proteins. We understand from equation (4) and from figure 3A that a high yield of active particles under conditions where rRNAs are synthesized in excess over r-proteins require a small number of assemblyinitiator proteins. Why then, are there two initiator proteins rather than one? As already mentioned, the mechanisms regulating the mutual adaptation of the synthesis of rRNA and that of r-proteins uncouple during extremely unfavorable growth conditions leading to an excess synthesis of rRNA. At this point feed-back mechanisms and auto-regulatory circles become increasingly important, such as the translational regulation of the rproteins [4]. The principle of the translational regulation is demonstrated in figure 6. Usually the second or third cistron of a polycistronic mRNA of r-proteins codes for an RNA-binding protein which can also bind to the Shine-Dalgarno region of the first cistron from its own mRNA thus competing with initiating 11 ~-r-'--r- .... "--'T .......

1

3

i

.~

20

6

pulse time [mini

Fig 5. Time course of the 14C/3H ratio of the various Lproteins determined according to the strategy outlined in figure 4B. Ta'-en from [40].

one cistron

SD AUG potycistronic mRNA !. . . . . . . . . . . . . . . . . . . . . .~

~

binding region

I

t

I low offinity I I

r-protein ,,,

I

high offinity rRNA

- binding region

~---=~

Regutotive r-proteins: RNA binding proteins St,, S?. 58, $21, L1, L6, LIO

Fig 6. Translational regulation of the ribosomal proteins [4]. Usually the second or third cistron of a polycistronic code for an rRNA-binding protein which can also bind to the Shine-Daigamo region of the first cistron thus competing with initiating 30S subunits. This regulatory protein will only inhibit the translation of its own polycistronic mRNA when a significant free pool of the protein is in the cell. 30S subunit and reducing the frequency of translation of its mRNA. If there would be only one assembly-initiator protein in the presence of excess rRNA all the rproteins would flow in the formation of active particles leaving no free pool of r-proteins for the regulatory tasks. Only the fact that two initiator proteins exist is responsible for assembly-dead ends from which r-proteins might be provided for the translational control. Therefore, two initiator proteins seem to represent an optimum, the number must be small in order to allow the formation of significant amounts of active ribosomes in the presence of rRNA excess, on the other hand the number must be larger than one in order to enable translational control under unfavorable growth conditions, When we consider the binding sites of the assembly-initiator proteins a further aspect of the highly differentiated system of the initiation of ribosomal assembly becomes evident. L24 is that RNA-binding protein which binds nearest to the 5'-end of 23S rRNA [41 ] and is thus ideally suited as an assembly initiator protein. Two binding regions of L24 have been identified, one in the region from nucleotide 280 to 360 (E coli numbering) and one from nucleotide 480 to 510 which have been implicated in the primary binding and in 'organizing' the rRNA structure for assembly, respectively. In contrast, L3 binds far away from the 5'-end in the 3'-half of the 23S rRNA molecule [42]. It follows that as long as rRNAs and rproteins are synthesized stoichiometrically the in-

747

The assembly of ribosomes

itiation of the assembly of the large subunit is solely governed by L24; the protein L3 becomes effective only when it is needed, namely when an excess of L3binding sites is exposed under unfavorable growth conditions thus generating assembly-dead ends. An E coli mutant with an altered L24 (Gly 84 Asp) produces assembly-defective 50S subunits [43] in agreement with the findings described above. Although it was already known that L24 was only essential for the early assembly but dispensable in the late assembly events and not involved in the functions of the ribosome [44], the report of a mutant lacking L24 was a surprise [45]. The mutation is conditionally lethal, the corresponding mutant is temperature sensitive, ie it does not grow at temperatures above 36°C. It shows severe growth defects even at permissive temperatures (doubling time five to seven times longer than that of wildtype), and the molar ratio of 30S: 50S in the cell is only 1:0.5. An in vitro analysis of the assembly of the mutant ribosomes could satisfyingly trace back all the phenotypic features to the lack of L24. L24 was absolutely required for the total reconstitution of active 50S subunits when the incubation temperature of the first step (normally 44°C, see equation (3)) was above 40°C. Below 40°C active particles could be formed in the absence of L24, the maximal output of active 50S subunits was only 50% at permissive temperatures as compared to optimal conditions, and the activation energy of the rate-limiting reaction during the first step incubation was twice as large as in the presence of L24 [37]. Obviously, another protein replaces L24 at 'permissive temperatures' (below 40°C) with reduced efficiency, and a systematic study with isolated Lproteins revealed that this protein was L20 [46]. With the 30S subunit from E coil ribosomes equivalent results were found. Here also two proteins initiate the assembly process, namely $4 and $7 [47]. $4 binds near the 5' end, $7 distant from the 5' end

[48]. Proteins essential for the early assembly: the assembly gradient The transition of the reconstitution intermediate RIs0(1) ~ Rlso*(l) is marked by a drastic S-value shift from 33S to 41S (see Precursor particles and reconstitution intermediates and table IV). RIs0*(1) is the essential product of the early assembly during the first-step incubation which cannot be by-passed during the second step. Both intermediate particles consist of 23S and 5S rRNA and 20 different proteins. Are all these many components needed for the critical transition RIs0(1) ~ RIso*(1)? The following strategy was used to explore this question (fig 7). Selected proteins were incubated with

23S and 5S rRNA in the first step of the total reconstitution, then TPso was added and Mg2+ increased to 20 mM, and the mixture was subjected to the secondstep incubation. The RIso*(1) conformation could only be formed when all the components essential for the transition xvere present during the first-step incubation, and only in this case the assembly could be completed to active 50S subunits in the second step. The activity of the resulting particles can be assessed by poly(U) dependent poly(Phe) synthesis. The results of a systematic analysis with purified ribosomal proteins were surprising [35]. Only 23S rRNA and five proteins (L4, L13, L20, L22 and L24) were necessary and sufficient for establishing the functionally important conformation RIs0*(1), L3 stimulated the formation, 5S rRNA and the other ribosomal proteins were not required. The comparison of the known binding sites [42] of the proteins important for the early assembly revealed that all the essential proteins have a binding site near the 5'-end of the 3S rRNA, only the stimulating L3 binds near the 3'-end. Since all polymerase-dependent synthesis of nucleic acids start at the 5'-end, this observation has an important consequence. Those proteins which determine the early-assembly reactions bind in vivo already just after the onset of the synthesis of 23S rRNA and before the synthesis of rRNA has been completed. This coupling of rRNA synthesis and ribosomal assembly was termed 'assembly gradient' [36] and means that 'the progress of rRNA synthesis dictates the progress of assembly'. Therefore, the entropic situation of the in vivo assembly is much simpler than that of the total reconstitution: in vivo the assembly starts with a relatively short 5' sequence of the rRNA and the five proteins essential for the early assembly, whereas in vitro the mature 23S rRNA is exposed to all 33 L-proteins. This entropic advantage of the in vivo assembly over the in vitro reconstitution cannot be overestimated. It might be one important reason why in vivo the 50S assembly of E coli ribosomes occurs in a couple of min at 37°C, whereas in vitro we need 90 min at 50°C. Early assembly proteins

(23S+5S) rRNA + selected proteins ,

1. step incubation

,~>

RiVal1) formation ?

+TPSO

2.step incubation ~ >

poty(Phe)

Fig 7. Experimental strategy for the identification of the early-assembly proteins essential for the formation of the RIs0*(1)-conformation.

748

KH Nierhaus

The longer the rRNA the more important will be the assembly gradient for an energetically favorable assembly process. The 'assembly gradient' is until now the only explanation for the unbelievably complicated process of ribosomal assembly in eukaryotes, where the r-proteins are imported to the nucleoli, the loci of rRNA transcription except those of 5S rRNA, and from where the more or less mature ribosomal subunits are exported to the cytoplasma. We assume that this mechanism is required by the necessity to couple rRNA transcription and ribosomal assembly and thus to retain the entropic advantage of the assembly gradient. The identification of the early assembly proteins responsible for the transition Rl.~0~ RI30* on the way to active 30S subunits had to be performed with the one-step reconstitution technique (equation (1)). Selected S-proteins were incubated at 37°C for 20 , i n , then TP30 were added at 0°C and the activity of the 30S subunits formed were assessed. The proteins essential for the transition were $4, $7, $8, S 16 and S 19 [29], but here the corresponding binding sites do not show such a clear preference for 16S rRNAregions near the 5'-end (R Brimacombe, Biochimie 73, in press). 16S rRNA might be too short so that an assembly gradient cannot become effective (about 1500 nt as compared to the about 3000 nt of 23S rRNA). The relatively short incubation times at 37°C required for the total reconstitution of 30S subunits are not very far from the assembly-time in vivo

thus supporting this view (active 30S subunits are already observed after a couple of minutes in the total reconstitution system). The assembly gradient requires a contiguous rRNA for the assembly process and the importance of the assembly gradient for an ordered assembly is more pronounced with increasing length of the rRNA. For ribosomal subunits with shorter rRNA the importance of the assembly gradient drops and thus eventually even the requirement for one contiguous rRNA molecule. The shortest rRNAs are found in mitochondria of some organisms. In fact, the 23S-type of rRNA from the mitochondria of Chlamydomonas reinhardtii is not coded in one contiguous gene but separated into at least 8 'subgenes' ([49], table V) which are scattered over the whole genome and might give clues towards 'assembly domains' which combine as rRNA-protein complexes to form the ribosomal subunit. An assembly gradient is obviously not effective in these mitochondria. The medium-sized E coli ribosome shows clear indications for the existence of an assembly gradient concerning the 50S assembly. However, the assembly gradient is not obligatory for the formation of active 50S particles since this subunit can be totally reconstituted. The 5S rRNA with its binding proteins L5, L 18 and L25 might be a remnant of early times in evolution when the rRNA was short enough to allow an assembly of separated assembly domains of rRNAprotein complexes.

Table V. Corresponding regions of rRNA species of the small subunit in mitoehondria of Chlamydomonas reinhardtii and in E coil (from [491). Subunit

Small subunit

Mito-rRNA species in C reinhardtii

Si $2 $3 $4

Coordinates in C rh

1-115 116--332 333-742 743-1200

Length

115 217 410 458

Corresponding region in E coli rRNA type 16S

no

1-61 112-403 500-992 1045-1542

1200 Large subunit

Li L2a

L2b L3a L3b

L4 Ls L6 L7 Ls

1-207 208-296 297-516 517-630 631-822 823-949 950-1089 1090-1247 1248-1926 1927-2419

207 89 220 114 192 127

140 158 679 493 2419

440-536 557-598 23S

659-856 914-1015 1030-1169 1180-1347 1598-2403 2413-2788

The assembly of ribosomes We find that the assembly gradient is not effective for short rRNAs, and that it is important but not obligatory for rRNA of intermediate length. If we are allowed to extrapolate this argument to the longest rRNAs present in the eukaryotic 60S subunits, then here the rRNA synthesis might be necessarily coupled to the ribosomal assembly. If so, the total reconstitution of 60S subunits from mature rRNA and TP60 would be impossible in principle. The recent finding that even the large subunit of eukaryotic cytoplasmic ribosomes from Euglena gracilis contains fragmented rRNA (the 23S-type rRNA appears in 13 fragments! [50]) does only superficially contradict this interpretation. The various gene stretches corresponding to the fragments are aligned in the correct order, connected by 'internal transcribed spacers' (ITS) and form together with the ITS one transcriptional unit. The excision of the ITS might occur during or after assembly and thus does not necessarily hinder an assembly-gradient process. (For intervening sequences in bacterial 23S rRNA and evolutionary implications see [51 ].)

Late-assembly components When we define those components of the 50S subunit as 'late-assembly components', which can be added to the second-step incubation yet yielding active particles, then 5S rRNA and all L-proteins (except those five proteins essential for the formation of RIs0*(1) particles) would belong to this category. However, here we define 'late-assembly components' in the narrow sense as those components which play a decisive role in the late assembly process, regardless of whether or not they are in addition important for stabilization of the structure and/or participate directly in ribosomal functions. Hitherto, only two L-proteins have been identified in this respect, namely L I 5 and L16, the main task of which is the organization of the late assembly. A mutant exists which lacks L15 ([52], see also next section); whereas a mutant without L I6 has not yet been described. However, ribosomes lacking either L15 or L 16 or even both proteins can be reconstituted and are active in poly(U) dependent poly(Phe) synthesis. Either protein accelerates the assembly process by a factor of 2 to 4; the proteins act synergistically since both proteins together increase the assembly rate by a factor of 20. The effects of both proteins are also observed when they were added after the two-step reconstitution during a short third incubation under the conditions of the second step. The latter feature underlines their involvement in the late assembly [53]. The L16 dependent activation energy has been measured as 42 kcal/mol [54]. 5S rRNA also fulfills the criteria of a late assembly component, viz it can also be added after the two-

749

step reconstitution to a third incubation [55]. The activation effect of 5S rRNA is heat dependent, ie it induces a conformational change. However, in contrast to L15 and L16, fully active particles without 5S rRNA cannot be reconstituted [55, 56]. Therefore, a direct or indirect involvement of 5S rRNA in ribosomal functions is likely in addition to its assembly activities. A comparison of the reactions of the early with those of the late assembly reveals that the sequences of the early reactions are better defined than those of the late assembly. For example, L24 can be replaced as assembly-initiator protein by L20 but only with severely impaired efficiency. In the presence of L24 therefore the L20 alternative practically does not exist (for more details see preceding section). In contrast, the order of pathways seems to be less rigid during the late assembly. Under standard reconstitution conditions the integration of L16 depends largely on the preceding incorporation of LI5 [57]. However, a slight change in the ionic conditions (240 instead of 400 mM NHnC1) allows the L I6 incorporation independent of LI5 [53]. It follows that the order of assembly reactions seems to be tightly defined during the early assembly, whereas alternative pathways exist in the process of the late assembly.

Mere assembly proteins A comparison of the secondary structures of rRNAs from organisms of the various kingdoms has shown that about two-thirds of the E coli 16S and 23S rRNAs are universally conserved ([58], fig 8). The regions of the remaining one-third, which are scattered over the rRNAs, can be shorter, longer or even absent in other organisms. Likewise, one-third of the r-proteins are dispensable in E coli, since mutants have been described lacking one or the other r-protein ([59], see also table VI). These dispensable rRNA regions and r-proteins are obviously not essential for ribosomal assembly, structure or functions, but they might accelerate assembly, stabilize structures or tune functions. Proteins which accelerate assembly represent one class of mere assembly proteins. S16 [60] and LI5 [53] belong to this class. Fully active 30S subunits can be assembled in the absence of 516, but the protein accelerates the assembly process. L15 is absolutely required for the formation of active particles under standard reconstitution conditions, but this requirement is relieved after reducing the NH4CI concentration from 400 to 240 raM. Now L15 accelerates the assembly process by a factor 2 to 4 which corresponds to the prolonged generation time (2- to 3-fold) of the mutant lacking L15. The relative dependence of both assembly rate and generation time on L15 is very similar. The conclusion at hand is that the production

750

KH Nierhaus Table VI. Mutants from E coli lacking r-proteins. Taken from [591.

Subunit

Missing protein

30S t

50S

Phenotype

SI $6 $9 S13 SI7 $20

cold-sensitive

LI LII LI5 LI9 L24

cold-sensitive

L27 L28 L29 L30 L33

temperature-sensitive

temperature-sensitive, very slow growth cold-sensitive cold-sensitive cold-sensitive

m

..iw,

Fig 8, The core structure (thick lines) common to all 16Stype rRNAs from ribosomes of various organisms. A. Eubacteria (E coli). B. Archaebacteria (Halobacterium volcanii). C. Eukaryotes (yeast, cytoplasmic ribosomes). D. Mitochondria of plants (maize). Taken from [581.

of the large ribosomal subunits is the rate-limiting factor of the generation time of E coli cells under optimal growth conditions. A second class of mere assembly proteins consists of proteins which are essential for achieving a distinct assembly stage which is a necessary intermediate in the path towards an active subunit. If mutants lacking such a protein exist at all they must be very sick. L20, L24, and probably L I6 belong to this class. L I6 is an assembly protein [53], and a mutant lacking L I6 has not yet been identified. Negative arguments are usually weak. But since mutants lacking a ribosomal protein were fished in a systematic way [59], we may take the fact that no minus-Ll6 mutant was detected as a hint, that minus-Ll6 mutants might be lethal. A mutant lacking L20 has also as yet not been described. The mutant lacking L24 is severely handi-

capped as expected, it is temperature sensitive and grows even at permissive temperatures very slowly ([45], see Assembly-initiator proteins). Both L20 and L24 are essential for the formation of the obligatory, early intermediate Rls0*(1), but they are involved neither in the late assembly nor in ribosomal functions. How can one test this? 50S subunits incubated with 4.2 M LiC! loose 5S rRNA and most of the L-proteins. A core particle '4.2c' remains which consists of 23S rRNA and mainly the proteins L3, L4, L 13 and L22. The point is that the 4.2c core particle can be reconstituted in the presence of TP50 exclusively under the conditions of the second step (see equation (3)). The fact that the first-step incubation can be omitted demonstrates that this particle still maintains the RIs0*(l) conformation which is the essential product of the first-step incubation (see Precursor particles and reconstitution intermediates). Obviously L20, L24, and other proteins are essential for the formation of the Rls0*(l) conformation, but if this conformation has been once achieved, at least L20 and L24 can be again removed without loosing the Rls0*(1) contbrmation. If now the 4.2c core particle is reconstituted with TP50 lacking either L20 or L24, fully active particles are obtained ([61] and [44], respectively). Therefore, both proteins are essential for the early assembly but play no role in either late assembly or ribosomal functions.

Assembly maps In the preceding sections elements of the formal assembly pathway were described: identification of

The assembly of ribosomes reconstitution intermediates, of rate-limiting steps and ribosomal components responsible for early and late assembly reactions, and of mere assembly proteins. In this section we shall discuss a new stage of resolution of the assembly, viz the precise sequence of binding reactions starting with the 23S (16S) rRNA. The experimental results of such binding analyses are accumulated in 'assembly maps' (see below). Figure 9 outlines the principle of assembly mapping experiments: 23S rRNA and a selection of r-proteins are incubated in the first-step incubation, then the rRNA-protein complex is separated from non-bound proteins by sucrose-gradient centrifugation and the proteins of the complex are identified via gel electrophoresis. An example is shown in figure 10. When L5 is incubated alone with 23S rRNA, no binding is observed, ie L5 is not an RNA-binding protein. However, the binding of L5 can be mediated by the rRNA-binding proteins L2 and L4; L2 confers a strong, L4 a weak binding. In the assembly map, 23S rRNA is drawn as an oblong (at the top of fig 12). An arrow going from 23S rRNA to a protein marks an rRNA-binding

751

protein (for e~ample, L2 and L4); strong assembly dependences z~:~-eindicated by thick arrows (eg, L2 ----> L5), weak independences by thin arrows (eg, L4 ---> L5). Assembly maps for the 30S [62] and for the 50S subunit [34] are shown in figures 11 and 12, respectively. Assembly maps give important topographical clues, since an assembly dependence is most likely due to a next-neighbour interaction rather than to a long-range effect. Assembly maps also demonstrate that the procedure of splitting off r-proteins with increasing LiCi concentrations reverses the assembly order (fig 12B). Two further implications of the assembly map should b~:~-mentioned. Firstly, the organization of most of the p r o t e i n genes in large operons [41 is highly consc~ d in pro- and archaebacteria [65, 66], the selective antage of these gene groupings is not yet fully un~ rood. The projection of the L-proteins present in on,: ~3peron onto the assembly map reveals a striking correspondence. All L-proteins of an operon are embedded in a network of assembly dependencies (fig 12C) thus possibly reflecting assembly domains. The co-regulation of members of an assembly domain would improve the efficiency of the assembly process and might ~herefore represent one factor for the

Assembly mopping experiments 280 nm

1. Reconstitution

23 S RNA+someproteins

6,ramMg2*/64"C

2. SW grodient Sucrose 10-30 % 2h 65rain 150000rpm

3. TEA precipitotion

Ut

11

unbound proteins RNA-protein complex

L2L~-O.~,,~ L5- " ~'~ ,,

5% TCA

I

/+. SDS gel onolysis of the bound proteins Fig 9. Principle of assembly-mapping ~xperiments.

0 /~ 8 frQction number

12

direction of sedimentofion Fig 10. Example of assembly mapping experiments. For details see ter~. Taken from [57].

752

KH Nierhaus

I

16S rRNA

I

. . . . . . . .

I I I I I I 1

,, -III

%

5 i

,sk6 S19

y

, .......

$18

$6"

" /

jr

//

S21 Fig 11. Assembly map of the small (30S) ribosomal subunit [62]. Supplemented with the results from [63] (broken arrows) and from [64] (S l).

conservation of the operon structures of r-protein genes. Secondly, single-omission tests have identified six components which are essential for the reconstitution of an active peptidyltransferase center: 23S rRNA, L2, L3, L4, L15 and L16. All of them are interconnected with strong assembly dependencies (fig 12D) thus representing a functional skeleton of the ribosome [38]. Since both L15 and LI6 are mere late-assembly proteins not involved in ribosomal functions ([53], see Late assembly components), they are not candidates for the peptidyltransferase. The most likely candidates for this essential function are L2 and 23S rRNA (see [34] for discussion). Outlook An assembly map gives a complete or near complete picture of the assembly dependencies of the ribosomal components, but one should not assume that the map appropriately reflects the assembly process. For example, the 50S assembly map (fig 12) implies that most of the RNA-binding proteins (thick arrows from

23S rRNA) bind independently to 23S rRNA. We have, however, seen in Assembly-initiator proteins, that only two out of the 20 RNA-binding proteins can bind independently to 23S rRNA thus initiating the assembly process. The same is true for the 30S assembly, where also only two rRNA-binding proteins are assembly-initiator proteins. Not only the assembly initiation is governed by two proteins. The whole assembly of the 50S subunit seems to proceed in four or five steps, each of which is directed by one or two 'assembly-leader proteins' which still need to be identified (T Niedenzu, KH Nierhaus, unpublished). An indication for this groupwise assembly might be seen in the fact that most of the ribosomal proteins are split away from the 50S subunit in cooperative groups with increasing salt concentrations in an all-or-nothing process (fig 12B). With the identification of the assembly-leader proteins the formal assembly will be well understood; we know the key reactions, the key components and the thermodynamic parameters involved in the total reconstitution. However, we have only a poor knowledge about the factors which dramatically facilitate the 50S assembly in vivo as compared to the total reconstitution (in vivo: a couple of minutes at 37°C; in vitro: 90 min at 50°C). What is the importance of the assembly gradient in this respect? Does the coupling of assembly and rRNA processing play a role? Are rRNA helicases involved which might favor the rRNA folding in the course of the assembly? Do 'chaperonins' exist for rproteins which keep the r-proteins in an unfolded state thus facilitating the assembly process? A severe conformational change of a ribosomal protein upon assembly has been observed in at least one case. Isolated L16 has a radius of gyration of a globular protein, whereas L I6 in situ (after reconstitution) is much more compact and has a radius of gyration of a near spherical protein (V Nowotny, R May, KH Nierhaus, unpublished). The question 'which factors reduce the activation energies of the in vivo assembly?' cannot be explored in standard reconstitution systems. Rather, the rRNA to be assembled must be transcribed in vitro from the corresponding gene and the transcription process must be coupled to rRNAprocessing and assembly. Effective transcription of the 16S rRNA gene in vitro has been reported by two groups, the transcribed 16S rRNA could be reconstituted to active 30S subunits post transcriptionem [67, 68], but the goal of coupling of in vitro transcription and assembly of active subunits has not yet been reached. The ultimate resolution is an understanding of the assembly process at the molecular level. A first step on the long way is the elucidation of the changes of the rRNAprotection patterns against modifying reagents in the course of the assembly [69]. According

The assembly of ribosomes

0

A

~

~I

~s

,

E

LJ

/..,~

........"~

17mini emlnl~

RI

ITJ

.,~

..

"

'-'-~

•

~"

,e,'.. ,..'x,..__.....--...~ ...

R%o(21

T. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

~

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

--1

B

C

3'

'1

m

753

to the assembly map, S-proteins were subsequently added to 16S rRNA and the increasing protection of rRNA-bases was determined. If a base protection is not caused by long-range effects (which might be true at least for some of the bases), it gives a topographical clue but is not necessarily related to the binding site of the corresponding protein. Furthermore, the complementary knowledge concerning the r-protein moiety is still lacking. Although the primary sequences of all components of the E coli ribosome are known (for review see [70]), we are far away from a molecular resolution of the assembly process, since it would require a precise knowledge of at least the essential protein-protein, protein-rRNA and rRNA-rRNA interactions in the course of the assembly. One may question whether such a detailed picture is a desirable scientific goal because the picture differs in various aspects from organism to organism. Therefore, concentration on the principles and more general features of the assembly process is necessary. In spite of the large body of knowledge, which has been accumulated in the past concerning the assembly of ribosomes, and which has been outlined here, many important and exciting questions are still open. Some of the key words to be addressed here have been already mentioned: assembly leader proteins; factors facilitating in vivo assembly such as assembly gradient, chaperonins, helicases; regulation of ribosomal assembly in organelles where the ribosomal genes are distributed between the organelle chromosome and the nuclear DNA; ribosomal assembly in halophiles ([71], molar concentrations of cations in the cell) and ribosomal assembly in the nucleolus and the export of the quasimature ribosomes into the cytosol.

'

Fig 12. Assembly map of the large (50S) ribosomal subunit RI~o(I )

'

"," ',

Je

I

. ' ~K_h

L l'

:1

,.&~ .....

,

(A, [34]). The proteins boxed in with a broken line are required for the early assembly. RIs0(l) and RI50(2) are reconstitution intermediates of the 50S assembly. B. Comparison with the order of proteins split off by increasing LiCI concentrations. From bottom to top: 1 M LiCI, 2 M LiCI, 4 M LiCI, proteins remaining on the 23S rRNA after incubation with 4 M LiCI (4.0 c core particle). For details see [34]. C. Projection of the operon structure [5] of the L-protein genes in E coil onto the 50S assemb!y map. An exception is L I 6 of the S 10 operon (L3, L4, L23, L2, L22, LI6, L29) which is not present in the corresponding operon in archaebacteria [64, 65] and thus is not a member of a conserved assembly domain related to the S 10 operon; L30 of the spc operon (regulated unit: L5, L6, LI8, L30, L15) and L1 of the L l l and LIO operon (Lll, LI, LIO, L12) are further exceptions; both L30 and LI can be absent in phenotypically normal E coli mutants (table VI) and thus are not essential members of putative assembly domains. D. The 'functional skeleton' of L-proteins essential for the reconstitution of the peptidyltransferase activity of E coli ribosomes under standard conditions.

754

KH Nierhaus

Acknowledgments This paper is dedicated to HG Wittmann who passed away in March 1990. It was Wittmann's friendly, direct and continuous support and help which enabled the research in my group and promoted my scientific development. I consider it as a great fortune to have worked under his influence in his department for more than 20 years. I thank C Prescott, B Lewicki and FJ Franceschi for many discussions. I am grateful to J Belart and E Philippi for their kind and patient manner to optimize the presentations of our results.

References I 2 3 4 5 6

7 8 9

l0 Il 12 13

14 15 16

Wittmann HG (1986) Structure of ribosomes. In: Structure, Function, and Genetics of Ribosomes (Hardesty B, Kramer G, eds) Springer-Verlag, New York Tissi~res A, Watson JD, Schlessinger D, Hollingworth BR (1959) Ribonucleoprotein particles from Escherichia coll. J Mol Biol I, 221-233 Blobel G, Potter VR (1967) Studies on free and membrane-bound ribosomes in rat liver. J Mol Biol 26, 279-292 Nomura M, Gourse R, Baughman G (1984) Regulation of the synthesis of ribosomes and ribosomal components. Annu Rev Biochem 53, 75-117 Lindahl L, Zengel JM (1986) Ribosomal genes in Escherichia coli. Annu Rev Genet 20, 297-326 Hemmingsen SM, Woolford C, van der Fries SM, Tilly K, Dennis DT, Georgopoulos CP, Hendrix RW, Ellis RJ (1988) Homologous plant and bacterial proteins chaperone oligomeric protein assembly. Nature 333, 330-334 Sachs AB, Davis RW (1990) Translation initiation and ribosomal biogenesis: Involvement of a putative rRNA helicase and RPL46. Science 247, 1077-1079 Nierhaus KH (1990) Reconstitution of ribosomes. In: Ribosomes and protein synthesis (Spedding G, ed) University Press, Oxford 131 p Srivastava AK, Schlessinger D (1990) rRNA processing in Escherichia coli. In: The Ribosome: Structure, Function. and Evohaion (Hill WE, Dahlberg A, Garrett RA, Moore PB, Schlessinger D, Warner JR, eds) American Society for Microbiology, Washington DC, 426 p Sirdeshmukh R, Schlessinger D (1985) Ordered processing of Escherichia coli 23S rRNA in vio'o. Nucleic Acids Res 13, 5041-5054 Apirion D, Gegenheimer P (1984) Molecular biology of RNA processing in prokaryotic cells. In: Processing of RNA (Apirion D, ed) CRC Press, Boca Raton, 36 p Skinner RH, Stark MJR, Dahlberg AE (1985) Mutations within the 23S rRNA coding sequence of E coli which block ribosome assembly. EMBO J 4, 1605-1608 Stark MJR, Gregory RJ, Gourse RL, Thurlow DL, Zwieb C, Zimmermann RA, Dahlberg AE (1984) Effects of site-directed mutations in the central domain of 16S ribosomal RNA upon ribosomal protein binding, RNA processing and 30S subunit assembly. J Mol Biol 178, 303-322 Sirdeshmukh R, Schlessinger D (1985) Why is processing of 23S ribosomal RNA in Escherichia coli not obligate for its function? J Mol Biol 186, 669-672 Brimacombe R, Stiege W (1985) Structure and function of ribosomal RNA. Biochem J 229, 1-17 Srivastava AK, Schlessinger D (1989) Escherichia coli 16S rRNA 3'-end formation requires a distal transfer RNA sequence at a proper distance. EMBO J 8, 3159-3166

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Srivastava AK, Schlessinger D (1988) Coregulation of processing and translation: Mature 5' termini of Escherichia coli 23S ribosomal RNA form in polysomes. Proc Natl Acad Sci USA 85, 7144-7148 18 Misra TK, Apirion D (1979) RNase E, an RNA processing enzyme from Escherichia coll. J Biol Chem 254,

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Feunteun J, Jordan BK, Monier R (1972) Study of the maturation of 5S rRNA precursors in Escherichia coli. J Mol Bio170, 465-474 Bjtirk GR (1987) Modification of stable RNA. hz: Escherichia coli and Salmonella typhimurium (Neidhardt FC, ed) American Society for Microbiology, Washington, 719 p Chelbi-Alix MK, Expert-Bezan~on A, Hayes F, Alix JH, Branlant C (1981) Properties of ribosomes and ribosomal rRNAs synthesized by Escherichia coil grown in the presence of ethionine. Normal maturation of ribosomal RNA in the absence of methylation. Eur J Biochem 115, 627--634 Stiege W, Stade K, Schtiler D, Brimacombe R (1988) Covalent cross-linking of poly(A) to Escherichia coli ribosomes, and localization of the cross-link site within the 16S RNA. Nucleic Acids Res 16, 2369-2388 van Buul CPJJ, Visser W, van Knippenberg PH (1984) Increased translational fidelity caused by the antibiotic kasugamycin and ribosomal ambiguity in mutants harbouring the ksgA gene. FEBS Lett 177, 119-124 Osawa S (1968) Ribosome formation and structure. Annu Rev Biochem 37, 109-130 Lindahl L (1975) Intermediates and time kinetics to the in vivo assembly of Escherichia coli ribosomes. J Moi Biol 92, 15-37 Nierhaus KH, Bordasch K, Homann HE (1973) hz vivo assembly of Escherichia coil ribosomal proteins. J Mol Bio174, 587-597 Traub P, Nomura M (1968) Reconstitution of functionally active 30S ribosomal particles from RNA and proteins. Proc Nati Acad Sci USA 59, 777-784 Traub P, Nomura M (1969) Mechanism of assembly of 30S ribosomes studied in vitro. J Mol Bio140, 391--413 Held WA, Nomura M (1973) Rate determining step in the reconstitution of Escherichia coli 30S ribosomal subunits. Biochemistry 12, 3273-3281 Mangiarotti G, Apirion D, Schlessinger D, Silengo L (1968) Biosynthetic precursors of 30S and 50S ribosomal particles in Escherichia coli. Biochemistry 7,456--472 Hayes F, Hayes D (1971) Biosynthesis of ribosomes in E coli. Properties of ~ibosomal precursor particles and their RNA components. Biochimie 53, 369-382 Nierhaus KH, Dohme F (1974) Total reconstitution of functionally active 50S ribosomal subunits from Escherichia coli. Proc Natl Acad Sci USA 71, 4713--4717 Dohme F, Nierhaus KH (1976) Total reconstitution and assembly of 50S subunits from Escherichia coli ribosomes Or vitro. J Mol Biol 107, 585-599 Herold M, Nierhaus KH (1987) Incorporation of six additional proteins to complete the assembly map of the 50S subunit from Escherichia coli ribosomes. J Biol Chem 262, 8826-8833 Spillmann S, Dohme F, Nierhaus KH (1977) Assembly in vitro of the 50S subunit from Escherichia coli ribosomes: Proteins essential for the first heat-dependent conformational change. J Mol Biol 115, 513-523 Sieber G, Nierhaus KH (1978) Kinetic and thermodynamic parameters of the assembly in vitro of the large subunit from Escherichia coli ribosomes. Biochemistry 17, 3505-3511

The assembly of ribosomes 37

38 39

40 41

42 43

44 45 46 47

48

49 50 51

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