Biochimie (1991) 73, 1093-1100 © Soci6t6 fran~aise de biochimie et biologie mol6culaire / Elsevier, Paris

1093

Functional aspects of ribosomal proteins W M611er

Department of Medical Biochemistry, Sylvius Laboratory, Faculty of Medicine, Universityof Leiden, PO Box 9503, 2300 RA Leiden, The Netherlands (Received 2 November 1990; accepted 14 March 1991)

Summary - - A short personal recollection of HG Wittmann is given with emphasis on his basic contribution to the structure of the ribosome, in particular the ribosomal proteins. With these considerations in mind, two interrelated problems are reviewed here. The first relates to the internal symmetry both in tRNA and in the tetrameric Ll2-protein complex. The second problem to be addressed relates to the dynamics of transfer RNA in the ribosome and the role of L12 proteins in this process. ~ e importance of electrostatic repulsion in the maintenance of the mutual spatial orientation of tRNAs and LI 2 in the ribosome is emphasized in relation to a pendulum model for how L12 may steer translocation. ribosomes / L12-proteins / GTP hydrolysis / elongation factors translocation / transfer RNA

A personal recollection HG Wittmann was raised at Gut Mari#nhof, district Sensburg, East Prussia, once a territory of Germany, stretching along the Baltic with old names, like Ki~nigsberg, now Kaliningrad. In 1945, East Prussia was passed in part to the USSR and the remainder to Poland. After the war young HG Wittmann went to the West to build up a new life there. Pondering about this, one understands perhaps better who HG Wittmann was and why he radiated order, extraordinary discipline, a sense for hard work and strength in his environment. The farmer's son, who he was, had a keen interest in the laws of nature and it was this unique combination of personal qualities and circumstances which led to his creation of much of the basis framework for ribosome research as we kow it today. He lined up ribosomal protein S1 to 821 and LI to 1.,34 on 2-Dgels and next succeeded to purify each in quantities sufficient for protein structural studies. It is doubOrul whether this job would have been done elsewhere as accurately and completely. Probably not in the USA, where in the late sixties many young scientists turned towards problems related to regulation of gene expression, embryology, immunology or tumor virology. For HG Wittmann, this protein isolation was only a beginning; he knew best that one must sew to reap and that it takes time for a tree to grow; indeed the tree he planted, is still growing and branching into many fields.

l first met HG Wittmann during the summer of 1966 at CoM Spring Harbor when he gave the shortest lecture on ribosomes I ever heard. Just a dry announcement that his institute wouM embark on ribosome research, especially ribosomal protein of the 30S and 50S subunit in E coli. At that time I worked at Johns Hopkins, Medical School, as a young faculty member, making the transition from a physical chemist to a biochemist. Head of the biophysics department there was Howard Dintzis who coined the word 'ribosome' as few people know. During my stay there, I managed to isolate and fingerprint a f e w ribosomal proteins among which a small, multiple, 70S acidic ribosomal protein which I had named A-protein and found to be motile. It was gratifying for me that HG Wittmann acknowledged these findings early and once called me Mr Acidic when we talked about this protein. As ! indicated before, the major question HG Wittmann was interested in during those years, was to resolve all ribosomal proteins from each of the two subunits and indeed in 1970 he and E Kaltschmidt published together their now famous paper on the number of proteins in the small and large ribosomal subunit of E coil as determined by two-dimensional gel-electrophoresis. Here I just want to add briefly ti~at HG Wittmann inspired me greatly to continue ribosome research, having returned to Holland in 1967. With K Kischa from Vienna, we found in Leiden that A-protein (LI2 in the nomenclature of Wittmann) was important for

1094

w MOiler

triggering EF-G factor-dependent GTPase on the ribosome; this GTPase activity was inhibited in the presence of anti-L12, as supplied to us by G St6ffler. It was a productive and also competitive period, leading quickly to the identification of the proteins L6, LIO and LI1 in the neighbourhood of the GTPase trigger. During this period, everybody benefited greatly from the 2-D-gel-electrophoresis system as developed by Wittmann. Concerning the primary structure of A-protein, its determination was speeded up by his help considerably. Although ! got some feeling for fishing out ribosomal proteins, and do their tryptic peptide maps, sequencing them completely was something quite different. On that score HG Wittmann and his wife B Wittmann-Liebold taught me in Berlin the ins and outs of protein sequencing, leading in 1972 to a joint publication of the first ribosomal protein sequence, namely Al /A2-protein being identical to L7/L12. &lost of the hard hand-work was done by C Terhorst (Leiden), while Richard Laursen (Boston) carried out Edman degradations on the automatic solid phase apparatus, and B Wittmann-Liebold operated the automatic Beckman sequencer. On all occasions, HG Wittmann offered me his personal friendship and hospitality. C Terhor~t and I even had the honor to find ourselves inadvertently inaugurating the new building which we had entered as guest-workers. The extraordinary enthousiasm of Wittmann for science created between the two of us a sphere of sympathy and mutual respect throughout

the years. The primary sequence of A(L12) protein, involved in both EF-G and EF-T dependent GTP hydrolysis led to many new studies for instance on the phylogenetic relationship between prokaryotes, eukaryotes and so-called archaebacteriae. For one thing, HG Wittmann stood for 'Griindlichkeit' and a distaste for rapid conclusions and speculations. He felt that the components of the ribosome had to be isolated first [11 followed by an accurate description of their architecture [21. As a tribute to a scientist who was well aware that the work he started would never be finished by him, I quote from his own words: 'Without insight into the structure of the ribosome and its components at high resolution our knowledge of the process of protein biosynthesis will remain at the descriptive level, and many important questions eg on the molecular mechanism of the translocation process or the peptidyl transferase reaction and on the interaction of ribosomes with factors, mRNA, tRNA and antibodies will remain unanswered or at a hypothetical level'. Hopefully, future generations will see the wisdom of his words.

Introduction

specific role for individual ribosomal proteins in ribosome function. For this reason, I confine myself mostly to protein L12 and offer a plausible way in which this protein may facilitate the transloeation reaction in protein synthesis. This should not be construed to mean that ribosomal RNA would be a bystander in this reaction. On the contrary, in a popularity contest for ribosomal proteins (vs ribosomal RNA [2, 3]), the noes would have it and they may be right for the time being. However, evolution has tinkered on the proteinRNA machinery for a long time so that even visualizing a functional protein-free ribosome may remain a fiction. Therefore, I would like to plead for continuing work on both ribosomal RNA and ribosomal proteins, independently of recent trends in biochemistry seducing many students to clone and sequence only nucleic ~cids irrespective of the information yielded on the function of a protein per se. As someone who studied the integrity of ribosomal RNA in the period when messenger was discovered [4] and who focussed on ribosomal proteins while at Hopkins later on [5, 6], I can only say

Ever since the discovery of elongation factors, their transient interaction with the ribosome has been compared to some type of mechano-chemical reaction using GTP instead of ATP. When it was found that 2 mol of GTP were needed for binding of the elongation factors EF-G and EF-Tu to the ribosome, attention turned towards the ribosomal components responsible for this binding and thus for triggering hydrolysis of GTP. In this paper I shall describe, firstly where elongation factors bind to the ribosomes and, secondly how they may induce transitions in the ribosome and what the effects of these transitions may be on tRNA movements. A more complete description of this part of the subject can be found elsewhere [1 ]. Perhaps with exception of the acidic ribosomal protein LI2 (1.,7 is the N-terminal acetylated form of L12; in this review L7/L 12 proteins are treated as one (L12) unless stated otherwise), there are few data proving a

References 1 WittmannHG (1982) Components of bacterial ribosomes. Annu Rev Biochem 51,155-183 2 WittmannHG (1983) Architectureof prokaryotic ribosomes. Annu Rev 8iochem 52, 35-65

The ribosome and translation factors that that science goes through cycles and that it is good to refine the step of the cycle one is interested in, irrespective of the glitter of the moment. Sites of interaction between factors and ribosomes

GTP hydrolysis as induced by elongation factor EF-G and EF-Tu requires an intact ribosome, including L12. Particular striking were the early findings of a functional interdependence of protein LI0, L11 and LI2 for reconstitution of factor-dependent GTPases [6]. This behaviour is in line with their mapping together in a restricted domain of the 50S particle and with their in tandem arrangement in one operon of the bacterial chromosome. The discovery of an L12 stalk [7] strengthened the idea that factors bind at or near L 12 and indeed crosslinking of factors had led to labelling of combinations of LI0, L11 and LI2 [6]. Immuno-electron microscopy studies substantiated this and the binding site for EF-G and EF-Tu was mapped at the base of the L I 2-stalk [8, 9]. The limited resolution of electron-microscopy prevented really an at-or-near L I2 statement but the localization agreed with the cross-link found between EF-G and L l l [10] when one considers that LI1 is known to map at the base of the L12-stalk [11]. Also a photoreactive azido-GTP derivative cross-linked in an EF-G dependent manner to protein L I 1 [12]. Thus the picture emerged that factors and GTP meet in a special domain of the 50S ribosome, but how 23S ribosomal RNA distributed through it was less clear. Therefore it was not surprising that elongation factor EF-G protects 23S ribosomal RNA in the 1067 region [13] where the GTPase related LI 1 protein and the (L12)4L10 complex both bind at the same 23S RNA site [14]; moreover this 1067 region is the site of interaction of the antibiotic thiostrepton, which prevents formation of an EF-G-GTP-ribosome complex [15]. Does this mean that ribosomal RNA and not ribosomal proteins are the real target of the factors? This is hard to say with the present techniques used and any firm choice would seem premature. Definite answers may require higher resolution methods with less interference like NMR, neutron diffraction or atomic mapping of factor-ribosome complexes possibly by X-ray crystallography. Perhaps non-invasive methods in which a small segment of a particular ribosomal protein or ribosomal RNA could be enriched for certain isotopes and thus studied in the ribosome by NMR might provide a new approach. This may be relevant also to the acidic 'blobs' in protein L12 which, judged from phylogenetic criteria, must have an ancient and important function in translation. Given the overall negative charge of RNA, the contribution of electrostatic repulsions between L12 and tRNAs in orienting transfer RNAs towards their reaction sites, may be important in this context.

1095

Ribosomal protein L12 resembles a motile protein

In an early attempt to understand the function of the factors, attention was drawn to L12 showing properties reminescent of mobile proteins [16]. There is no doubt of a formal analogy between ATP-linked stepwise interaction of actin and myosin and the GTP-linked stepwise interaction of EF-G with the ribosome region comprising proteins L10, L11 and LI2; especially the structural properties of L I2 including its multimeric occurrence, alanine-rich hinge region and strong acidity is typical for contractile systems acting on ATP instead of GTP. Both myosin and actin behave as ATPases, while ribosomes and factors, when added together, possess GTPase activity. Thus the family of GTP- and ATP-binding proteins shows family traits in the mode of partner interaction at the level of nucleotide hydrolysis. The mutual interplay between ribosotnes and factors therefore may contain elements of an ancient mechano-chemical system from which ATPdependent contractile systems have been derived later on. In this context, it is worth mentioning that tubulin itself is also a GTPase and it would be interesting to know how protein synthesis and spindle formation compete for the same GTP pool during the cell cycle. Thus in molecular terms, elongation factors may possess motile properties which induce cyclic changes in the orientation of ribosomal complexes like (L12)4. LI0 leading to the facilitation of movements of transfer RNA in the ribosome. This raises the question how transfer RNAs move through the ribosome before they bind to mRNA and react during protein synthesis. Do different amino acyl transfer RNAs follow the same trajectory on their way through the ribosome or do they diffuse along different pathways before being selected on the basis of their correct codon-anticodon interaction on the messenger [17]? In this respect, energy transfer measurements between different aminoacyl tRNAs and defined domains of ribosomal RNAs would be of a great help. Orientation of the LI2 chains in dimers and tetramers

The structural properties of protein LI2 have been reviewed recently. Here we want to make two comments, one about whether the dimer structure is parallel or antiparallel, the other about the orientation of the two dimers in the ribosome. There is consensus that dimers of L12 dissociate only in strongly denaturating solvents like 8 M urea or 5 M guanidiniumchlofide [18]. By far the most plausible model for the arrangement of the two chains is a parallel orientation. It is based on cross-linking [19], X-ray crystallography of the C-terminal part [20] and binding experiments of the N-terminal part of L I 2 [21, 22] as follows. 1) The presence of a 9 A, high yield

! 096

W MOiler

Table I. Phosphoryi binding loop of guanine nucleotide binding proteins. For references see [30]. Elongation factor EF-I ct (EF-Tu) Artemia Yeast Human Mucor racemosus

Mouse Drosophilia Xenopus laevis E coli T thermophihls Euglena chloroplast

Yeast mitochondrial E coil IF~

Hormone receptor Go-protein Transduction Ras protein Oncogen P2t Proto-oncogen P2~ H/K viral oncogen P~I Adenylate kinase Consensus sequence

GHVDS G KST GHVDS G KST GHVDS G KST GHVDS G KST GHVDS G KST GHVDS G KST GHVDS G KST GHVDS G KIT GHVDH G KIT GHVDS G KIT GHVDH G KIT GHVDH G KIT GAGEG G KTS GAGEG G KTS

partial endgroup determination would have been of particular value in this case. In the past, we have observed also higher molecular weight forms of L12 but those were found in the absence of 13-mercaptoethanol and residual L10 could not be excluded at that time [24]. Granted the truth that in salt L I 2-tetramers can be extracted, it should also be stated that CsCi or saltethanol extraction does only yield stable dimers which yield on reconstitution a fully, functional 70S particle. There is also no doubt that L12 dimers are capable to form reversibly a functional, tetramerie structure in the ribosome [25]. The second remark we like to make is that in the ribosome itself dimeric L I2 structures diverge from L I0 and that roughly four N-termini are found at the basis of the stalk [22]. Their internal orientation is based on fluorescence energy transfer [26] and also proposed on the basis of thorough immuno-electron microscopy in several independent studies [22, 27], As a result an alternating parallel-antiparallel orientation of 4 LI2 chains [23] seems to us of doubtful physiological significance.

EF-I~ switches its conformation around Arg-68 under influence of GTP hydrolysis GAVGV G KSA GAGGV G KSA GARGV G KSA GGPGS G KGT GXXXX G KS/T

cross-link between residue 51 in one chain and residue 29 in the other chain supports a parallel rather than an antiparallel arrangement; 2) the two-fold symmetry axis in the crystal structure of a C-terminal fragment of L I2 (CTF47-120) relates one molecule to another and the contacts made in the crystal are of a conservative nature when compared for several eubacteria. This two-fold symmetry probably reflects the true symetry in the intact dimer; 3) an N-terminal intact dimeric fragment (NTF1-73) can bind to L I2-minus ribosomes whereas a C-terminal fragment (CTF74-120) is totally inactive [21, 22, 251. Therefore a parallel, two-headed L 12 [20] dimer conforms most closely to a number of different observations on this protein. Recently L I2 proteins have been reported to occur as a tetramer after salt extraction [23]. At first sight this result seems questionable because salt extraction leads easily to aggregation of LI2 due to residual LI0 or LI0 fragments. However, the absence of L10 was checked reportedly by SDS-polyacrylamide gel-electrophoresis although citing of a simple amino acid composition or

X-ray crystallography and comparative sequence analysis of a number of GTP binding proteins have supplied several determinants for the binding of guanine nucleotide to such proteins [28]. Of particular interest is the presence of a typical phosphoryl binding loop GXXXXGK in most guanine nucleotide-binding proteins [29, 30]. The conservative nature of this glycine-rich loop is given as illustration in table I. We have searched for well-defined changes in the EF-IGt structure of Artemia on replacement of GDP by GTP in trypsin digestion studies [31 ]. An unique local unfolding of EF-1o~ in the region of Arg-68 takes place on replacement of GDP by GTP as judged from the increase in vulnerability of Arg-68 for trypsin; amino acyi tRNA increases this apparent local exposure; the response is specific for GTP and not for ATE A comparison of the sequence around the trypsinsensitive Arg-68 in Artemia EF-Ia is made for elongation factors from different sources (table II). During this search, we found that a particular stretch of amino acids has been conserved only for elongation factors. In IF-2, G-protein, transducine or ras p21 proteins no such sequences are present thereby supporting a specific role of 'RGITI'-sequences in polypeptide chain elongation. An interpretation of the trypsin effect is that the protein region encompassing Arg-68 of EF-1 o~ interacts with the ribosome [31]: in the GTP form this region behaves as exposed to the particle and tRNA enlarges this effect; conversely in the GDP form, the Arg-68 region seems to fold back and as a result EF-I~

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The ribosome and translation factors

GDP may dissociate from the ribosome. Recently elongation factor E F - l a from Artemia has been specifically labelled at Lys-63 by the guanine nucleotide analog pyridoxal-5'-diphospho-5'-guanosine in high yield [32]. The vicinity of the binding site of pyridoxal GDP to the GDP-GTP sensitive trypsin cleavage site of Artemia E F - I a is highly intriguing and in line with the suggestion that region 63-74 of EF-I a may comprise a flexible site (see fig 1). Interestingly this region in EFl a corresponds to the region 53-64 in EF-Tu where it is part of a tetradeca peptide missing in the protein crystals used for X-ray analysis of EF-Tu [28]. As a result the local folding in this region of EF-Tu is unknown. Recently the effector region in Thermus thermophilus EF-Tu has also been mapped and the region 52-60 related to the intramolecular signal transduction for the GTPase activation [33]. In still another study interaction between EF-2 and ribosomes has been proposed on the basis of loss of finding to pretranslocation ribosomes of EF-2 cleaved at Arg-66 [34].

L12 as a pendulum facilitating transiocation and expulsion of tRNA It is possible to attach fluorescent p.robes to defined sections of LI2 and L10 and to incorporate these proteins back into the ribosome under recovery of EF-

Table !I. Amino acid sequence around common tryptic cleavage site in EF-I a, EF-Tu, EF-2 and EF-G proteins. Protein

Sequence

Ref

64-75 63-74 64-75 63-74 64-75

KAERERGITIDI KAARERGITIDI KAERERGITIDI KAERERGITIDI KAERERGITIDI

[43] [44] [45] [46] [47]

64-75

KAERERGITIDI

[48]

92-103

PEERARGITIST

[49]

54-65 53-64

PEERARGITINT PEERARGITINT

[50] [51 ]

Hamster EF-2

61-72

KDEQERCITIKS [52]

E coli EF-G

53-64

EQEQERGITITS

Human EF-Ia Artemia EF-Ia Xenopus EF-Ia Drosophilia EF-la Yeast EF-la Mucor racemosus EF-Ia Yeast mitochondrial EF-Tu Euglena gracilis chloroplast EF-Tu E coil EF-Tu

Residue number

[53]

Ar|emia EF 1,k

nucleotide tdnding of semdllw pylldoxal GDP hypsln cleavage

1!

IL vJo

I

||

III

Fig 1. Schematic presentation of the tryptic cleavage site at Arginine-68 in Artemia elongation factor EF-Ia. The position of Lysine-63 where pyridoxai GDP binds [32] is

also indicated. Roman numbers I, II and III refer to the three consensus sequences found in most guanine nucleotide binding proteins [55]. G-dependent, uncoupled GTPase activity ([26] and references therein). Notwithstanding the problem inherent to fluorescence measurements, it is probably safe to say that in the ribosome the two dimers lie not parallel to each other but diverge from LI0. Furthermore, that 4 N-termini of LI2 are situated at the comer and two C-termini of L 12 are located at each of the two ends of the letter L [35]. Localization of Ll2 by immuno-electron microscopy strongly indicates that the domains of L12 can occupy different positions in the ribosome [27]. There are carboxyl-terminal L12 epitopes at the top of the stalk but also at positions more towards the body of the ribosome [27]. The precise orientation of the (L12)4L10 complex relative to the body of the 50S ribosome has remained undecided due to the lack of electron microscopic resolution in the LI2 stalk region and the possibility of positional heterogeneity of the (LI2)4LI0 complex in the ribosome. When we compared by fluorescence the distances between fixed locations in the two dimers of L12 of ribosomes in pre- and post-translocation state, we could never observe any changes in distances between the two states ([26] and unpublished work of Thielen). Neither EF-G, EF-Tu nor occupation of A/P site with tRNAs in the presence of GTP or GDPNP induced any conformational change within the (L 12)4L10 complex. The model we favour would therefore be in agreement with a tetrameric LI2 structure in which the two dimers have fixed a position with respect to each other during the elongation cycle. Any changes corresponding to more than 5 A we should have detected. A functional movement of tetrameric L12 may occvr on factor interaction, as judged from the published data on the varying sensitivity of L12 for trypsin during elongation [36, 37]. Upon interaction of EF-G with ribosomes in the pre-GTP hydrolysis state, the L12 molecules are sensitive to digestion, whereas ribosomes in the post-GTP hydrolysis state become trypsin resistant [36]. The opposite effect with respect to the action of trypsin has been found with EFTu [37]. An attractive interpretation of the data [7, 26, 36, 37] would be that on GTP hydrolysis by EF-G, the stalk moves inwards and returns to its old position once

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GTP hydrolysis under influence of EF-Tu has taken place. However, direct prove that outside the stalk region L12-proteins are trypsin resistant has not been given. Proton NMR of ribosomes [38, 39] shows a large mobility of L I2, especially in the C-terminal domain. The conformational changes L12-proteins go through during the elongation cycle are believed to be related to an hinge flexibility which gives the C-terminal domain special freedom to rotate [38]. The NMR and trypsin proteolysis experiments could imply therefore that parts of the (LI2)4LI0 complex change their orientation with respect to the body of the ribosome, for instance the A-site and P-site.

Currently we prefer to emphasize L12 rotation and wish to advance an Ll2-pendulum model which accounts for the presence of certain symmetry elements in tRNAs [ 1] including the remnants of a second code at position 3-5 in transfer RNAs for primordial amino acids [54]. When looking at the acceptor and anticodon stem structure of tRNAs, we took symmetry properties of tRNAs into serious consideration. As a result it becomes understandable why there is such a tetramerie Ll2-structure in the ribosome: the particle harbours two tRNAs, each having a pseudo-symmetry which may be related to the symmetry in the L l2-tetramer.

(a)

(b)

(c)

(d)

_.(2) EF-Tu

EF-Tu

Fig 2. Diagram of a functional cycle of the elongation. In this tripartite representation nascent protein runs downwards. Adapted from MOiler [ 1].

The ribosome and translation factors

Figure 2 shows such a scheme of a functional cycle of elongation, in which L I2 plays a title role in the process. The quintessence is that L12 proteins are considered to drive coupled rotations of the two tRNAs when triggered by elongation factors. Thus the signal transduced by the elongation factors is considered to be propagated to tRNAs via L I2 proteins [1]. The force could derive from the electrostatic repulsion between tRNA and L12, both being negatively charged; simultaneously with the rotation of tRNA, mRNA could be dragged along in this process. Thus on EF-G dependent GTP hydrolysis, movement of tetrameric L12 would result in concurrent rotation of peptidyl tRNA and deacylated tRNA to the P-site and E-site respectively. Conversely after EF-Tu-dependent GTP hydrolysis, the tetrameric L12 structure would return to its old position under binding of a new aa-tRNA and expulsion of the deacylated tRNA from the E site. An attractive feature of this L12 pendulum model is that it gives a direct explanation for the bidirectional linkage between E- and A-sites as proposed for the allosteric three-site (A, P and E) model in the ribosomal elongation cycle [40]. Furthermore, according to the scheme of figure 2 50S ribosomes may be in a preor post-translocation state depending on whether the stalk is visible or not. Since in our EM study of 50S ribosomes, precisely half of the number of particles contained a visible stalk [25], according to the model of figure 2, half of the 50S ribosomes would then be in the pretranslocation and the rest in the posttranslocation state. A recent structural analysis of the position of the Esite, has mapped it on 23S ribosomal RNA between L1 and L27 which is on the opposite site of the stalk [41 ]. According to topographical data the distance between the E-site and the stalk would be at least 100 A. Considering, however, the dimensions of tRNA and the (L12)4L10 complex, this would not necessarily invalidate an A-site-E-site-L12 site-coupling as suggested, albeit that the distance between E-site and stalk site seems too large for a non-allosteric coupling. Much depends on how P-tRNA rotates on its exit route and where on this trajectory the E-site precisely lies. In any case, the simplicity and inherent properties capable of explaining many features of ribosome function and structure, make a rotation model for tRNAs as presented here a useful extention of the conventional model based on linear displacement of tRNAs [42]. In addition, our suggestion that protein L12 mimicks the shape of transfer RNA, may be a nice example of a protein resembling a nucleic acid. Acknowledgments I thank Dr J Dijk for his constructive comments and thorough criticism. This work was supported in part by grants from the Netherlands Foundation of Chemical Research (SON/NWO).

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44 45 46 47 48 49 50 51

52 .53 54 55

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