boc 0950179

Review

The puzzling lateral flexible stalk of the ribosome

Philippe Gonzalo *, Jean-Paul Reboud

Institut de Biologie et Chimie des Protéines – UMR 5086 Centre National de la Recherche Scientifique,Translational Mechanisms and Regulation in Mammals, 7, passage du Vercors, 69367 Lyon cedex 07, France

Received 13 March 2003; accepted 16 March 2003

Abstract

The lateral flexible stalk of the large ribosomal subunit is made of several interacting proteins anchored to a conserved region of the 28S(26S) rRNA termed the GTPase-associated domain or thiostrepton loop. This structure is demonstrated to adopt puzzling changes ofconformation following the different steps of the elongation cycle. Some of these proteins termed the P-proteins in eukaryotes and L10 andL7/L12 in bacteria, present little structural similarities between Eubacteria on one side and Archae and Eukaryotes on the other side. However,up to now, these proteins seem to present a similar macromolecular organisation and they have been involved in the same functions.Convincing evidence attests that these proteins participate in elongation factor binding to the ribosome, and it has been suggested that theseproteins might be evolved in a GTP hydrolysis activating protein activity. Involvement of these proteins in the translational mechanism isdiscussed. Moreover, in eukaryotes, small P-proteins are also found as isolated proteins in a cytoplasmic pool that exchanges with theribosome-associated P-proteins. Moreover, a part of the ribosomal proteins is phosphorylated (hence their P-protein names). The biologicalsignification of these particularities is discussed.

© 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.

Keywords:Ribosome; Translation; Acidic proteins; Lateral flexible stalk; GTP hydrolysis

1. Introduction

Most studies of the translation apparatus have been carriedout in prokaryotic cells. This is the case for the ribosome aswell as for the translation factors. The reasons are that theeukaryotic system appeared more complicated to study(availability of the components, ability to obtain mutants,etc.) and that the translation was thought to operate in asimilar way in all the cells. Indeed, it appears that the generalmechanisms of translation exhibit a great similarity in allorganisms. However, significant differences in componentsof the translation apparatus have been observed. Eukaryoticribosomes are bigger with a more complex structure. Theycontain an internal core similar to that in prokaryotic ribo-somes with external extension thought to support specificfunctions (Dube et al., 1998a, 1998b; Verschoor et al., 1998).Concerning the region in which we are interested, it is veryinteresting that it has shifted its 3D position during evolution,as some other peripheral regions that have been demon-

strated to play a main function in the ribosome. Two mainRNA structures are concerned by this shift at the base of thelateral flexible stalk, the L11 binding domain at the thiostrep-ton loop and the sarcin ricin loop (SRL) involved in bindingelongation factors. The reason for this displacement may beto conserve the distance to the central core that is made of thetRNA binding sites and of the peptidyltransferase centre. Bythis mean, the ribosome would accommodate an increasingsize during the course of the evolution (Mears et al., 2002;Spahn et al., 2001). Eukaryotic translational factors are alsogenerally bigger, although they also seem to have the samegeneral structure with additional domains (and functions?).The newly released 3D structure of yeast EF-2 in the ProteinData Bank (pdb code 1N0U and 1N0V) is expected to illus-trate this paradigm (Joergensen et al., 2003). Factors are alsogenerally more numerous, mostly at the initiation step whichrequires only three factors in bacteria vs. more than30 polypeptide chains in eukaryotes.

This increased complexity of the eukaryotic system can beattributed to specific requirements of the eukaryotic cells,one of them being supplementary mechanisms for transla-tional regulation needed for a differential expression in dis-tinct cell types and particular physiological conditions.

* Corresponding author. Tel.: +33-4-72-72-26-45;fax: +33-4-72-72-26-25.

E-mail address:[email protected] (P. Gonzalo).

Biology of the Cell 95 (2003) 179–193

www.elsevier.com/locate/bicell

© 2003 Éditions scientifiques et médicales Elsevier SAS. All rights reserved.DOI: 10.1016/S0248-4900(03)00034-0

Among things to consider is the fact that coupled transcrip-tion and translation is not observed in eukaryotic cells. Arequirement for mRNA processing results in a space–timeseparation of these steps of the genetic expression, whichresults in the impossibility to synthesise proteins in an emer-gency from newly transcribed mRNA. Accordingly, contraryto what is observed in bacteria, some mRNAs are stored ashnRNPs without being actively translated, which supposes apossibility to regulate the genetic expression at the transla-tional step. This difference between bacteria and eukaryotesis shown by mRNA half-lifes in bacteria (less than 3 min)compared to higher eukaryotes (hours or more). Anothertheoretical reason supporting the requirement for an efficientregulation at the translational step in eukaryotes is that,otherwise, non-mature mRNAs might be translated duringthe cell cycle steps when nuclear membranes are disrupted.Transcription is then inhibited and translation is nearlystopped. Residual translation is made possible only throughspecific pathways for proteins specifically required at thesesteps (Pyronnet et al., 2000).

These general considerations are well illustrated by thelateral flexible stalk of the large ribosomal subunit. This verypeculiar region is found among all the ribosomes and plays abasic role in translation. This role, though it is not yet entirelyclarified, seems similar in eukaryotes and prokaryotes. How-ever, very important differences are observed between thecomponents of this stalk in prokaryotic and eukaryotic ribo-somes and even among the different eukaryotic ribosomes, asexemplified by yeast, plant or rat ribosomes. Furthermore, acommon feature distinguishes eukaryotic ribosomes: theprotein components of the stalk are phosphorylated, whereasno such phosphorylation is observed in bacteria. This obser-vation supports the notion that specific mechanisms of regu-lation mediated by these proteins have appeared in the courseof evolution.

2. Structure of the stalk

2.1. Components

The eukaryotic lateral flexible stalk of the ribosome ismade of four protein components (two P1 and two P2) linkedto another protein (P0) connected itself to a sixth protein(RL12) and to the 28S rRNA at the “thiostrepton loop orGTPase centre” that constitutes the base of this ribosomalprotuberance (Fig. 1; Egebjerg et al., 1990). It is the onlystructure made of multicopy elements and involving protein–protein interaction without apparent rRNA contact (Ban etal., 2000). This is appealing since rRNA shape is nearlyfilling the tridimensional structure of every other part of thewhole ribosome. Thus, the lateral flexible stalk of the largesubunit constitutes a distinct domain only made of proteins.This supports the idea that it is the product of the evolutionfrom an archaic ribosome, only made of rRNA at the time ofthe RNA world times (Spirin, 2002a).

The ribosome contains another symmetric lateral protu-berance called the L1 protuberance that has an importantflexibility as demonstrated recently. This protuberance ismade of only one large protein (L1) anchored to the 28SrRNA and involved in the release of the deacylated tRNAfrom the exit site (E-site) of the ribosome (Nikulin et al.,2003).

2.1.1. The stalk proteins among the biological kingdomsEukaryotic ribosomal proteins are not termed by the same

name as their bacterial analogues. To prevent confusion,

Fig. 1. Classical representation of the components of the lateral flexiblestalk of the eukaryotic ribosome. In this drawing, association of the compo-nents of the stalk is displayed as deduced from several biochemical experi-ments. P0 interacts with the 28S rRNA at the thiostrepton loop or GTPase-associated domain. This protein anchored two dimers P1/P2 to the ribosome.The C-terminal domain of the P-proteins is exposed and the hinge region isextended. No difference is made between the two dimers P1/P2. P2 exhibitsno direct contact with P0 and is linked by an interaction between itsN-terminal domain and that of P1. RL12 is located at the base of the stalkand interacts with the thiostrepton loop. Both intermediary domains of P0and the C-terminal domains of the small P-proteins have been shown tointeract with the elongation factors, but that other parts of the stalk compo-nents contact them cannot be excluded. This entire region is flexible and theprotein components are regarded as able to induce modifications of confor-mation of the thiostrepton loop and reciprocally, signals mediated by therRNA are supposed to result in modifications in the conformation of theproteins.

180 P. Gonzalo, J.-P. Reboud / Biology of the Cell 95 (2003) 179–193

eukaryotic proteins will be preceded by an “R”, except forthe P-proteins. Archae proteins are preceded by “a”.

The eubacterial equivalent of P1 and P2 is a unique proteintermed L7/L12 and sequence analysis fails to identify anyhomology between these related proteins (Wool et al., 1991).There are four copies of L7/L12 in active bacterial ribosome(Koteliansky et al., 1978; Moller et al., 1983; Subramanian,1975). The only difference between L7 and L12 is that L7 isthe N-acetylated form of L12 (Terhorst et al., 1973; Terhorstet al., 1972). The ratio between L7 and L12 seems to dependon cultural conditions (Deusser, 1972), and N-acetylation isgenerally considered as without any functional consequences(Tokimatsu et al., 1981). However, it cannot be excluded thatit might have a specific role in some bacterial species. Con-trary to L7/L12, eukaryotic P1 and P2 are very differentproteins based on their sequences (Shimmin et al., 1989).Moreover, the number of these proteins is not the sameamong eukaryotes. There are four proteins termed P1A, P1Band P2A, P2B in yeast (Shimmin et al., 1989) instead of twoin mammals (Wool et al., 1991). P1A interacts with P2B,whereas P1B interacts with P2A (Ballesta et al., 2000).However, no precise function for yeast P1 and P2 variants inribosome is known, although the stalk content in P-proteinvariants depends on yeast cultural conditions (Saenz-Robleset al., 1990). Three small P-proteins (P1, P2 and P3) can befound in plants (Szick et al., 1998). No specific function forP3 has been identified yet. In archae, the protein equivalent toP1/P2 is termed aL12, although it is closer to P1/P2 than to itseubacterial equivalent (Ramirez et al., 1994).

The eubacterial equivalent of P0 is L10 and that of RL12is L11. L10 is a much shorter protein than P0 and it does notcontain a domain common to L7/L12, contrary to P0 thatshares its C-terminal domain with P1 and P2. In archaebac-teria (a), aL10 is larger than in eubacteria and it has theC-terminal domain that is found in aL12, which illustratesthe paradigm, that archaebacteria ribosomal proteins andtranslational factors are much closer to their eukaryotic coun-terparts than to their eubacterial ones (Ramirez et al., 1994).P0 and L10 seem to play the same structural function inbinding P1/P2 (L7/L12, respectively) (Gudkov et al., 1978b),but the additional domain of P0 seems to support additionalfunctions (Remacha et al., 1995).

L11 and RL12 bind to a very conserved rRNA motif andthey share a strong homology both in sequence and in 3Dstructure.

2.1.2. Modular structure of the P-proteins and bacterialequivalents

2.1.2.1. Structure of the eukaryotic P-proteins. Sequenceanalysis of the different P-proteins reveals the presence of acommon C-terminal domain made of about 20 mainlycharged residues. This domain that contains two serine resi-dues that have been shown to be phosphorylated in cellulo, is

preceded by a long sequence of uncharged residues (Ala andPro essentially) known as the “hinge”. The hinge is found inthe three P-proteins P0, P1 and P2.

The N-terminal domains of about 65 residues of theseproteins are quite different. The N-terminal domain of P1(termed N1 in this review) is mainly hydrophobic, whereasthe N-terminal domain of P2 (N2) is very hydrophilic(Gonzalo et al., 2001). Several regions of the much largerN-terminal domain of yeast P0 have been identified usingseveral approaches. The N-terminal sequence is involved inthe binding to rRNA; another sequence interacts with elon-gation factor EF-2 while the C-terminal part of the domaininteracts with the small P-proteins.

2.1.2.2. Prokaryotic equivalents of the P-proteins. Interest-ingly, the unique protein L7/L12, the prokaryotic equivalentto P1 and P2, is of similar size but has quite a differentprimary structure. L10, equivalent to P0, is much smallerthan P0 (18 kDa vs. 34 kDa). Three domains were predictedin L7/L12; a short alpha helical N-terminal domain, an inter-mediary domain (the “hinge”) and a C-terminal globulardomain (Gudkov et al., 1978a; Leijonmarck and Liljas,1987). In another comparative analysis of L7/L12, P1 and P2,Ramirez et al. (1989) predicted also a very short N-terminaldomain and a long C-terminal one in the prokaryotic protein.This is exactly the contrary of the eukaryotic situation andthis suggests that a simple transposition of functional do-mains from the C-terminal to the N-terminal region may haveoccurred between the prokaryotes and the eukaryotes. Adimerisation domain was also predicted in the N-terminaldomain for eukaryotic P-protein and in the C-terminal do-main for the prokaryotic L7/L12 (Ramirez et al., 1989).However, additional data have suggested that the N-terminaldomain of L7/L12 also plays an important role in dimerisa-tion and in its attachment to the ribosome (Koteliansky et al.,1978). NMR studies of L7/L12 in solution predicted thatdimers of the ribosomal protein L7/L12 from Escherichiacoli had a parallel (head-to-head) orientation and that theN-terminal domain had no contacts with the C-terminal do-main. The hinge region was predicted as an unordered struc-ture (Bocharov et al., 1996). However, the 3D high resolutionstructure of L7/L12 as a dimer linked to two proteolysedfragments confirmed the existence of the three predicteddomains but revealed a totally different organisation (Fig. 2;Wahl et al., 2000). Interacting parts of L7/L12 are not re-stricted to one domain. The hinge region is very structuredcontrary to what was expected. Moreover, the hinge is a maininteracting domain of the dimer but additional contacts aremade by contacts between the C-terminal domains, theN-terminal domains and the opposite N-terminal andC-terminal domains. L7/L12 is therefore, a very compactstructure in which all the domains are intricate. This structureis therefore, very different from the classical model in whichproteins are associated by their homologous regions to form adimer and in which N-terminal and C-terminal domains areseparated by a stretched and flexible region to form an ex-

181P. Gonzalo, J.-P. Reboud / Biology of the Cell 95 (2003) 179–193

tended stalk with the C-terminal domain as the external tipand N-terminal one as the anchoring point (Marquis et al.,1981). Which one of these structures is found in the ribosomeis still unclear. However, discrepancies between differentobservations of L7/L12 (mostly described in terms of a rigidstructure despite the repeated observation of high flexibility)had been pointed out previously (Liljas and Gudkov, 1987).The discrepancy between these two representations of thestalk proteins is shown by comparing P1/P2 representationand its suspected mode of dimerisation (Fig. 1) and L7/L12high-resolution structure (Fig. 2).

This 3D structure of L7/L12 also reveals that a large helixof about 20 residues decorates the two C-terminal domains ofL7/L12 and exhibits charged residues on its external face.This helix might be the functional equivalent of the 20charged residue C-terminal domain of the P-proteins that canbe phosphorylated. Then, it might be involved in the interac-tion with elongation factors belonging to the GTP bindingand hydrolysing protein superfamily.

2.1.3. Intrinsic flexibility of these proteins

The intrinsic flexibility of these proteins has been estab-lished for many years by several groups using different ap-proaches. RMN of the proton have been used to locate theresidues involved in the flexibility of L7/L12, and the resultsmatched the previously recognised hinge region (Bushuev etal., 1989). RMN experiments on full length P2 made by ushave also revealed a high flexibility of the molecule thatimpeded its 3D determination using this technique (unpub-lished results). Cross-linking of the extremity of these pro-teins to several and distant proteins in the ribosome alsosuggested an important mobility of this structure (Dey et al.,1998).

2.2. RL12 and the GTPase centre rRNA structure

2.2.1. Conservation of these elements amongthe biological kingdoms

The equivalent of RL12 in prokaryotes is L11. Theseproteins share an important homology among the differentkingdoms. These proteins bind a conserved rRNA sequenceregion termed the GTPase-associated rRNA domain or thios-trepton loop (Schmidt et al., 1981). This name is due to thefact that antibiotic thiostrepton inhibits GTP hydrolysis byEF-G and interacts with this rRNA region. (Pestka, 1970).

The rRNA involved in the binding of L11 or RL12 ishighly homologous among the different biological king-doms. Among the nucleotides forming this region (1050–1108 in E. coli sequence), 24 are conserved in more than95% of eubacteria, archaebacteria, eukaryote, and chloro-plast sequences and among them, 12 nucleotides are univer-sally conserved (>95%) after inclusion of mitochondrial se-quences.

These conserved nucleotides are involved in long-rangebase pairs or base triple interaction and allow the constitutionof the universal secondary and 3D structure of the GTPase-associated RNA domain. The 3D structure of L11 in complexwith the thiostrepton loop has been deposited in the proteindata bank under the pdb code 1mms (Wimberly et al., 1999).This precisely folded RNA structure is indeed stabilised bythe extensive tertiary contacts and contains an unusuallylarge core of stacked bases. Several positions are different insome species, but appear in the 3D structure to compensateeach other in allowing equivalent-based pairing (Uchiumi etal., 2000). This identical 3D structure authorises the bindingof rat proteins to E. coli ribosome, which yields hybridribosomes (Uchiumi et al., 1999, 2002b).

L11 is made of two globular domains connected by a shortbut quite rigid linker. Each domain of L11 covers differentfaces of the thiostrepton loop. The C-terminal domain wouldbe bound with a higher affinity to the rRNA than theN-terminal domain that has a higher mobility. This wouldexplain that it had to be deleted to solve one of the 3Dstructures of this complex (pdb code 1qa6; Conn et al., 1999).

2.2.2. 3D structure modulation by L11 and agonistsThiostrepton loop is supposed to adopt two alternative

conformations and therefore to be one of the moving ortrigger regions of the ribosome (Uchiumi and Kominami,1997 ; Wilson and Noller, 1998b). Thiostrepton and ana-logues act by increasing the folding and the stability of thisloop and probably prevent it from adopting the conformationinvolved in the GTP hydrolysis promoting activity (GAPactivity; Draper and Xing, 1995; Draper et al., 1995; Laingand Draper, 1994; Laing et al., 1994; Xing and Draper,1995). Thiostrepton acts cooperatively with L11 in increas-ing the stability of this rRNA loop and it would mimic anormal component of the translational apparatus interactingwith L11 N-terminal domain at the base of the lateral stalk ofthe ribosome (Xing and Draper, 1996). However, action of

Fig. 2. L7/L12 dimer of Thermotoga maritima from the Wahl et al. (2000)crystal structure (pdb code 1DD4). Proteolysed fragments are not shown.The first molecule (A) is red and the second one is blue. The N-terminaldomains (N), the hinge regions (H) and the C-terminal domains (C) aredepicted in different tones. a6 is the amphipathic helix that might be theequivalent of the short eukaryotic charged C-terminal domain that is sup-posed to interact with elongation factors.


the L11-rRNA complex as a GAP is still discussed and it wassuggested that thiostrepton would rather act by preventingthe dissociation of EF-G after GTP hydrolysis (Wintermeyer,non-published). It has to be pointed out that thiostrepton isnot the only antibiotic acting at this region: micrococcin wasshown to stimulate GTP hydrolysis of EF-G by binding theL11-rRNA complex (Cundliffe and Thompson, 1981).

2.3. Correlation between biochemical data and structuralresults

2.3.1. X-ray crystallographyNo crystals have been obtained for eukaryotic ribosomes

up to now and no high-resolution structure of eukaryoticribosome is available yet. Crystals diffracting at high resolu-tion of bacterial ribosomal subunits and whole ribosomehave been obtained for a long time, but solution of thediffraction data has taken years. Several high-resolutionstructures of the large subunit of the bacterial ribosome isnow available, either alone (Ban et al., 2000) or in complexwith the small subunit, tRNAs at the A-, P-, and E-sites and afragment of mRNA (Yusupov et al., 2001). None of thesestructures allows the placement of both the components ofthe stalk. The more resolved structure for this part of theribosome is that of Yusupov et al. (2001) comprising thethree tRNAs and the messenger RNA (Fig. 3; pdb code 1giy).The corresponding pdb file includes a dimer of L7/L12 forwhich the dimerisation mode is similar to that described byWahl et al. (2000). No electronic density was obtained for the

second dimer of L7/L12 and a poorly defined density that isattributed to L10 is mentioned but not represented. Twohelices of L10 were recently defined in the 50S subunit ofHaloarcula marismortui. These helices interact with a newsecondary structure motive called the K-turn. These helicescorrespond to L10 residues located at the N-terminal part ofL10 (pdb code 1JJ2; Klein et al., 2001). In this structure,electronic density for L11 and the GTPase-associated do-main of the 26S rRNA is sufficient to allow their clearplacement in the large subunit, which corresponds to whatwas predicted from the high-resolution X-ray structure ofisolated L11 in complex with this rRNA domain (pdb code1mms; Wimberly et al., 1999). In conclusion, at the momentno model describes the full bacterial stalk: at least one dimerof L7/L12 and the major part of L10 are lacking. However,available data suggest a direct interaction of the two mol-ecules of the dimer L7/L12 with both the 26S rRNA at theGTPase-associated centre and with L11 (Yusupov et al.,2001). This alternative to associate these proteins has neverbeen suggested, but it would explain the large flexibility ofthis structure. It is noteworthy that a direct interaction be-tween RL12 and a dimer of P1/P2 can also be postulated ineukaryotes from data indicating that the association of P1Aand P2B to the ribosome is drastically reduced in ribosomelacking L12 (Briones et al., 1998).

2.3.2. Cryoelectronmicroscopy

Location of L7/L12 in the stalk region was first estab-lished by immune microscopy (Strycharz et al., 1978). Elec-

Fig. 3. The stalk components in the crystal structure of the 50S ribosome subunit at 5.5 Å resolution (Yusupov et al., 2001; pdb code 1GIY). The original pdbfile has been deleted from all the atomic coordinates but those corresponding to the components of the stalk. Proteins are depicted by their Ca backbone andrRNA by the phosphor atom chain. L11 and L7/L12 are the bacterial equivalents of RL12 and P1/P2, respectively. No clear electronic density is obtained neitherfor L10 (P0 in eukaryotes) nor for the second dimer of L7/L12. This structure displays a previously unrecognised direct interaction of L7/L12 with thethiostrepton loop and supports the hypothesis of an alternative mode of association of these proteins to the ribosome.


tronmicroscopy also indicated that each dimer bound to theribosome at different sites and that only one dimer wasresponsible for the elongated visible stalk (Moller et al.,1983). More recently, outstanding images have been ob-tained of the prokaryotic ribosome (and also, at a lesserextent, of the eukaryotic ribosomes (Dube et al., 1998b;Gomez-Lorenzo et al., 2000; Rawat et al., 2003; Spahn et al.,2001)) in several steps of the translation process: free sub-units, 70S ribosomes complexed with three tRNAs (Agrawalet al., 1996), 70S ribosomes complexed with EF-Tu–aminoa-cyl tRNA in a GTP-like conformation (Stark et al., 1997),70S ribosomes complexed with EF-G in either a GTP-like ora GDP conformation (Agrawal et al., 1998, 1999; Frank andAgrawal, 2000; Stark et al., 2000), 70S ribosomes duringinitiation (Gabashvili et al., 2000; Malhotra et al., 1998;McCutcheon et al., 1999) or termination (Klaholz et al.,2003; Rawat et al., 2003). These images gave a dynamicimage of the translation, confirmed the high mobility of thelateral L7/L12 stalk and gave birth to many new ideas. How-ever, attribution of specific proteins or rRNA domains todensities obtained by the combination of cryo-electron mi-croscopy and single particle reconstruction turned out to besomewhat complicated. (Fig. 4).

The precise location of the two dimers of L7/L12 withinthe structure of the 70S ribosome by cryo-electron micros-copy and single particle reconstruction was made possibleonly after using two reconstitution approaches. Differencemapping between ribosomal core particles depleted inL7/L12 and L7/L12-reconstituted ribosomes was required tolocate the L7/L12 shoulder next to protein L11. Difference

mapping between ribosomal core particles depleted inL7/L12 and ribosomes reconstituted with L7/L12 having ananogold modified-C-terminal domain helped the placementof the C-terminal domains of L7/L12 at four distinct posi-tions (Montesano-Roditis et al., 2001).

By comparing a 3D cryo-EM reconstruction of the 70Sribosome isolated from a mutant lacking ribosomal proteinL11, with the 3D map of the wild-type ribosome and byfitting the X-ray coordinates of L11-23S RNA complex, theL11 protein was located at the base of the L7/L12 stalk of the50S subunit of the E. coli ribosome. Additional fitting ofEF-G into the cryo-EM maps revealed that the N-terminaldomain of L11 was likely to be responsible for the arc-likeconnection with the G' domain of EF-G that was previouslybelieved to be the L7/12 C-terminal domain (Agrawal et al.,1998, 2001).

Concerning the involvement of L7/L12 in the binding ofEF-Tu in a kirromycine stabilised EF-Tu-aminoacyl-tRNA-GTP-70S complex, a connection was observed between thebase of the stalk and EF-Tu and this was attributed to L7/L12(Stark et al., 1997). This connection was not observed in anew reconstruction of this complex (Valle et al., 2002).

These examples show that CEM and single particle recon-struction have to be interpreted cautiously taking into ac-count that material has to be very homogenous in structureand conformation, which is not evident even after the use ofantibiotic as blocking agents, that mobile parts are poorlydefined and that attribution of a component to a volumerequires interpretation and hypotheses concerning conforma-tion changes.

Fig. 4. Visualisation of the bacterial lateral flexible stalk by cryoelectronmicroscopy and single particle reconstruction at several main steps of the elongationcycle. These pictures show the elongation factors either EF-Tu (panel A) or EF-G (panels B and C) bound to the bacterial ribosome. Elongation factors arelocated at the vicinity of the lateral flexible stalk that is shown at different positions (termed L7/L12 inA, bifurcated St in B and Stalk in C).Attribution of specificproteins to density results from an interpretation of the data. In A, the ternary complex (EF-Tu-GTP-Phe-tRNA) is kirromycin-locked at the A-site (reproducedwith permission of Nature and the authors (Stark et al., 1997)). In B, EF-G is bound to the ribosome in complex with a non-hydrolysable GTP analogue and inC, EF-G is bound to the ribosome in the presence of fusidic acid (GDP-like conformation?) (reproduced with permission of Nature Structural Biology and theauthors (Agrawal et al., 1999).


2.4. Association of the compounds: structure relationshipin the stalk

We saw that no structural data gave precise informationconcerning the way by which eukaryotic P1, P2, and P0 orprokaryotic equivalents associated to RL12 and 28S rRNA.We also described how the crystallographic structure of iso-lated L7/L12 challenged the classical model for the associa-tion of the dimers of L7/L12.

2.4.1. Binding of P0 and equivalent to the rRNATwo helices at the N-terminal domain of L10 (residues

12–29 and 63–73) have recently been shown in the refinedstructure of the 50S subunit of H. marismortui (pdb code1JJ2; Klein et al., 2001). These helices constitute a L10binding site to the 26S rRNA through a novel class of RNAsecondary structure motif termed the kinky turn or K-turn.The K-turn is found around the A1150 protruding nucleotide(in E. coli nomenclature), a conserved and predicted interact-ing motive for L10 (Egebjerg et al., 1990). It offers animportant surface of interaction with L10 and buries a sig-nificant hydrophobic surface of the protein. In yeast, residues185–230 have been shown to be required for the interactionof P0 with the rRNA (Santos and Ballesta, 1995).

2.4.2. Elements involved in the dimerisation of P1/P2(and equivalents)

Several lines of evidence indicate that the N-terminaldomains of P1/P2, and bacterial equivalents are involved indimerisation (Gonzalo et al., 2001; Gudkov and Behlke,1978; Kopke et al., 1992; Tchorzewski et al., 2000; Zurdo etal., 2000) and in interaction with P0 or L10 (Gonzalo et al.,2001; Kopke et al., 1992; Pettersson et al., 1976; Zurdo et al.,2000).

2.4.3. Binding of P1/P2 dimers (and equivalents) to P0(and equivalent)

Most of these laboratories agree that dimers bind to differ-ent sites of P0 or its bacterial equivalents and play a differentfunction even if evidences exist for a close location of the twodimers in the stalk (Shimizu et al., 2002). There is also aconsensus for an anchorage of the P1/P2 dimer by a directinteraction of the P1 N-terminal domain, but not the P2N-terminal domain with P0 (Gonzalo et al., 2001; Shimizu etal., 2002; Zurdo et al., 2000).

Domains of P0 and L10 involved in P1 and L7/L12 bind-ing have been delineated by deletion experiments. In yeast,P0 is made of 312 residues. The region of P0 around posi-tions 230–290 is involved in the binding of proteins P1/P2(Santos and Ballesta, 1995). The last 20 C-terminal aminoac-ids constitute the common charged domain also found in P1and P2 and the preceding 40 aminoacids (260–300) form thealanine-rich region with hydrophobic properties that hasbeen called the hinge. Therefore, the P0 hinge hydrophobicregion plays a major part in P1 binding, which is not surpris-ing since P1 N-terminal domain is also very hydrophobic

(Gonzalo et al., 2001). In E. coli, L10 contains 164 aminoacids. Deletion experiments at the C-terminus of L10 haveshown that the last 10 residues were responsible for thebinding of one of the two L7/L12 dimer, whereas the 10preceding ones were involved in binding the second L7/L12dimer (Griaznova and Traut, 2000). Interestingly, in thisexperiment, the mutant ribosome depleted of one L7/L12dimer supported elongation factor G dependent GTP hy-drolysis and in vitro protein synthesis with the same activityas that of two-dimer particles. In yeast, a similar observationhas been made indicating that the dimers were not equivalent,that they bind to two different sites in P0 and that they mightplay a different function (Briones et al., 1998; Remacha et al.,1992). This is not the only report of an apparently uselessredundancy of the proteins belonging to the P1/P2 family:this has also been shown by yeast supporting slow growth inthe absence of P1 and P2 (Santos and Ballesta, 1995) and inrat ribosomes reconstituted with truncated forms of P1(Gonzalo et al., 2001). However, there is also convincingevidence for an increased efficiency of ribosomes containingthe four functional components (Moller et al., 1983).

2.4.4. Functions of the C-terminal domain

Several approaches indicate that the C-terminal domain ofP1/P2 and their eu- and archae-bacterial equivalent are in-volved in the interaction with elongation factors (Kopke etal., 1992; Koteliansky et al., 1978). In rats, phosphorylationof a seryl residue in the C-terminal charged and commondomain strongly modulates both in vitro protein synthesisand EF-2 dependent GTPase activity (Bargis-Surgey et al.,1999; Vard et al., 1997 ). In the latter work, direct interactionsbetween EF-2 and P1 or P2 have been shown by limitedproteolysis and surface plasmonic resonance. These interac-tions are not observed using the N-terminal domain of P2,which suggests that the interacting part of P2 is in theC-terminal or/and in the hinge-domains (unpublished result).

2.4.5. Functions of the hinge

The hinge domain is functionally essential for the P-pro-teins and their equivalents (Gudkov et al., 1991). Mutations ordeletions in these domains result in decreased rates of growthand translational elongation in vitro and in increased nonsensecodon read-through in vivo and missense error rates in vitro(Kirsebom et al., 1986). Length of the stalk also seems impor-tant for L7/L12 functions. What matters more than the exactsequences is the repetition of a small group of aminoacids (A,V, P, G in eubacteria) and (G,A, P in eukaryotes) (Bubunenkoet al., 1992). The hinge is generally regarded as an extendedand flexible region, which contrasts with the rigid and verystructured helix observed in the L7/L12 crystallographicstructure (Wahl et al., 2000). Such a discrepancy can arise forseveral reasons. The observed L7/L12 dimers (Wahl et al.,2000) could be artefacts of crystallisation. This is very un-likely, especially since this fold corresponds to that of theL7/L12 dimer found in the 70S ribosome (Yusupov et al.,2001). Another possibility is that the observed fold corre-


sponds to a relaxed state of L7/L12, and that another confor-mation exists in which the hinge is extended after a helix-coiltransition (hypothesis developed in (Wahl and Moller, 2002)).This would explain the different aspects of this region underthe different steps of the elongation cycle (Agrawal et al.,1998; Frank and Agrawal, 2000; Valle et al., 2002).

3. Functions of the stalk components in translation

When concluding the last Cold Spring Harbor Meeting on“The Ribosome” (May 31–June 5, 2001), Peter Moore statedthat two main unsolved questions about the ribosome werethe stalk region structure and function and the translocationmechanism. Since then evidence has accumulated to connectboth the questions, we will develop a somewhat theoreticalapproach to the translation to help define what might be thefunctions of the stalk proteins within the ribosome.

3.1. Some data on ribosome function

3.1.1. Factor free translationThe three main steps of the elongation cycle in the trans-

lation process can be performed without using any externalprotein factors or GTP. The only components needed are theribosome, aminoacylated-tRNAs and an RNA matrix thatwill direct the cognate peptide synthesis (Gavrilova et al.,1976; Gavrilova and Spirin, 1971; Pestka, 1969). This “factorfree translation” demonstrates that the ribosome contains allthe structural elements required to perform the decoding ofthe aminoacylated-tRNA at the A-site, the transpeptidationreaction at the peptidyltransferase centre, and the co-translocation (i) of the mRNA to bring a new codon to theA-site, (ii) of the peptidyl-tRNA from the A-site to the P-site,(iii) of the deacylated-tRNA from the P-site to the E-site.Concerning the decoding phase, rRNAs have proved to play amajor part in this process (Yusupov et al., 2001). Peptidebond formation is also the result of the rRNA catalyticproperties, establishing that the ribosome is a ribozyme (Nis-sen et al., 2000). Translocation is complex, which seemslogical taking into account the number of external elementsthat are set in movement. It is also a risky operation since itcan be the occasion of a frameshift of the mRNA. Frameshiftis normally prevented by the fact that the anticodon–codonbonds should not be disrupted during translocation. How-ever, it should be pointed out that anticodon–codon recogni-tion is weak and that it represents little of the total bindingenergy that has to be redistributed during the translocation. Apart of the catalysis of this step probably originates from theE-site that possesses a high affinity for deacylated-tRNA,which of course facilitates the translocation in driving themovement of all the tRNA in a unidirectional progression(Rheinberger and Nierhaus, 1986). Movement of the mRNAseems at least partly due to the rotation of the small subunittowards the large one during the translocation step (Frankand Agrawal, 2000). Reorganisation of the contact betweenthe two subunits also seems to involve mainly rRNAs(Yusupov et al., 2001).

It is therefore likely that rRNAs in the ribosomes supportmost of the elementary ribosomal functions, which supportsthe idea of an archaic RNA world (Spirin, 2002a). The factthat the ribosomal machinery is able to perform the factor-free translation indicates that all elongation steps are thermo-dynamically spontaneous and that elongation factors contrib-ute kinetically rather than thermodynamically. In otherwords, they act as enzymes.

3.1.2. Role of elongation factors

3.1.2.1. Effıciency and accuracy require energy consump-tion. Factor-free translation is slow. It presents an increasedrate of incorporation of non-cognate aminoacyl-tRNA and itis subjected to a higher number of frameshift errors. The useof translational factors results in an increase in the translationrate up to about five residues per second per eukaryoticribosome (faster in prokaryotes; Lodish and Jacobsen, 1972;Palmiter, 1974; Sonenberg et al., 2000). Factors also increasethe accuracy of translation, which reduces error incorpora-tion to less than 1/10,000 (Ibba and Soll, 1999; Yarus, 1979).

Accuracy depends on three main steps, one is the aminoa-cylation process that uses two energy-rich bonds, the secondone relies on a correct recognition between the codon in themRNA and the anticodon in the tRNA, the third one occursduring translocation where mRNA advances exactly onecodon. The action of the translational factors, bringing bothspeed and accuracy, relies on an increase in energy consump-tion of 2 GTP by residues but it is unclear how this GTPhydrolysis energy is coupled to the translational process effi-ciency.

3.1.2.2. The place of GTP hydrolysis in elongation factormechanism of action. The classical theory to describeG-proteins supposes that they alternate between two confor-mations that are stabilised either by GTP- or GDP-binding.The GDP state is the product of GTP hydrolysis that occursin the presence of a GTPase Activating Protein (GAP) activ-ity. Reformation of the GTP state requires the release of GDPfrom the factor and this step is promoted by a Guanylicnucleotide Exchange Factor (GEF) activity. The replacementof GDP by GTP in the nucleotide-free factor is favoured bythe high cellular GTP/GDP concentration ratio (Sprang,1997). Both of the GAP and GEF activities have been pro-posed for the stalk proteins.

Elongation factors have an intrinsic GTP hydrolysis activ-ity, but it is low in the absence of large ribosomal subunits.This GTPase activity is unmasked in the presence of anappropriate physicochemical environment (De Vendittis etal., 1986; Raimo et al., 1996). In cellulo, GTPase activity isstimulated by the presence of empty ribosomes (Kawakita etal., 1974; Nishizuka and Lipmann, 1966) and this stimulationis increased by tRNA-bound ribosome (Chinali and Parmeg-giani, 1982; Voigt and Nagel, 1993). GTPase activity in-creased in a synergistic manner when both elongation factors


are present (Mesters et al., 1994). These results support theidea that elongation factors hydrolyse GTP only when theribosome is in a precise conformation and therefore that aribosomal component playing the GAP function is unmaskedin response to the appropriate position of the tRNAs at theA-,P- and E-sites. GTP hydrolysis is therefore the result of asuccessful progression in the elongation cycle, and con-versely, GTP hydrolysis constitutes the irreversible eventdriving elongation unidirectionally (Spirin, 2002b).

3.1.2.3. The translocation step in elongation. The translo-case mechanism is still puzzling, and that at least one cycle ofelongation can be catalysed by EF-G GTP binding in theabsence of GTP hydrolysis is not the least perplexing result(Kaziro, 1978). However, it is noteworthy that energy neededfor this step might also be partly given by the preceding andthe following exergonic steps, namely, transpeptidation anddecoding. Transpeptidation is performed by approaching the3' end of the aminoacyl-tRNA to that of the peptidyl-tRNA atthe P-site. This intermediate state in which the codon arm ofthe aminoacyl-tRNA is still at the P-site is called theA/P statein the hybrid sites theory (Moazed and Noller, 1989). In theA/P state, aminoacyl-tRNA has a lower affinity for the ribo-some than in the A/A state (Semenkov et al., 2000). Then,stabilisation of the peptidyl-tRNA at the P/P site aftertranspeptidation is promoted. A part of the energy involved inthe translocation might therefore originate from theaminoacyl-tRNA hydrolysis during the transpeptidation re-action. Further promotion of translocation is also brought bythe existence of the E-site that drives the deacylated-tRNAout of the P-site as indicated above. However, E-site releaseof the deacylated-tRNA is supposed to be energetically con-nected with A-site occupancy by a cognate aminoacyl-tRNAin complex with EF-Tu-GTP (Rheinberger and Nierhaus,1986). Displacement of the equilibrium to drive the translo-cation is therefore partly linked to the energetic contributionof GTP hydrolysis by EF-Tu in the decoding step (allostericsites theory). In contrast to this model, another theory pro-poses that the binding and release of translocase is not simplybringing the activation energy required to catalyse the trans-location. Rather, in coupling the free energy of GTP hydroly-sis to translocation, EF-G serves as an authentic motor pro-tein and drives the directional movement of transfer andmessenger RNAs on the ribosome (Rodnina et al., 1997;Stark et al., 2000; Wintermeyer and Rodnina, 2000).

3.1.2.4. The decoding step in elongation. Decoding at theA-site of the ribosome is controlled by several mechanismsthat are out of the focus of this review but GTP hydrolysis byEF-1A or EF-Tu is the final step that results in the release ofthe aminoacyl-tRNA. This is due to the fact that the GDPconformation is “relaxed”, whereas GTP conformation is“tight”. When GTP is bound, the acceptor arm of theaminoacyl-tRNA is enclosed between two domains of theelongation factor that are separated in the GDP conforma-tion.

3.2. P-proteins involvement in elongation factor functions

3.2.1. Whatever the molecular mechanism supportingthe translocation, GTPase triggering plays a decisivefunction. P-proteins and equivalents are involved inelongation factor binding

Interaction of P-proteins with elongation factors and prob-ably with most of the translational factors has been estab-lished. This is the case for initiation factor 2 (IF-2), EF1-A(EF-Tu in prokaryotes), EF-2 (EF-G) which are GTP hydrol-ysing factors (Fakunding et al., 1973; Kischa et al., 1971;Weissbach, 1972 #226), but also the termination factors RF-1and RF-2 in E. coli that do not belong to the G-proteinsuperfamily were demonstrated to be dependent on L7/L12presence and to a lower extent on L11 binding (Brot et al.,1974; McCaughan et al., 1984; Tate et al., 1984, 1990).Recently, this interaction of the release factors with the stalkhas been shown by cryo-electronmicroscopy, but no directcontact was observed with L7/L12 (Klaholz et al., 2003;Rawat et al., 2003). The importance of the proteins constitut-ing the stalk in binding elongation factors was shown bysubstituting the L11-L10-(L7/L12)4 complex by its rat coun-terpart RL12-P0(P1-P2)2 on E. coli ribosome (Uchiumi etal., 2002b). This experiment was made possible by the factthat the rRNA region to which the P-protein complex binds ishighly conserved. The hybrid ribosomes obtained were ac-tive in an in vitro protein synthesis test using EF-1A and EF-2in place of EF-Tu and EF-G, respectively. This demonstratedthat proteins of this complex constitute the specific motive ofrecognition of the elongation factors in the translating ribo-some. It does not however exclude that other ribosomalmotifs do not participate in elongation factor binding, pro-vided that they are conserved in both the ribosomes. This, forexample is the case for rRNA motifs such as the Sarcin-RicinLoop and the thiostrepton loop for which accessibility ismodulated by elongation factor binding (Guillot et al.,1993a, 1993b; Uchiumi et al., 2002a). Conversely, bacterialL7/L12 have been shown to be able to partly reconstitute theproperties of yeast ribosomes depleted of P1/P2 (Sanchez-Madrid et al., 1981b).

3.2.2. Are P-proteins GAP proteins?

In prokaryotes, L7/L12 have been observed for years toplay a central role in the ribosome-dependent stimulation ofGTP hydrolysis by elongation factor (Kischa et al., 1971).Our research team has postulated that P-proteins might beinvolved in this GAP activity towards EF-2 during the trans-location. P-protein presence is required for both in vitroPoly(Phe) synthesis activity and ribosome-dependent-EF-2GTPase activity (Bargis-Surgey et al., 1999; Vard et al.,1997). Another set of experiments was made with the recon-stituted core particles used to test the ribosome-dependentEF-2 GTPase activity (unpublished data; Cf. Table 1). Ex-amination of the results indicates that among the ribosomalproteins that are removed by the chemical treatment of theribosome in the deproteinisation–reconstitution experiments,


EF-2 GTP binding depended only on the addition ofP-protein, whereas GTP hydrolysis and EF-2 GDP bindingdepended also on the presence of additional proteins. Weinterpret this data as an indication of the existence of the tworibosomal states involved in EF-2 function; there would be aGTP state involved in EF-2 GTP binding and dependingstrongly on P1 and P2, and another one involved in EF-2GTP-hydrolysis activation and EF-2 GDP binding dependingon P1, P2 and additional ribosomal proteins. It should bepointed out that RL12 is found among the proteins partlyremoved by the DMMA extraction.

A direct interaction has been shown between EF-2 and theP-proteins in solution (Bargis-Surgey et al., 1999): P-proteininteraction induced an EF-2 fold that rendered it more sensi-tive to limited proteolysis by V8 protease. It was postulatedthat P-proteins induced an EF-2 fold that was related to atransition state catalysing GTP hydrolysis since the sameproteolytic pattern was observed in the presence of GDP butnot GTP. However, no GTPase activity was observed afterincubation of EF-2 GTP with isolated P1, P2, or bothP-proteins, whatever their state of phosphorylation, even forconcentrations for which an interaction was demonstrated(0.5 µM) (unpublished data). It is noteworthy that concerningEF-G, it has been reported that isolated L7/L12 was able tostimulate EF-G GTPase activity contrary to what we reporthere for the eukaryotic counterparts (Savelsbergh et al.,2000). This discrepancy can originate from the fact thatisolated L7/L12 dimer conformation can be closer to theconformation adopted in the ribosome than that of isolatedP1/P2 heterodimer.

3.2.3. Are P-proteins involved in other stepsof the GTPase cycle?

Taken together, these results suggest that P-protein mightpossess a GAP activity on EF-2 but that they are probably notthe only determinant responsible for it. Also, it cannot be

excluded that P-proteins might be involved in other steps ofthe GTPase activity, besides the GAP catalytic activity itself.Indeed, in a multiround kinetic assay, abolition of the GT-Pase activity can originate from a lack of several steps, EF-2GTP binding to the ribosome, GAP activity, EF-2 GDPrelease, and even GDP exchange with GTP since it is unclearhow this exchange is performed by EF-2. Some data indicatethat P-proteins might be involved in GEF function since afterEF-2 GTP hydrolysis, GDP was found associated with aribosomal protein that was identified as P2 (Lavergne et al.,1992).

3.2.4. Other candidate for the GAP activityAnother main motive involved in the elongation step is the

conserved SRL in the 28S rRNA and in the 26S rRNA. SRLand thiostrepton loops are two rRNA motives located at thebase of the stalk (Fig. 5). The crystal structure of the largesubunit has confirmed that both these rRNA loops are closedones (Guillot et al., 1993a; Wilson and Noller, 1998a). SRLwas identified by several groups as interacting with elonga-tion factors. Our team laboratory has shown that the terminaladenine of this loop, that is suspected to exist under twoconformations, is protected only by the GTP conformation ofEF-2 and that this interaction involved a specific residue ofthe G domain of EF-2 (Guillot et al., 1993a, 1993b).

3.2.5. The stalk as a polyvalent adapter for translationalcomponents

Stalk proteins are not the only determinant involved inelongation factors binding, but all these data tend to supportthe idea that the stalk is flexible so as to adapt its conforma-tion to fit with the whole set of translational factors andtRNAs that bind at its base. The lateral flexible stalk functionat the entry site would then be very close to that of thesymmetrical L1 protein protuberance that is located at theE-site. Both sites have to adapt to allow the entry and binding

Table 1Reconstitution experiments of DMMA cores (CDMMA) were performed with a two molar excess of each of the P-proteins and in a condition in which unboundproteins are removed by ultracentrifugation as described by Vard et al. (1997) and Bargis-Surgey et al. (1999). In these studies, phosphorylated proteins arebiphosphorylated. Poly(Phe) synthesis was measured in the presence of a limiting amount (2.5 pmoles) of large subunits or CDMMA as in Reboud et al. (1980).100% corresponded to 16 pmoles of [14C]-Phe incorporated. GTPase activity was measured using a kinetic method in the presence of 30 pmoles ribosomes (orindicated CDMMA reconstitutions), 30 µM GTP and 80 pmoles EF-2 (Gonzalo et al., 1995). Intrinsic GTPase activity of the ribosome (or indicated CDMMA

reconstitutions) has been subtracted. EF-2 preparation contained no GTPase or contaminating activity. 100% represents 180 pmol/min GTP hydrolysed. Thedetection limit of this technique in these conditions is less than 10 pmol/min, which represents less than 5% of the 80S ribosome dependent- EF-2 GTPaseactivity. Ternary complexes [EF-2/ribosomes/GDP] and [EF-2-/non-hydrolysable GTP analogue GDPNP/80S] were estimated by measuring the [3H] labellednucleotides retained on a nitro-cellulose filter after an incubate with 30 pmoles EF-2, 30 pmoles ribosomes (or indicated CDMMA reconstitutions) and saturatingconcentrations of nucleotides (40 µM [3H] GDPNP or 1 mM [3H] GDP) as in Dumont-Miscopein et al. (1994)

Preparation Poly(Phe) synthesis (%) GTPase activity (%) EF-2-GDP–80S complex (%) EF-2-GDNP–80S complex (%)80S ribosome 100 100 100 100CDMMA 5 <5 16 3CDMMA + Ethanol extract 82 48 40 100CDMMA + P1 + P2 14 <5 / /CDMMA + P1p +P2p 85 37 22 97CDMMA + P2p + P1 63 51 38 97CDMMA + P1p + P2 10 <5 0 0CDMMA + P1p 8 <5 0 0CDMMA + P2p 8 <5 0 0


of the aminoacylated-tRNA and the release of thedeacylated-tRNA. This symmetry is also appealing since theallosteric three sites model proposes that at the E site-deacylated-tRNA is only released when a cognateaminoacylated-tRNA binds to the A-site (entry site), whichsupposes a coordinated function (Nierhaus, 1993).

4. Experimental support for an involvementof the stalk in translational regulation

4.1. Phosphorylation state of the ribosomal proteins P

In contrast to the prokaryotic proteins L7/L12, theP-proteins have been found to be phosphorylated in practi-cally all types of eukaryotic cells from the vegetal as well asthe animal kingdom, which is the reason for their denomina-tion. The phosphorylated residues have been identified as thetwo serines present in the C-terminal peptide common to P0,P1 and P2 (Hasler et al., 1991). The functional significance ofsuch a universal presence of phosphoproteins in the eukary-otic ribosomal stalk has been debated since their discovery.Their involvement in translational regulation mechanisms issuspected from data obtained in vitro and in cellulo.

4.2. P-protein kinases

The existence of protein kinases bound to rat liver poly-somes and able to phosphorylate ribosomal proteins wasdemonstrated a long time ago. Two distinct protein kinaseactivities have been characterised: one was released at lowionic strength and could phosphorylate basic ribosomal pro-

teins, mainly S6, another one needed a higher ionic strengthand could phosphorylate the acidic P-proteins. This secondactivity was attributed to an enzyme of the “Casein kinase”class (Cenatiempo et al., 1978, 1981; Genot et al., 1978,1979). The phosphorylation sites of the P-proteins were iden-tified on Ehrlich ascites cells and were found to be identicalin in vivo [32P]-labelled cells and in isolated P-proteinstreated by Casein kinase 2 (Hasler et al., 1991). However, theprecise nature of the endogenous enzyme(s) responsible forthe phosphorylation in cellulo was not determined. Interest-ingly, an increase of P2 phosphorylation was observed re-cently in HEK 293 cells following activation of G protein-coupled receptor kinase 2 (GRK2) that is known tophosphorylate also non-receptor substrates (Freeman et al.,2002; Pitcher et al., 1998). This suggests that in this systemGRK2 is at least one of the kinases responsible for P2phosphorylation. In yeast, several other protein kinases havebeen described as potential candidates for the in vivo phos-phorylation of the stalk proteins (Ballesta et al., 1999). Re-cently, the existence of a phosphorylated pool of P-proteinson the cellular wall was established, which raises the ideathat a kinase activity is present at this compartment (Bo-guszewska et al., 2002). In conclusion, until now, no consis-tent and indubitable cellular pathway has been establishedfor the phosphorylation or the dephosphorylation of theseribosomal proteins.

4.3. Hypotheses concerning the role of P-proteinphosphorylation

4.3.1. Binding of the P-proteins to the ribosomeP1 and P2 are the only ribosomal proteins found not only

bound to the ribosome but also free in the cytoplasm; this isnot the case for the prokaryotic L7/L12 (Mitsui et al., 1988;Tsurugi and Ogata, 1985). Data indicate that P-proteins arein rapid exchange between the cytoplasm and the ribosome,and that phosphorylated P-proteins are only found in theribosome. It was first hypothesised that these proteins joinedthe ribosome at a late stage of its synthesis; this was con-firmed recently (Boguszewska et al., 2002; Tchorzewski etal., 2003). It was also hypothesised that phosphorylation ofP1 and P2 could allow their binding to the ribosome(Naranda and Ballesta, 1991; Sanchez-Madrid et al., 1981a).However, such hypothesis has been discarded for severalreasons, in particular because both the unphosphorylated andphosphorylated proteins have been found to bind to ribo-somes (Ballesta et al., 1999; Vard et al., 1997).

4.3.2. Specific functionsA specific role for the phosphorylation of P2 in protein

synthesis regulation has been suggested by in vitro reconsti-tution experiments of rat liver ribosomes. When recombinantP1 and P2 were added to partially deproteinised inactive coreparticles, in which these proteins were lacking, active ribo-somes could be reconstituted only if P2 had been phoshory-lated by either CK2 or GRK2 (Freeman et al., 2002; Vard et

Fig. 5. Thiostrepton loop and SRL vicinity at the elongation factor bindingsite. This image is made of some of the atomic coordinates of the pdb file1ffk that contains crystal structure of the large ribosomal subunit fromH. marismortui at 2.4 Å resolution (Ban et al., 2000). It demonstrates thatthe two main 28S rRNA motives involved in the binding of elongationfactors are located in close vicinity at the base of the lateral flexible stalk.These rRNA motives have been proposed as potential GTP hydrolysispromoting components.


al., 1997). P1 phosphorylation was not required. The fact thatP1 and P2 would play different roles in the function of thestalk is in agreement with recent structural studies, that pointout a dissymmetry between P1 and P2 showing that P1, butnot P2 interacts with P0. Taking into account the observationalready mentioned that GRK2 could phosphorylate P2 inHEK 293 cells, one can hypothesise the existence of a trans-lational regulation mechanism through an adrenergic path-way.

In yeast, some results suggest that phosphorylation of thestalk proteins could modify the expression of some mRNAand therefore the nature of the proteins synthesised (Rem-acha et al., 1995). A similar hypothesis has recently proposedthat ribosome might participate in the discrimination of themRNAs recruitment for translation (Mauro and Edelman,2002).

Other more recent studies using these cells demonstrate apeculiar effect of P1 phosphorylation on its stability, thisshould also have functional consequences (Nusspaumer etal., 2000).

5. Conclusion

The stalk proteins present a similar general organisationacross all biological kingdoms even the primary structures ofP-protein (and bacterial equivalents) have considerablyevolved. Recent results demonstrate that this structural evo-lution occurred in parallel with that of elongation factors, andtogether with other arguments, this promotes the idea that thelateral flexible stalk has been merged to an archaic coreribosome to improve translation efficiency. The ribosomalstructure has been visualised at outstandingly different posi-tions at different steps of the translation process. Our hypoth-esis is that this malleability is required to fit the whole set oftranslational components that pass through this strategic siteof the ribosome. It is not easy to determine the precisefunction of each of this component since the properties ofisolated and ribosome bound components are probably notthe same. Some data suggest that P-proteins might be apotential target for a regulation mechanism involving phos-phorylation. However, physiological meaning of this phos-phorylation remains to be demonstrated in order to validatethis hypothesis.

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