Current Protein and Peptide Science, 2002, 3, 133-141 133 Is tRNA Binding or tRNA Mimicry Mandatory for Translation Factors?

Ole Kristensen, Martin Laurberg, Anders Liljas*, and Maria Selmer

Molecular Biophysics, Centre for Chemistry and Chemical Engineering, Lund University, Box 124, Se-221 00 Lund, Sweden

Abstract: tRNA is the adaptor in the translation process. The ribosome has three sites for tRNA, the A-, P-, and E-sites. The tRNAs bridge between the ribosomal subunits with the decoding site and the mRNA on the small or 30S subunit and the peptidyl transfer site on the large or 50S subunit. The possibility that translation release factors could mimic tRNA has been discussed for a long time, since their function is very similar to that of tRNA. They identify stop codons of the mRNA presented in the decoding site and hydrolyse the nascent peptide from the peptidyl tRNA in the peptidyl transfer site. The structures of eubacterial release factors are not yet known, and the first example of tRNA mimicry was discovered when elongation factor G (EF-G) was found to have a closely similar shape to a complex of elongation factor Tu (EF-Tu) with aminoacyl-tRNA. An even closer imitation of the tRNA shape is seen in ribosome recycling factor (RRF). The number of proteins mimicking tRNA is rapidly increasing. This primarily concerns translation factors. It is now evident that in some sense they are either tRNA mimics, GTPases or possibly both.

INTRODUCTION tRNA in the neighbouring ribosomal P-site. When the structures of elongation factor G (EF-G) [14,15] and the tRNA is the adaptor in the translation process [1]. This ternary complex of elongation factor Tu (EF-Tu) with remarkable molecule has two functional parts, the anticodon aminoacyl-tRNA and a GTP analogue were solved, it recognising the mRNA codon and the amino acid acceptor became clear that their shapes were very similar (Table 1). end. These two sites are separated by as much as 75 Å [2,3]. Thus part of EF-G mimics tRNA [16]. This was the first The ribosome is known to contain three sites for tRNA, the A-, P-, and E-sites [4,5]. With the recent progress of cryo- Table 1. Eubacterial Translation Factors EM and macromolecular crystallography, the structure of ribosomal subunits and whole ribosomes alone and in complex with tRNA or other ligands has clarified much of Initiation the way the tRNAs are bound and moved between the ribosomal sites [6-11]. The tRNAs bridge between the IF1 Assists IF2 in initiation. Binds to the A-site. ribosomal subunits with the decoding site and the mRNA on the small or 30S subunit [12] and the peptidyl transfer site on IF2 Binds initiator-tRNA to the P-site. Aids in joining the two the large or 50S subunit [8]. The length of the tRNA may ribosomal subunits. GTPase. have evolved together with the two functional sites of the IF3 Assists in dissociation of subunits. Binds to the subunit ribosome to achieve the fidelity of translation. The tRNA interface side of the small subunit. sites, A, P, E, are placed next to each other in the intersubunit space [11]. Elongation

tRNA-like motifs are extensively found in biology. Thus EF-Tu Binds aatRNA to the A-site. e.g. viral genomic RNAs may contain tRNA mimicry EF-Ts Nucleotide exchange factor for EF-Tu. regions that are functionally important and that can be aminoacylated by tRNA synthetases [13]. The tRNA motif EF-G Translocates peptidyl tRNA from A-to P-site. seems to have originated early in evolution and used in EF-P Unclear function. Homologous to eucaryotic initiation factor multiple ways. eIF5A.

The possibility that some proteins could mimic tRNA has Termination been discussed. The most evident are the translation termination factors (Table 1). These proteins are able to, RF1,2 Recognizes termination codons and hydrolyses the peptide directly or indirectly, identify stop codons of the mRNA from P-site tRNA. presented in the decoding site of the ribosome and as a RF3 Releases RF1 and RF2 from ribosome. consequence hydrolyse the nascent peptide from the peptidyl Recycling

RRF Dissociates the terminated ribosomes. *Address correspondence to this author at the Molecular Biophysics, Centre for Chemistry and Chemical Engineering, Lund University, Box 124, Se- EF-G Needed for the recycling reaction. 221 00 Lund, Sweden; E-mail: [email protected]

1389-2037/02 $35.00+.00 © 2002 Bentham Science Publishers Ltd. 134 Current Protein and Peptide Science, 2002, Vol. 3, No. 1 Liljas et al. observation of tRNA mimicry in a protein. During the last Obviously this part of the molecule is then also interacting few years the number of proteins known to mimic tRNA has with the small subunit. IF2 also has a G-domain and is active increased. This is primarily true for translation factors (Table in complex with GTP. The N-terminal domains thus must 2), but at least one additional case is identified. The field has interact with the GTPase center of the large ribosomal been reviewed recently [17]. The current state is summarised subunit. There is no suggestion that the factor mimics tRNA. in this paper. The tRNA mimicry has simplified our view of eubacterial translation factors. Essentially all of them are IF3 binds to the small ribosomal subunit and has two either tRNA mimics or GTPases or possibly both. distinct functions. The first role is specific binding to the 30S to prevent premature association of the large subunit, a function attributed to the C-terminal domain [25]. Secondly, INITIATION IF3 is involved in the selection of initiator tRNA, and recognises the three unique G:C base pairs of the anticodon Three proteins are normally identified as initiation factors stem [26]. The structure of IF3 is characterised [25, 27]. It in eubacteria, IF1, 2 and 3 (Table 1). Their functions are consists of two domains separated by a linker. The binding primarily related to the small subunit. In the case of IF1 the site of its C-terminal domain on the small subunit is also function has been least obvious. Observations of binding to identified [28-30]. It is in the area where the platform the A-site have been reported [18]. It has also been suggested contacts the 50S subunit. The C-terminal domain overlaps that the protein may mimic the anti-codon stem and loop with the position of a protruding element of the 23S rRNA (ASL) of a tRNA [19]. In fact, the structure [20] of IF1 (a 9K that interacts with the small subunit [28, 30]. Furthermore protein) can easily be fitted into the ASL of a tRNA. the position of the N-terminal domain corresponds to the Recently Carter et al. [21] determined the structure of the tRNA E-site [30]. Thus in a sense it can be said to mimic 30S subunit from Thermus thermophilus with a bound tRNA. molecule of IF1. The binding is clearly to the decoding part of the A-site (Fig. 1). The role of IF1 may be understood EF-P is a factor that may be active in the formation of the from this location together with previous observations that first peptide bond, in the step between the initiation and IF1 prevents aminoacyl tRNAs from binding at the A-site. elongation cycles [31]. It has a homologue in eucaryotes Probably IF1 assists IF2 in binding the initiator tRNA to the called eIF5A [32,33]. The crystallographic structure of EF-P P-site by blocking the A-site. [34] shows that the factor is a clear tRNA mimic, but it is still unknown where it binds. The eubacterial factor IF2 is the factor that brings the formylmethionine tRNA into the P-site and assists in joining the two ribosomal subunits. The complete structure of ELONGATION IF2/eIF5B was recently determined [22]. It is a protein composed of four domains and has the shape of a chalice, The elongation cycle is the main mode of protein where a long α-helix connects the C-terminal domain to the synthesis. In bacteria there are three main elongation factors. rest of the protein. The C-terminal domain interacts with One of them, elongation factor Ts (EF-Ts) is a nucleotide fMet-tRNAfMet [23] as well as with IF1 on the ribosome [24]. exchange factor for elongation factor Tu (EF-Tu). It does not

Table 2. tRNA mimicry in Translation Factors

Factor GTPase tRNA mimic Binding site Role

IF1 + A Anticodon stem loop

IF2 + P Binds initiator-tRNA to the P-site

IF3 + E Disassembly

EF-P +? ?

EF-Tu + A Binds aminoacyl tRNA to the A-site

EF-Ts Nucleotide exchange

EF-G + + A Mimics EF-Tu:tRNA

RF1,2 +(?) A Hydrolysis of peptide

RF3 + Removal of RF1,2

RRF + A Ribosomal recycling

Additional protein in translation

TL5 / CTC + ? Ribosomal protein/ general stress factor Is tRNA Binding or tRNA Mimicry Mandatory Current Protein and Peptide Science, Vol. 3, No. 1 135

A

B

Fig. (1). a). IF1 (yellow) binds to the decoding site of the small subunit and is overlapping with the binding site for the anticodon stem and loop of the A-site tRNA [21]. The penultimate helix of the 16 S RNA is shown in purple. (RIBBONS, [66]). b). tRNA in the A-site is shown in the same view, from the E-site towards the A-site [11]. bind to the ribosome and is therefore not of further interest determined [16,35]. The structure of the complex is quite for this discussion. elongated with the amino acid bound to the tRNA enclosed by EF-Tu and the anticodon exposed at the other extreme of EF-Tu is a three-domain protein, which binds aminoacyl- the complex (Fig. 2a). There is also a cryo-electron tRNA and catalyses its binding to the ribosome. microscopic structure of a ternary complex bound to the Crystallographic structures of ternary complexes between ribosome at 13 Å resolution [36]. In this structure the tRNA EF-Tu, aminoacyl-tRNA and a GTP analogue have been molecule of the complex remains bound to EF-Tu due to the 136 Current Protein and Peptide Science, 2002, Vol. 3, No. 1 Liljas et al. presence of an antibiotic, kirromycin that prevents expected to have totally different structures [48]. Thus we dissociation of EF-Tu from the tRNA and the ribosome. The cannot immediately assume that eubacterial RF1 and –2 also factor is bound to what can be called the GTPase site on the have the L-shape of a tRNA. ribosome inside the so-called L7/L12 stalk, primarily interacting with the large subunit. The anticodon of the tRNA is located in the decoding site of the small subunit RECYCLING probably base-pairing to the mRNA. The acceptor end and the aminoacyl part of the tRNA are far away from the The termination reaction results in a ribosomal post- peptidyl transfer site due to its attachment to EF-Tu. When termination complex consisting of the 70S ribosome bound EF-Tu dissociates from the ribosome, the tRNA is obviously to an mRNA, with a deacylated tRNA in the P-site. allowed to swing into the peptidyl transfer site. There it can Ribosome recycling factor (RRF), EF-G and GTP are needed engage in peptidyl transfer whereby the peptide is transferred for disassembly of this complex [49,50]. Depending on the from the tRNA in the P-site to the tRNA in the A-site. For mRNA used, this results in monosomes or 70S ribosomes protein synthesis to proceed the peptidyl-tRNA needs to be separated from the mRNA [49] or 50S subunits and 30S shifted over to the P-site. This is catalysed by elongation subunits bound to mRNA and tRNA [50]. From its factor G (EF-G). competition for binding with RF1 [51] RRF was suggested to bind to the ribosome in the A-site region. The crystal The crystallographic structure of EF-G has been structure of T. maritima RRF [52] as well as more recent determined in a few somewhat different conformations structures of RRF from other bacteria [53-55] show that RRF [14,15,37,38]. In addition a number of cryo-EM studies have consists of two domains that together create a characteristic been made of EF-G bound to the ribosome under different L-shape. The shape of RRF agrees nicely with that of a conditions [6, 39-41]. EF-G was the first real observation of tRNA molecule as is shown in Fig. (2d). The two molecules tRNA mimicry. In fact this factor mimics the ternary have the same shape except that the part corresponding to the complex of EF-Tu, aminoacyl-tRNA and a GTP analogue amino acid-binding 3’ end is missing in RRF. The close [16]. Domains I (or the G-domain) and II are homologous mimicry leads to the hypothesis that RRF binds to the A-site between these proteins sharing distinctive sequence motifs similarly to a tRNA and is translocated to the P-site by EF-G [42]. Domains III, IV and V of EF-G mimic the tRNA of the [52]. This is supported by recent experiments where ternary complex as shown in Fig. (2b). The structural details Mycobacterium tuberculosis RRF and EF-G were of the translocation process remain unclear. However, it is overexpressed in E. coli [56]. Here it is clear that both understood that EF-G by binding to the ribosome in a partly proteins need to originate from the same species for proper similar manner as the EF-Tu.tRNA complex and undergoing recycling. Furthermore with a deficient peptide hydrolase the specific conformational changes catalyses the translocation presence of both proteins led to an accumulation of released of the peptidyl-tRNA from the A-site to the P-site as a result peptidyl-tRNA. Another hypothesis is that EF-G drives the of its GTPase activity. In particular it has been observed both ribosome to a post-translocational state, which is not stable in the comparison of EF-G with and without bound GDP as with RRF bound, and leads to 50S-dissociation [50]. well as in the case of a mutant form of EF-G that the two blocks of EF-G are mobile with regard to each other. These RRF has a hinge between the two domains, which leads two blocks are domains I and II versus the tRNA mimicry to a functionally important flexibility [54]. This flexibility is domains III, IV and V. not found for tRNA. The RRF sequence is not conserved in the anticodon region of the molecule and RRF is not believed to make any codon recognition. Therefore it could TERMINATION also bind to the ribosome when a non-stop codon is exposed as is evidenced by the release of peptidyl-tRNA [56]. This When one of the three stop codons is exposed in the may explain the small but significant error-reducing activity decoding part of the A-site this leads to termination of of RRF [57]. protein synthesis. These stop codons are directly or maybe indirectly recognised by release factors 1 and 2 (RF1 and RF2) [43,44]. As a response to their binding to the ribosomal OTHER CASES A-site they hydrolyse the peptide from the peptidyl-tRNA in the P-site. This catalysis must be performed in the peptidyl Ribosomal proteins are to a large extent conserved transfer site of the ribosome where the bond between the P- between eubacteria, archaea and eucarya [58]. For several site tRNA and the nascent peptide is situated. The release proteins, where the sequence homology has not been factors have been expected to mimic tRNA [45,46]. Recently identified, we expect that homologies may be identified from the structure of the human release factor 1 (eRF1) was their structures and locations within the ribosome. determined [47]. The protein is composed of three domains in an extended conformation (Fig. 2c). The decoding and In the case of ribosomal protein L25 (nomenclature from catalytic sites are identified in the two extremes of the Escherichia coli) there are two versions. The smaller molecule. Even though the molecule is not a perfect tRNA version, found in E. coli with approximately 90 amino acid mimic one can easily imagine that the hinge between the residues, is unusual. The only other available example is domains is flexible enough to permit a conformation more from Hæmophilus influenza [59]. The normal version, found like the L of a tRNA. The volume of the factor corresponds in all other eubacteria, has slightly less than 200 amino acid well with that of a tRNA. Eukaryotic and eubacterial release residues and has been called TL5 [60]. Both versions of the factors have essentially no sequence homology and are protein bind to the ribosomal 5S RNA and structures of both Is tRNA Binding or tRNA Mimicry Mandatory Current Protein and Peptide Science, Vol. 3, No. 1 137

A C

B D

E

Fig. (2). Ribbon diagrams [66] of tRNA mimicing proteins. a). The ternary complex of EF-Tu with tRNA and a GTP analogue [16]. b). EF-G [38] mimics the ternary complex of EF-Tu with tRNA and GTP. c). Human release factor 1 (eRF1) [47] reminds of a tRNA. d). Ribosome recycling factor [52] is a close tRNA mimic. e). Ribosomal protein TL5 from T. thermophilus has a structure closely similar to a tRNA molecule [63]. The protein binds to the 5S rRNA but probably has a second receptor since the protein in Bacillus subtilis is a general stress factor, called ctc. 138 Current Protein and Peptide Science, 2002, Vol. 3, No. 1 Liljas et al.

A

B

D C

E

Fig. (3). The electrostatic surface potential of the proteins mimicking (GRASP, [67]). For each protein the left view shows the side facing the P-site if the protein binds as a tRNA to the A-site (orientation from Fig. (1) and Fig. (2) and the right view shows the opposite side. a). IF1. b). EF-G. c). eRF1. d). RRF. e). TL5. versions bound to fragments of the 5S RNA are known [61- negative. One possible understanding of the two roles of the 63]. The residues involved in the binding to the RNA are protein is that it has one high affinity-binding site, the 5S highly conserved [63]. RNA of the ribosome. When it is expressed at higher concentrations as a response to stress it is able to also bind at In Bacillus subtilis this protein, also called CTC, is a site with lower affinity, the stress response receptor. This expressed in high amounts as a response to general stress. site may identify the tRNA shape and be a normal tRNA Thus CTC was originally identified as a general stress binding component of the cell. What this receptor might be protein [64]. It is not presently known whether this protein is and whether the tRNA mimicry has any functional role are a stress protein in other species. Since there is always one currently not known. gene for the protein this appears to be yet another ribosomal protein with multiple functions [65]. The structure of the CTC /TL5 version reveals that the protein is yet a tRNA SUMMARY AND CONCLUSIONS mimic [63]. It not only has the shape of an L but also the volume agrees surprisingly well as is shown in Fig. (2e). In The tRNA binding sites, A-, P- and E-, are naturally the addition the overall electrostatic potential of the protein is prime functional sites on the ribosome. The ribosomal Is tRNA Binding or tRNA Mimicry Mandatory Current Protein and Peptide Science, Vol. 3, No. 1 139

Table 3. Probable Occupancy of the tRNA Sites of the Ribosome During the Different Steps of Translaion

Phase Catalyst* A P E Comment

Initiation** IF2 IF1 f-Met-tRNA IF3

Elongation binding EF-Tu aa-tRNA pp-tRNA (tRNA) Aminoacyl-tRNA

pp-tRNA tRNA Peptidyl transfer

EF-G EF-G pp-tRNA tRNA Translocation

Termination RF1/2 pp-tRNA

RF3 tRNA

Recycling RRF tRNA

EF-G EF-G RRF tRNA

* The catalysts are GTPases that bind to the GTPase site on the ribosome. ** The initiation cycle only involves the small ribosomal subunit. activity is assisted by a number of proteins, translation factor has different interactions from a tRNA. For example factors. The old expectation that the eubacterial release G530 and A1492 of 16S RNA have hydrogen bond factors 1 and 2, should be tRNA mimics both structurally interactions in the minor groove of the second base pair of and functionally has not yet been fully verified. However a the codon-anticodon helix in the tRNA complex. In the IF1 eukaryotic release factor, human eRF1, has been shown to be complex G530 is stacking with Met42 and A1492 sits a relatively good tRNA mimic and a number of other unordered in a cavity between IF1 and S12. translation factors show surprising structural similarities to tRNA. In several of these cases it is clear that this similarity If one regards the occupancy of the three tRNA binding is paralleled by a capacity to bind to some tRNA binding site sites on the ribosome during the different functional states, it on the ribosome. Some of the translation factors do not have turns out that proteins occupy the A-site in all stages of the shape of a tRNA molecule, but they nevertheless bind to translation other than elongation (Table 3). Also during one of the binding sites for tRNA. Here the mimicry is more elongation EF-G enters into the decoding part of the A-site functional than structural. This is the case for IF1 [21 ] and during translocation. The P- and E-sites on the other hand are IF3 [28-30 ]. No doubt the charge distribution of the tRNA rarely binding proteins mimicking tRNA. mimicry proteins is important. However, as Fig. (3) shows the charge distribution varies greatly and no general conclusion can be drawn at this stage. ABBREVIATIONS

One surprising aspect of the tRNA mimicry of translation Å = 0.1nm factors is the fact that all of them have different folding patterns. Thus these proteins must have evolved individually EF-G = Elongation factor G from different precursors. This may suggest that they are EF-P = Elongation factor P later additions to a primordial RNA based translation system where the tRNA molecules were already more or less fully EF-Ts = Elongation factor Ts developed. EF-Tu = Elongation factor Tu One interesting question is if tRNA mimics really imitate tRNAs when bound to the ribosome, and thus interact with eIF5A,B = Eucaryotic initiation factors 5A and B the same counterparts of the ribosome or if they just bind to a tRNA binding site through unique interactions with the eRF1 = Eucaryotic release factor 1 site. Since several different protein structures interact with the A-site it would seem unlikely that they all use the same GDP = Guanine nucleotide diphosphate interactions as tRNA. The more likely situation is that they have a shape that fits the site (tRNA mimicry), but have GTP = Guanine nucleotide triphosphate different interactions with the ribosome in its different conformational states and may trigger conformational IF1,2,3 = Initiation factor 1,2,3 changes. So far the only complete information at high- resolution for comparisons are the complexes of a tRNA RF1,2,3 = Termination factor 1,2,3 anticodon stem-loop and IF1 with the 30S subunit [12, 21]. Even though IF1 interacts with the same ribosomal parts, the RRF = Ribosome release factor 140 Current Protein and Peptide Science, 2002, Vol. 3, No. 1 Liljas et al.

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