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Evolutionary Conservation of Reactions in Translation

C. H. Best Institute, Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario, Canada M5G 1L6

SUMMARY

INTRODUCTION

TRANSLATIONAL INITIATION

Eukaryotic Initiation

Initiation Reaction

Function of the RNA Helicases in Initiation

Structure of eIF4A

Eubacterial IF4A (W2) Protein

PEPTIDE BOND SYNTHESIS

Peptidyl Transferase

rRNA-tRNA Interactions and the Peptidyl Transferase Active Center

Limited Capacity of Peptidyl Transferase To Synthesize Certain Dipeptides

efp Gene in Eubacteria

Eukaryotic eIF5A

Structure of aIF5A

Possible Role of EFP in the Peptidyl Transferase Reaction

PROTEIN CHAIN ELONGATION

Decoding

Translocation

Elongation Factor 3 of Fungi

Eubacterial EF3

Possible Function of EF3

TRANSLATIONAL TERMINATION

CONCLUDING COMMENTS

ACKNOWLEDGMENTS

REFERENCES

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INTRODUCTION

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There is overwhelming evidence that the genetic code is, with relatively minor variations, conserved throughout species. It is thus fully expected that the apparatus that deciphers this code should be correspondingly conserved. The intermediates and the products of the translation reaction are conserved, giving further credence to this idea. Current X-ray diffraction analysis of the eubacterial ribosomes at 5.5-Å resolution has resulted in the direct visualization of the tRNA substrates in the ribosomal A, P, and E sites (27, 204). Since the sites are conserved (173), this argues for conservation of the mechanisms in translation as well.

The ribosomal proteins, the tRNAs, and the aminoacyl-tRNA synthetases have remarkably conserved structures throughout the species, as have the protein factors that are involved in initiation, elongation, and termination (149, 150). The only apparent exception to the fact that all of the components of translation are conserved is the initiation reaction (113). The initiation processes are functionally similar in all cells, but the components of the reactions appear to differ between archaebacteria/eukaryotes and the eubacteria. Thus, three factors are assumed sufficient for initiation in eubacteria, IF1, IF2, and IF3. In contrast, eukaryotic initiation involves additional proteins, many of which are made up of several subunits (83, 84). This observation implies that the evolution of these processes differed, an issue that has been elegantly addressed by Kyrpides and Woese (113).

Here, we review observations that several initiation factors and one elongation factor of Escherichia coli, first isolated as required to reconstitute translation (55, 58, 65, 74), bear a surprising degree of similarity in structure and function to the eukaryotic initiation factors eIF4A and eIF5A and to the elongation factor eEF3, previously described as a fungus-specific protein. The possible mechanism of action of these proteins is discussed after a brief review of the initiation and elongation reactions. Reviews are cited whenever possible.

TRANSLATIONAL INITIATION

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In all cells, translation initiation establishes the reading phase of the genetic code. The start codon in the mRNA interacts with a special methionyl-tRNA (fMet-tRNA_f^Met or Met-tRNA_i) in the peptidyl donor center of the small subunit of ribosomes. The start codon is usually AUG or GUG; rarely, other codons such as UUG, AUU, and AUA can also initiate synthesis (70, 76, 185). Start codons are ambiguous since they also specify insertion of methionine, valine, or other amino acids at internal positions. These triplets also occur out of phase in the genetic transcript. Therefore, features of the mRNA, apart from the start codon, are necessary to phase translation of the genetic transcript. Several reviews are available (70, 76, 185).

In most eubacteria, a second determinant of specificity is an mRNA polypurine tract 5' of the start triplet. This sequence complements the 3' end of the 16S rRNA of the 30S subunit and is called the Shine-Dalgarno (SD) sequence (176). If the SD sequence is truncated or deleted in an mRNA, normal ribosomes cannot translate the encoded proteins (88). However, if the 16S rRNA is altered so as to anneal to these defective mRNAs, translation is possible (88). Biochemical, genetic, and statistical data emphasize the importance of this mRNA-16S rRNA interaction but indicate that the SD sequence is not always necessary and is often not sufficient to specify the start of protein synthesis (70, 76, 185). Other regions of the 16S rRNA, i.e., bases 1471 to 1480 and 458 to 466, have been proposed to bind the mRNA 3' or 5' of the initiation codon (184). These sequences occur in stem-loop structures that are not exposed on the 30S subunit (184). Mutations in the 16S rRNA complementary to the mRNA downstream sequences do not significantly alter synthesis (147, 160). Deletion of these "downstream" elements is necessary to establish their function (147).

Early studies of the binding properties of the bacteriophage Qß-A protein ribosome-binding site and analogues of this sequence indicated the following. (i) The intact start triplet is strictly required to bind the mRNA to the ribosome in the presence or in the absence of the fMet-tRNAfMet. (ii) The upstream SD signal confers stability to mRNA binding. (iii) The number and the type of bases between the SD signal and the start triplet affect initiation efficiency (60, 139). Identical requirements have been reported for a variety of mRNAs (71, 80-82, 161). It is thought that the SD sequence in most transcripts may help transiently to anchor the mRNA on the ribosome, allowing for the kinetic selection of the start triplet in closer proximity to the ribosomal P site (76, 77).

It has been suggested that an extended anticodon of the fMet-tRNA_f^Met (i.e., bases 3' or 5' of the tRNA anticodon that base pair with bases 5' or 3' of the mRNA start codon) enhances the rate of formation and the stability of initiation complexes (60, 63, 80, 123). The bases 5' of AUG markedly modulate initiation complex formation (60). In addition, the start codon itself is frequently found embedded in stretches of longer sequence homology to the anticodon loop (62). Mutations that disrupt the potential complementarity of the extended anticodon with the mRNA reduce translational rates, suggesting that they may be involved in the AUG recognition process. Examples of such features are given in reference 62. Similar results have been obtained with point mutations in the start triplet of the lacZ gene (89, 132).

mRNA secondary structure can affect translational initiation (40, 80, 118, 196). In some cases, the secondary structure masks initiation sites, e.g., AUG in the case of the bacteriophage R17 or MS2 RNA replicase or the SD sequence in the case of the lamB protein of E. coli (40, 70, 80, 185). In other cases, disruption of the secondary structure increases the ability of the ribosomes to recognize correct (and a few incorrect) initiation sites (118). Mutations that disrupt the secondary structure of ribosome-binding sites increase the expression of various genes or exhibit polar effects, presumably by exposing or creating initiation signals (70, 76, 79, 118, 185). The expression of the MS2 coat protein gene depends quantitatively on the thermodynamic stability of the secondary structures neighboring the AUG start codon (40).

Computer-assisted phylogenetic sequence comparisons and mutational studies suggest that the coding regions of several hundred mRNAs fold into a uniform, almost periodic pattern of secondary structure. In contrast, sequences 5' of start codons exhibit a relative lack of potential secondary structure (61). Many mutations 5' of the start site of prokaryotic and eukaryotic mRNAs were found to impair initiation by sequestering the start triplet in secondary structures (70, 71, 111). These studies suggest that the differences in mRNA secondary structure of the start and of the coding sequences, particularly the transition in such structures, may also be an important determinant of initiation site selection (53, 61). This, in turn, implies that an active process may be required to unwind such structures so that elongation can proceed.

An important difference in the mechanisms of initiation of eukaryotic and eubacterial cells reflects the fact that eukaryotic mRNAs are usually modified by a 5'-terminal ⁷mG cap structure (111) and have a 3' poly(A) extension (95). A special mechanism exists for recognizing the 5' cap structure. In similar fashion to eubacteria, the eukaryotic initiation codon is flanked by sequences that favor a specific consensus. Thus, an A is favored 3 bases 5' of the AUG, although many transcripts have a G at this position. Also, a unique G is usually found 3' of the start codon. The consensus sequence, i.e., bases near the 5' and 3' borders of AUG, is known to be important in start site recognition. Mutation of a nonstart AUG to the proper sequence context results in initiation of a new protein at that site (111).

The eukaryotic start site is generally devoid of stable secondary structures, as is the case for the eubacterial mRNAs. However, the length of the region that is free of secondary structure is smaller in the eukaryotic transcripts, probably reflecting the relative shorter length of the eukaryotic start site (53). Secondary structures that occlude recognition of the ⁷mG cap impair initiation (111). In general, recognition of the 5' terminus of the mRNA and of the ⁷mG cap structure precedes binding of the 40S subunit to the mRNA. These interactions, in turn, precede scanning of the transcript up to the start codon (111).

More proteins are involved in the process of initiation in eukaryotic cells than in eubacteria, and several of these proteins are polymeric, amplifying the possibilities for translation regulation. The genes encoding initiation proteins have been cloned and sequenced, and the structures of most of the encoded proteins have been determined (84). The details of their structure and function have been reviewed recently (84).

The structures of IF1 and IF3 have been determined (76). IF1 and IF2 form a complex that resembles the structure of domains IV and V of the EFG-GDP complex (19). Domains IV and V of the EFG-GDP complex, in turn, resemble the EFTu ternary complex and have been postulated to mimic the structure of tRNA. Such structural similarity also appears to aid binding of the EFTu-GTP-aminoacyl-tRNA complex to the ribosomal A site. There is considerable evidence, apart from this structural resemblance, that the initiation factors ensure the stable entrance of the initiator RNA into the ribosomal P site (80, 81).

Two forms of IF2 and IF3 occur in E. coli (169). It is possible that these forms act on alternate pathways for initiation in which tRNA_i interacts first with the 30S particle or with the 30S-mRNA complex (76, 77, 196).

Similarly, the eukaryotic proteins, eIF3 and eIF1A, bind to the 40S subunit and prevent the association of the 60S subunit. Although the exact mechanism is not known, it is suggested that subunit antiassociation may occur by an allosteric transition (84), by steric hindrance, or perhaps by preventing the displacement of the eIF2-GTP-Met-tRNA from the 40S by the 60S subunit (84). In addition, other factors, eIF6 and eIF3A, have been reported to impede the association of the subunits by binding to the 60S particle, thus further preventing an inappropriate joining of the subunits (84). The exact mechanism of eIF6 action has not yet been established.

One expects that a function such as the joining of the ribosomal subunits should remain a conserved process. In eubacteria, the junction of the ribosomal subunits is composed mostly of rRNA sequences, and it is assumed that these base pair with the 50S particle in some fashion. However, it appears that the process requires a protein, the "rescue" factor, to join properly in a functional complex devoid of IF3 (66). Direct evidence that the "rescue protein" competes with the eubacterial IF3 to foster 70S subunit formation suggests that this protein is indeed the counterpart of IF5. A more precise assignment depends on determining the structures of these proteins. What is clear is that the joining of the ribosomal subunits is also dependent on common functions carried out by nonribosomal proteins. For the case of the eubacterial ribosomes, the "rescue" factor may help unmask or align the rRNA sequences in the 30S and 50S subunits which may help join these particles.

IF1A has a significant (21%) sequence identity to the eubacterial factor IF1 and acts in analogous fashion to this protein. On the other hand, eIF3 is composed of 11 subunits and thus differs from the single eubacterial protein that displays a similar mechanism. The ternary complex that is formed, Met-tRNA_i-GTP-eIF2 in eukaryotes, is catalyzed by eIF2, although this protein is composed of three subunits; the subunit binds the Met-tRNA_i and GTP. The sequence of the eubacterial IF2 is, however, not related to the eIF2. The comparable protein is instead eIF5B (84, 113), which is a ribosome-dependent GTPase, as is the eubacterial IF2.

In eukaryotic cells, the binding of the mRNA to the tRNA_i-ribosome complex requires factors that are not represented in eubacterial cells or have not yet been identified. Among these factors are (i) eIF2A, a protein that acts only with AUG in formation of the tRNA_i-40S-AUG complex; (ii) eIF2B, a protein that stimulates the exchange of GDP for GTP in eIF2—the phosphorylation of eIF2B by a variety of means is involved in a unique series of reactions that are the targets for a negative regulation of translation; (iii) eIF3A and eIF6, which prevent subunit association by binding to the 60S particle; and (iv) the ⁷mG cap, which occurs as a singular feature of most eukaryotic mRNAs—a special mechanism is used to recognize the cap structure. The eukaryotic eIF4F is composed of a 25-kDa cap-binding protein designated eIF4E, a 220-kDa protein (eIF4G) that acts as chaperone for the action of eIF3, eIF4A, eIF4B, and eIF4E. The eIF4F complex unwinds the secondary structure of the mRNAs 5' proximal to the ⁷mG cap. This protein complex acts as an RNA helicase and is obligatory for translation in reticulocytes (84). For a summary of the nomenclature and function of the initiation factors, see Table 1 (see also reference 84).

Kinetic studies of the eIF4A protein indicate that the affinity of the enzyme is modulated by ATP · Mg²⁺ and ADP · Mg²⁺ such that the complex of the enzyme with the nucleoside triphosphate is much higher than that for the nucleoside diphosphate (120). The enzyme appears to bind RNA in a random fashion, and the hydrolysis of ATP is not required for eIF4A to bind to single-stranded RNA. The presence or absence of phosphate and the bound nucleoside has been postulated to act as a switch that modulates the structure of the enzyme and its ability to interact with single-stranded RNA. The binding and hydrolysis produce cyclical conformational changes in the enzyme that alter its affinity for the single-stranded substrate so that the energy of ATP hydrolysis can be converted into work (120).

The eIF4A protein dissociates faster from the single-stranded RNA substrate than it can hydrolyze ATP. Thus, the protein probably does not act alone as a processive helicase. Clearly, the helicase activity may depend on the binding of the eIF4B protein (84, 126). The structure of the complex between eIF4A and eIF4B has not yet been determined. Nevertheless, it is likely that the eIF4B or perhaps the newly described eIF4H, which acts in the same fashion as eIF4B, could cause a conformational change that forms a tighter structure around the RNA-binding site. The higher affinity of the eIF4B for the RNA may cause the molecule to transiently dissociate from the complex, enabling the movement of the RNA substrate. This movement is likely to be coupled to the hydrolysis of ATP through the cyclical conformational changes described above.

In the case of eukaryotic initiation, it appears necessary that the ribosomes scan from the 5' terminus to the position of the start codon of most mRNAs (111). It has been postulated that the intrinsic helicase activity of eIF4A could act as a "clamp" on the 5'-terminal region of the mRNA. ATP, which is necessary for scanning, could be hydrolyzed and result in the opening and closing of the active site dependent on the cycles of ATP hydrolysis (111). The merit of this idea depends, in part, in proving definitely whether scanning occurs by the systematic and unidirectional movement of the ribosome relative to the mRNA 5' terminus (111). The discovery that eukaryotic ribosomes can initiate at internal sites suggests that scanning is not restricted to starting at the 5' terminus of the mRNA (94).

A provocative idea has been put forth to explain the results of introducing a start codon 3' or 5' to the termination codon of the coat protein of MS2 or fr bacteriophages (1). The lysis protein, which occurs within the coat protein-coding region, is not expressed unless the upstream coat gene is translated. The lysis start site was positioned by mutation 3' or 5' of the termination codon of the coat protein. It was observed that if the coat protein termination codon occurred 3' of the two competent lysis start sites, the 3' start site was selected, whereas if the termination occurred 5' of both of the inserted starts, the 5' start site was utilized (1). These results suggest that ribosomes scan the mRNA in both directions after they terminate translation until they encounter a functional start site (1). Other early experiments by Sarabhai and Brenner (172) had proposed that ribosomes drift from the termination site to the restart site of the rIIB gene of bacteriophage T4.

Thus, a common mechanism may underlie the reinitiation of synthesis in eubacteria and the internal entry site of eukaryotic ribosomes. It also appears that scanning of the eukaryotic mRNAs need not be restricted to recognition of the 5' start site of the transcript.

View larger version (52K): [in this window] [in a new window]   FIG. 1. (A) Ribbon representation of the structure of the full-length eIF4A. The amino-terminal end is shown in brown, and the carboxyl-terminal end is shown in gold. The connecting flexible II residue linker is shown in black. The following domains are colored as indicated: the N-terminal domain, which harbors motif I, which has the Walker A motif, ASQSGTGKT (residues 65 to 72), blue; the Ia motif, PTRELA (residues 97 to 102), yellow; the GG motif (residues 125 to 126), orange; the TPGR (residues 145 to 148), pink; motif II, which harbors the Walker B DEAD motif (residues 169 to 172), red; and motif III, which harbors SAT (residues 200 to 202), green; the C-terminal motif IV, VIFCNTRR (residues 263 to 270), green; the conserved R motif, arginine-298, purple; the RGID motif in motif V (residues 321 to 324), magenta; and the HRIGRGGR (residues 345 to 352) of motif VI, cyan. (Reprinted from reference 25 with permission of the publisher.) (B) Ribbon representation of the structure of the full-length aIF4A from the archaebacterial M. jannaschii derived from the X-ray diffraction data at 1.8-Å resolution. The C-terminal end of the molecule is shown to the left of the figure. The color designation of domains I to IV is given below the figure. (Reprinted from reference 186 with permission of the publisher.) (C) Ribbon representation of the computer-derived structure of the eubacterial IF4A (W2) protein. Approximately 10% of the amino acid sequence of the N terminus of the protein is missing. The E. coli K-12 protein was patterned after the archaebacterial structure in panel B. The C-terminal end of the molecule is shown to the left of the figure. (D) Surface representation of aIF4A of M. jannaschii with the relative distribution of charged amino acid residues. Positively charged residues are shown in blue, and negatively charged residues are shown in red. The molecule is shown with the N-terminal domain to the left and the C-terminal domain to the right. The figure shows that the C-terminal end of the molecule and the bottom of the N-terminal end and the linker that joins the two domains are basic. This figure was generated by the Swiss Plot program.

The two domains that comprise the "dumbbell" structure of eIF4A occur in other helicases, as do the conserved helicase motifs. This is not surprising, since all these enzymes interact with ATP and with polynucleotides. The compact structure suggested by the work of Caruthers et al. (25) also occurs in other helicases whose structures have been determined. It has been noted, however, that the eukaryotic enzyme has a weak helicase activity which is strongly potentiated by other proteins of the eIF4F complex. These proteins may, in fact, stabilize a more compact structure of the eIF4A helicase.

The structures of several helicases, PcrA DNA helicase, hepatitis C virus RNA helicase, and Uvr DNA helicase, have been compared (25). These models have common features that may be relevant to the mechanism of eIF4A action. These include an extended single-stranded oligonucleotide binding region within domains 1 and 2. Motifs I and II of domain 1 of the molecule are juxtaposed with motifs V and VI of domain 2, as shown in Fig. 1A for the eIF4A structure. The orientation of the domains may differ somewhat in the different helicases.

The features of the eIF4A structure have been interpreted to suggest the following. (i) Several groups in the amino and carboxyl termini of the molecule which occur along the interface harbor the bound single-stranded oligonucleotide. Motif IV, which harbors the oligonucleotide-binding motif, the "QXXR" sequence, is specific to the DEA(D/H) box helicases and may directly interact with oligonucleotides. (ii) Several amino acid side chains in motifs V and VI contact the ATP-binding site and the DEAD motif of the amino-terminal domain. This arrangement suggests a role in coupling interactions with ATP or ADP to conformational changes in the protein (25). Mutations in the three arginines in motif VI of the mouse eIF4A reduce the ATPase activity of the protein, supporting this suggestion. (iii) The His-345 of eIF4A is conserved in this position in all helicases and forms a salt bridge with the last aspartic acid residue of the DEAD motif. The DNA helicases studied have altered residues at this (DEXX) site. It is clear that all helicases have structurally conserved motifs and regions that vary depending broadly on their substrate specificity.

The structure of aIF4A from Methanococcus jannaschii has been determined by X-ray diffraction at the 1.8-Å resolution (186). The structure harbors two domains and bears a striking resemblance to the "dumbbell" structure of the eukaryotic protein. The structure also has a relatively long linker joining the two domains (Fig. 1B).

The computer-derived structure of 90% of the eubacterial IF4A (W2) sequence (Fig. 1C) reveals that the structure indeed resembles the "dumbbell" feature of the eIF4A and aIF4A proteins. The C-terminal end of the eubacterial protein appears larger than the corresponding domain of the eIF4A. The linker between the two domains of the molecule and the relatively disordered carboxyl-terminal end suggests that this molecule might also be flexible as is the eIF4A structure.

Thus far, it is unclear whether helicases catalyze the unwinding of RNA and DNA structures through a "rolling" model which requires that the subunits of the proteins bind alternatively to single- and double-stranded RNA or DNA or by an "inchworm" model which requires binding of the helicase to single-stranded regions of RNA or DNA. The "rolling" model is best visualized if the helicases are dimers when complexed to their substrates. However, this is not always the case. Crystal structures of the RNA or DNA helicase complex may resolve this important mechanistic issue as well as the problem of where the RNA or DNA binds to the proteins. As a first approximation, the charge distribution of the archaebacterial aIF4A (W2) is such that relatively more basic groups occur on the surface of the C-terminal end. Some basic residues also occur on the interface on the N-terminal domain of the molecule (Fig. 1D). Thus, it is possible that the RNA substrates bind to the C-terminal surfaces and to the N-terminal junction. The RNA could unwind by the relative movement of the domains as proposed for the eukaryotic eIF4A.

View larger version (127K): [in this window] [in a new window]   FIG. 2. Alignment of amino acid sequence of the conserved domains of eIF4A and IF4A (W2) proteins from representative species of eubacteria and archaebacteria. Identical residues within the conserved domains are shown in blue, and highly conserved residues are shown in light blue and green. The sequences were aligned using the Swiss Plot program.

Study of the helicase activity of the eubacterial protein indicates that it acts as a helix-destabilizing protein (122). Unlike the eukaryotic protein, the IF4 (W2) protein unwinds double-stranded RNA past the 3 to 5 residues that are acted upon by the eukaryotic protein (100, 122) as a result of interactions with eIF4B. Thus, an eIF4B-like protein may not be required for the processive action of the eubacterial enzyme.

The sequence of the IF4A (W2) protein has a motif (HRIGRXXR) that is involved in binding RNA and in hydrolysis of ATP (41). Unlike the eukaryotic protein, however, the eubacterial protein does not hydrolyze ATP in the presence of several polynucleotides (122). Since the protein can stimulate the melting of an RNA duplex in the absence of added ATP, the IF4A (W2) protein from eubacteria may be a helix-destabilizing protein rather than an ATP-dependent RNA helicase as is the case for the eukaryotic eIF4A (100, 122).

Many different functions have been attributed to the IF4A (W2) protein. An elegant genetic study in which the host RNA polymerase was replaced by more efficient T7 polymerase indicated that the ß-galactosidase yield was markedly decreased but that the overexpression of the DEAD box, IF4A (W2), stabilized the yield of the mRNA and increased the amount of the encoded proteins. Several mRNA transcripts behaved in similar fashion, and it was suggested that the protein stabilizes mRNA structures (92). Binding of IF4A (W2) to ribosomes promotes initiation, which, in turn, could stabilize the mRNAs in question (122).

The eubacterial IF4A (W2) may exert certain interesting modes of regulating synthesis. The eubacterial protein is strongly induced under cold stress (100). A 15-fold increase in the activity is found upon a shift in temperature from 0 to 15°C (100). The protein induced by cold stress occurs bound to fully assembled 70S ribosomes.

The W2 protein stimulates synthesis programmed by templates that harbor secondary structures but is not required for the synthesis of polypeptides programmed by mRNAs devoid of secondary structures, e.g., poly(rU) (55). The N-terminal sequence of the W2 protein was found to be identical to that of the E. coli deaD gene product. The deaD gene product, in turn, has 54% identity and 87% sequence similarity to a eukaryotic factor, eIF4A, known to be involved in unwinding the secondary structure of mRNAs that impede the formation of initiation complexes. Polyvalent anti-eIF4A antibody cross-reacts with W2 (122).

To test if W2, in its putative role as an RNA helix-destabilizing factor, affects initiation per se, initiation was studied in a defined in vitro system. Two types of mRNA templates (each harboring the known determinants of initiation) were studied. On the one hand, synthetic mRNAs that bore little secondary structure and, on the other, the highly structured native mRNA were examined to assess what effect, if any, W2 might have on initiation. Initiation complexes using unstructured mRNAs are readily formed, and W2 does not appear to enhance their formation. Initiation complex formation using the structured MS2 RNA template, on the other hand, was markedly stimulated by the addition of W2 (Fig. 3). Since the unstructured mRNA harbored the SD region and a suitably spaced AUG start codon, it is unlikely that the W2 protein is involved in the recognition of the SD sequence. The protein could conceivably be involved in unwinding a secondary structure that masks the SD sequence, but this is unlikely because ribosomes can unmask stronger secondary structures which sequester SD sequences (162).

View larger version (24K): [in this window] [in a new window]   FIG. 3. (A) Secondary structure of the initiation site of the MS2 RNA coat protein cistron, sequence of the start site of the 200-bp unstructured mRNAs, and sequence of the start site of the unstructured 20-bp mRNA. (B) Ribosome-mRNA complexes with unstructured mRNAs or with the structured MS2 RNA. (A) W2 binds unstructured mRNAs (lanes 1 and 2, 20 and 200 bp of [32P]mRNA; lanes 4 and 5, 20 and 200 bp of [32P]mRNA plus 0.2 µg of W2). (B) Binding of 200 bp of unstructured [32P]mRNA to ribosomes with 0, 0.1, 0.2, and 0.4 µg of W2 (lanes 1 to 4). (C) Ternary initiation complexes, f[35S]Met-tRNA-ribosome-MS2 RNA, require W2. (D) Ternary initiation complexes, fMet[35S]Met-tRNA-ribosome-unstructured mRNA, do not require W2 (lane 1, no mRNA; lane 2, 200 bp of mRNA; lane 3, 200 bp of mRNA plus 0.1 µg of W2; lane 4, 20 bp of mRNA; lane 5, 20 bp of mRNA plus 0.1 µg of W2). See reference 122 for experimental details.

PEPTIDE BOND SYNTHESIS

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The crystal structure of the 50S subunit at the 2.4-Å resolution has revealed that the peptidyl transferase domain is protein free. The active site of this region has been cocrystallized with analogs of the CCA amino acids which act as inhibitors of peptide bond synthesis and a truncated aminoacyl-tRNA (141). These substrates bind to the A and P sites. The nucleotides that bind these substrate analogs are highly conserved within domain V (167). The residue that may be involved in catalysis is A2451, whose N-3 is about 3 Å from the phosphoramide oxygen of the bound peptide bond synthesis inhibitor and about 4 Å from the amide nitrogen of the peptide bond being formed (141). The unusual pK_a of A2451, which may be the essential catalytic base, derives in part from the fact it can form hydrogen bonds with G2447, which in turn interacts with a buried phosphate (135). Further, A2451 is essential for peptide bond synthesis (73). It has been suggested that the -amino group of the aminoacyl-tRNA attacks the carboxyl carbon that acylates the 3' hydroxyl group of the peptidyl-tRNA, resulting in the formation of an oxyanion intermediate at the carboxyl atom. The oxyanion dissociates to add an additional amino acid to the nascent protein chain esterified to the tRNA and bound to the A site. The deacyl-tRNA remains in the P site (141). Thus, it appears that the peptidyl transferase is a ribozyme and that the proteins L2, L3, and L4 help form the locus where A2451 can form the essential oxyanion (141). The region indeed catalyzes peptide bond synthesis as the reaction products are reported to cocrystallize at this site and result in a pre-translocation intermediate (173a). Mutants of A2451 are conditionally lethal but retain some peptidyl transferase activity in vitro. Further, mutants in G2447 are viable and have little or no effect or synthesis (154, 191a). The residual activity of the peptidyl transferase in the A2451 mutants might be compensated by perhaps another functional group in the rRNA. Alternatively, it is possible that the intermediate only enhances the rate of peptide bond synthesis. Substrate binding may be affected by the A2451 mutants, suggesting that this elegant mechanism remains to be proven.

Chemical footprinting has also identified nucleotides protected from chemical attack which are bound to the P, A, or E sites of the 23S rRNA within the sites depicted in the secondary structure of domain V (170, 182). Protection of the E site-bound tRNA is restricted to the 50S particle, whereas protection of the P and A site-bound tRNAs is scattered throughout other ribosomal domains on the 50S and 30S particles (73).

Mutations in G2585 of the 23S rRNA exhibit effects far away from the site where the peptidyl-tRNA substrate binds, suggesting that conformational changes affect the site of peptide bond formation. Indeed, a large number of mutations selected randomly on residues 2493 to 2606 in the central loop of domain V affect peptide bond formation (reviewed in reference 167). Thus, domain V, which binds the tRNA substrates, is also very likely to be involved in catalysis of peptide bond synthesis. The function of this center appears to be affected by distal rRNA elements.

Reconstitution studies indicate that the assembled peptidyl transferase of the 70S ribosome catalyzes peptide bond synthesis at a higher rate than does the peptidyl transferase of the 50S subunit but does not efficiently form peptide bonds with most amino acids (69). A soluble protein, EFP, stimulates peptide bond synthesis from several aminoacyl-tRNAs by the peptidyl transferase (52, 69) and restores this specificity. The general properties of the EFP protein are discussed below.

View larger version (89K): [in this window] [in a new window]   FIG. 4. Alignment of amino acid sequences of the EFP proteins from eubacterial, archaebacterial, and eukaryotic sources. The Lys-31 motif in the eubacterial sequences bears an unusual modification. Lys-54 of the eukaryotic sequences is modified by hypusine. Identical residues in the eukaryotic sequences are shown in yellow, sequences unique to eubacterial sequences are shown in blue, and conserved residues are shown in green.

The altered expression of eIF5A is correlated with several disease states. Thus, the expression of the protein is elevated in human immunodeficiency virus type 1-infected patients. Hypusine [N-(4-amino-2-hydroxybutyl)-L-lysine] formation is also significantly elevated in Ras oncogene-transfected mouse cells (31). In contrast, human carcinoma cells treated with alpha-2 interferon exhibit a reduced level of hypusine synthesis as well as an increase in the production of epidermal growth factor at the tumor cell surface, which decreases the proliferation of the tumor cells (24).

A requirement for eIF5A in cell proliferation is well established. Thus, deletion of the eIF5A gene in Saccharomyces cerevisiae reduces protein synthesis (174) and depletion of eIF5A in budding yeast arrests cells at the G₁ stage of growth and results in the production of nonviable spores (104).

The genes encoding these proteins have been sequenced. The eIF5A sequences have the strongest region of homology near the site of hypusine modification that is essential to the function of these proteins (151). Interestingly, eIF5A is the only protein that has been reported to be modified by hypusine. S. cerevisiae has two essential genes that encode eIF5A; they exhibit 26% identity in a 108-amino-acid sequence near Lys-54 of the eIF5A, where the hypusine modification occurs. The homologous region in the E. coli EFP near Lys-31 is modified by an unusual group of 148 ± 1 Da (molecular mass measured by mass spectrometric analysis). This group differs from hypusine but may also be essential for the activity and stability of the native protein (H. Aoki, M. Yaguchi, D. Watson, M. Pearson, and M. C. Ganoza, unpublished observations). The Lys-31 region has a special motif, VKPGK, that is conserved in many different bacteria and in the aIF5A of the archaebacterial M. jannaschii.

The EFP proteins have three highly conserved motifs. It is clear that a significant proportion of the total amino acids are similar in the eukaryotic and prokaryotic proteins (Fig. 4). Domain I, containing the longest contiguous stretch of conserved amino acids, harbors the Lys-31 modified residue in the E. coli EFP protein. Each domain appears to have highly conserved amino acid residues unique to eubacterial EFP proteins (blue in Fig. 4). The eukaryotic sequences also have unique motifs (yellow) which are interspaced between the other conserved domains of the proteins. The structure of the eubacterial Staphylococcus aureus proteins derived by X-ray diffraction at the 1.9-Å resolution (T. E. Benson, M. C. McCroskey, J. I. Cialdella, G. Choi, and J. D. Pearson, Proc. Second Symp. Struct. Aspects. Protein Synthesis, p. 15, 2000) has three main domains, whereas the archaeal and eukaryotic proteins have two such domains (108). The presence of this third domain in the eubacterial sequences creates a protein fold that suggests that EFP might function as a tRNA mimic (Benson et al., Abstract) This difference, particularly the absence of the third domain, may be important in eukaryotic mRNA transport and/or stabilization.

Several eukaryotic genes that encode eIF5A encode fewer than 190 residues (Fig. 4). This in itself explains the gaps observed at, for example, the C terminus at residues 124 to 136. The eubacterial EFP sequences have a well-conserved C-terminal domain that does not occur in archaebacteria or eukaryotes, suggesting a functional difference in these proteins. Nevertheless, it is clear that many amino acids are similar in the eukaryotic and eubacterial proteins. Amino acids within similar domains are divergent between the bacterial and eukaryotic sequences, but there seems to be a pattern of conservation of discrete, perhaps critical, amino acids in all these proteins. Study of the phylogenetic relationships between the prokaryotic and eukaryotic sequences suggests that these proteins coevolved with the organism in question, emphasizing their essential character. Thus, the genes encoding the eIF5A and EFP proteins from different species are highly conserved, but more evidence is needed to establish whether they represent orthologous functions.

View larger version (34K): [in this window] [in a new window]   FIG. 5. (A) Ribbon diagram and representative sketch of the structure of aIF5A from M. jannaschii. The ribbon structure was derived from X-ray diffraction patterns at 1.8-Å resolution. The sketch (A) shows the eleven ß sheets in two domains of the molecule joined by flexible links that contain the Lys-54 site of hypusine modification. (Reprinted from reference 108 with permission of the publisher.) (B) Ribbon structure of the eIF5A, showing the two domains of the molecule linked by the flexible linker that harbors the hypusine residue. The molecule bears opposite charges in the C- and N-terminal ends.

Ribosome reconstitution experiments showed that L16, or its first 47-amino-acid N-terminal fragment, was required for the EFP-mediated peptide bond synthesis whereas L11, L15, and L7/L12 were not, suggesting that EFP operates at a different ribosomal site than used by most other translation factors (64) (Fig. 6).

View larger version (19K): [in this window] [in a new window]   FIG. 6. (A) Requirement for EFP and L16 in peptide bond synthesis by reconstituted 70S ribosomes (, no EFP; , with 2 µg of EFP); the cores were reconstituted with L16 (), and then EFP was added (•). (B) Synthesis of dipeptides from fMet-tRNA and 5' CCA amino acids; association constants in the presence or absence of EFP as a function of the length of the amino acid side chain. Data are from references 52 and 64. The sizes of the amino acid side chains are derived from X-ray diffraction data.

Previous studies have demonstrated that streptomycin markedly inhibits the EFP-mediated synthesis of peptide bonds (5). The footprints observed on contact of EFP with the 30S subunit are near one of the regions where streptomycin binds to the 16S rRNA of the particle. This is within the decoding center of the ribosome, which is engaged in the mRNA-tRNA, codon-anticodon interactions. The bases that are enhanced by interactions of EFP with the 30S subunit are in the vicinity of the S1 and S5 proteins, which also bind within the decoding center of the ribosome.

EFP also interacts with domain II of the 50S subunit (H. Aoki, J. Lang, A. Lang, and M. C. Ganoza, submitted for publication). Domain II also has binding sites for both EFTu and EFG. The stimulation of peptide bond synthesis by EFP does not require EFG. However, EFP may influence the activity of EFTu. As the incoming aminoacyl-tRNA-GTP-EFTu complex first approaches the A site, it is too far away from the aminoacyl-tRNA in the P site to form a peptide bond (27, 128). Consequently, EFTu accommodates the incoming aminoacyl-tRNA to a position where a peptide bond can be formed. The hydrolysis of GTP that results on binding of EFTu-GTP-aminoacyl-tRNA to the ribosome is essential for binding the aminoacyl-tRNA substrates to the peptidyl transferase active center. EFP could accelerate the ribosomal GTPase activity of EFTu so as to promote the approximation of the aminoacyl-tRNAs into the peptidyl transferase active site.

Interactions between EFP and the 23S rRNA on domain V are of especial interest because EFP has been predicted to enhance elongation by interacting with the peptidyl transferase center. Indeed, the protection analysis demonstrates that EFP is in contact with the A site of domain V of the 23S rRNA. In binding directly to the A site, EFP could directly increase the affinity of the incoming aminoacyl-tRNA (22, 181). The binding of EFP to the A site also enhances the reactivity of bases in the 50S E site (Aoki et al., submitted). These interactions would increase the rate of efficiency of translation, as EFP is known to do (52).

Since the EFP-binding domain is very near the site of EFTu and EFG binding on both the 30S and 50S subunits (Aoki et al., submitted), it is likely that the binding site may span both subunits. The oligonucleotide-binding domain of the EFP protein could bind the aminoacyl-tRNA while the hydrophobic residues near the central loop of the molecule might interact with the peptidyl transferase center.

The contacts observed on the 30S at G577 and A579 are near the 530 loop that forms part of the binding site for EFTu (156) as well as one of the streptomycin-binding sites (180). The proposed elongated structure of the protein could facilitate interactions with both subunits that may enable the proper approximation of the tRNA substrates required for peptide bond synthesis. We suggest that EFP functions by interacting with the A site of the 30S subunit as well as with the A site of the peptide bond-forming center of the large ribosomal subunit.

EFP stimulates the initial rate of translation programmed by certain synthetic templates when synthesis is initiated by N-blocked aminoacyl-tRNAs (52). EFP could function in the formation of the first peptide bond and, more probably, could promote the synthesis of certain dipeptides during subsequent elongation. The later idea is consistent with the fact that EFP occurs bound to 70S ribosomes as well as to polyribosomes. EFP was first isolated as a factor that stimulated the translation of a natural mRNA [in contrast to that of simple, synthetic messages such as poly(rU)]. Subsequently, purified EFP was shown to stimulate peptide bond formation by the ribosome in the puromycin reaction model when the concentration of puromycin was decreased (68). Several aminoacyl 5' CCA fragments were synthesized and tested as acceptors in peptide bond formation, using fMet-tRNA, bound to 70S ribosomes, as donor. These studies showed that the intrinsic ability of the ribosome to catalyze the synthesis of certain dipeptides was extremely poor while other dipeptides were efficiently synthesized. Notably, EFP stimulated dipeptide synthesis in the cases of those aminoacyl acceptors that are poor substrates for the intrinsic peptidyl transferase. For example, fMet-Gly synthesis on ribosomes was stimulated 50- to 60-fold by the addition of EFP whereas fMet-Phe synthesis was barely affected. Significant stimulation of the synthesis of fMet-Ala, fMet-Leu, fMet-Val, fMet-Lys, and fMet-Met also occurred on addition of the EFP protein (52, 69) (Fig. 6). These results suggest that the side chain of the aminoacyl acceptors is essential for the stimulation of synthesis and the amino acid charge is not necessarily the feature that is recognized by the EFP protein. As a first approximation, the ability of EFP to stimulate peptide bond synthesis correlates inversely with the relative size of the amino acid side chain of the aminoacyl acceptor (52).

Molecular modeling of the peptide bond synthesis reaction between puromycin and an analogue of the peptidyl-tRNA indicates that the bulky dimethoxy benzene ring in the puromycin structure allows the molecule to bend in a U shape (158). In such a conformation, puromycin makes a perfect steric fit with the NH₂ group of the peptidyl-tRNA, which facilitates the formation of a peptide bond. Alterations of the puromycin structure that distort the U shape of the molecule, such as the substitution of a Gly residue at this position, drastically reduce the reaction rate (reviewed in reference 189). This suggests that the side chains of the amino acids are important to the reaction rate and prompts the suggestion that the EFP protein may directly or indirectly promote the approximation of aminoacyl-tRNAs bearing small amino acid side chains into the active center of the peptidyl transferase. The fact that little, if any, peptide bond synthesis occurs with acceptors that have small side chains in the absence of EFP could explain the requirement for this protein in vivo, where initiation of many protein-encoding reading frames commonly begins with such amino acids.

The observation that EFP, a nonribosomal protein, can restore the ability of the peptidyl transferase to polymerize amino acids that are otherwise poor substrates points to the structural and/or dynamic complexity of this reaction. IF2 and EFTu are known to bind to the 3' terminus of the initiator or aminoacyl-tRNA, respectively, making it unlikely that EFP also interacts directly with the tRNA 3' terminus. On the other hand, elements regulating the active center of the peptidyl transferase may span a larger portion of the ribosome, as suggested by mutagenesis studies (73, 167). Reconstitution studies show that several 50S subunit proteins (apart from L2, L3, and L4) significantly stimulate peptide bond synthesis (45, 175). Experiments involving antibiotics that interact with peptidyl transferase indicate that the active center can exist in a number of functional conformers (167). Given such plasticity, we suggest that EFP may act to restructure the active site in a way that promotes interaction between the peptidyl transferase and its less favorable aminoacyl-tRNA substrates. Thus, EFP may be viewed as a critical regulatory molecule for peptide bond formation during chain elongation.

PROTEIN CHAIN ELONGATION

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Protein chain elongation entails a cycle consisting of alignment of aminoacyl-tRNAs by their specific codons in mRNA (decoding), peptide bond synthesis, and movement of mRNA relative to the ribosome (translocation). The eubacterial proteins, EFTu, EFTs, and EFG (eEF1, eEF1ß, and eEF2 of eukaryotes), facilitate these processes on ribosomes. Reviews are available (22, 36, 116, 144, 146, 202).

The movement (translocation) of the nascent peptidyl-tRNA from the A site to the P site is inherent to the ribosome (183) but is catalyzed by elongation factor EFG and GTP (183). This movement is substantial, involving changes in the order of 50 Å at the elbow of the tRNA during each step of the elongation cycle. The tRNA substrates must remain bound to the ribosome during this process in order to maintain the reading frame. The energy to drive this mechanism comes largely from hydrolysis of GTP that results from contact of the EFG-GTP complex with the ribosome (73, 165, 202). Presumably, GTP hydrolysis allows the release of EFG from ribosomes (73, 165, 202). The results of RNase protection assays and site-directed mRNA cross-linking data, before and after addition of EFG, indicate that translocation moves the mRNA by 3 nucleotides (13, 73, 128, 165, 202).

Chemical footprinting revealed that tRNAs bound in various partial elongation reactions protect specific bases on rRNA from chemical probes (13, 73, 165, 202). The principal observations are that the 3' and 5' ends of the tRNAs are in different states in the two ribosomal subunits, such that a single tRNA can be found on the 30S A site and on the 50S P site. These positions are termed the "hybrid" sites of the particle (128). These footprinting, chemical protection experiments of the translation reaction programmed by poly(rU) revealed that the tRNA, particularly the 3' terminus of the molecule, undergoes considerable movement during each step of the elongation cycle (128). The EFTu-GTP-aminoacyl-tRNA complex does not bind to the ribosomal A site directly (128, 164). The tRNA footprints suggest that this initial contact occurs with a hybrid A/T site in which the anticodon of the tRNA and the mRNA on the 30S subunit remain fixed and that the 3' terminus of the tRNA does not bind directly to the A site but to the A/T site of the 50S particle. Following GTP hydrolysis, the aminoacyl-tRNA is moved from the A/T site into the ribosomal A site (A/A hybrid site equivalent). Peptide bond synthesis follows after ejection of the EFTu-GDP complex from the ribosome, leaving deacyl-tRNA in the P site and the peptidyl-tRNA in the A site. The EFG-GTP complex binds at this stage of the process. The hybrid site model and the site of EFTu and EFG action are shown in Fig. 7. We also posit a possible site of action for EFP and RbbA in this scheme. The action of EFG may be preceded by movement of the deacyl-tRNA from the P site to the E site (163). This reaction is stimulated strongly by the RbbA protein, but it is not certain whether the RbbA protein ejects tRNAs only from the E site after peptide bond synthesis or from the P site also (57). In the former case, EFG may mediate translocation both of the deacyl-tRNA from the P site to the E site and of the peptidyl-tRNA from the A site to the P site.

View larger version (20K): [in this window] [in a new window]   FIG. 7. Requirement for RbbA and EFP in MS2 programmed synthesis reconstituted from homogeneous proteins. Data are from reference 52.

There is evidence that EFTu enters the ribosome at a site different from the A site that binds the aminoacyl-tRNA. EFTu binds to ribosomes that already carry an aminoacyl-tRNA (114, 164). There is also EM evidence that the binding site for EFTu differs from that of other ribosomal sites (114). The crystal structure of the 70S ribosome has revealed the position of the three tRNA-binding sites and the contiguous site for binding of elongation factors (27). We refer to this site as the T site. EFP acts in concert with the peptidyl transferase to bring about peptide bond synthesis with a variety of aminoacyl-tRNA acceptors. Peptide bond formation results in a large rearrangement of the ribosome, as predicted by the hybrid model and by the data obtained with the fluorescent energy probes. We propose an active mechanism, in which the W (RbbA) protein is required for the ejection of the tRNAs that must occur after peptide bond synthesis. Also, EFTu may be actively removed from the ribosome prior to this step. The binding of EFG could bring about translocation, and the hydrolysis of GTP could occur as a result of the EFG-ribosome interaction. The similarity in the tertiary structure of EFTu-aminoacyl-tRNA and EFG (both have domains that resemble tRNA [202]) may be important to bind the factors to the T site. The T site is probably near the L7/L12 protein dimers that act with EFTu and EFG on the ribosome. The ribosome itself could bring about the translocation reaction on interaction with EFG (183, 202).

The translocation mediated by EFG occurs by contact of the G domain of the molecule with 23S rRNA bases, which include the -sarcin loop on domain VI. This contact results in molecular rearrangements of EFG which apparently change the conformation of the ribosome to an unlocked state. The resulting loosening of the tRNA contacts may be coupled to movement in regions of the ribosome, which could, in turn, move the tRNA relative to the mRNA (202). A disruption of the contacts made by domain 4 of EFG would reestablish the locked state of the ribosome, and EFG would be released (202).

It is imperative that the translocation reaction overcome the energy barrier required to move the substrates while maintaining the codon-anticodon interaction. The footprinting data of Moazed and Noller (128) suggest that the peptidyl-tRNA should remain anchored on the 50S particle. This ensures that the peptidyl-tRNA remains stably bound to the ribosome. Recent data suggest that the 30S subunit is able to effect translocation in the presence of EFG (101). One possibility is that codon-anticodon interactions occur in a subsite of the 30S subunit that is protected from factor interactions and is regulated by the P-site-bound substrates. This, in turn, suggests that part of the 30S subunit may move relative to the 50S particle. The head and body of the 30S subunit are held together by a single-stranded RNA that could enable such movement. Three-dimensional cryo-EM maps of the E. coli ribosomes have revealed that the two subunits rotate relative to each other on binding of EFG to the ribosome. This effects a ratchet-like movement which opens the mRNA channel. After GTP hydrolysis, this rotation is followed by the advance of the mRNA-tRNA₂ complex. The original position of the subunits is reestablished after EFG-GDP is released from the ribosomes (46). This model is compatible with the footprinting data of Moazed and Noller (128) and with the model first proposed by Woese (203) that the structure of the anticodon stem-loop of tRNAs permits a reciprocating ratchet-like movement of the tRNA-mRNA complex (46). One of the proteins, required for reconstitution of synthesis, the RbbA protein, is a ribosome-dependent ATPase (105, 106, 107). The RbbA protein has features in common with EF3 of yeast (29, 30, 110). The possible mode of action of these proteins in chain elongation is discussed below.

The gene encoding EF3 in S. cerevisiae, yef3, was cloned from a genomic library (171). The gene is present in a single copy, but recent analysis of the genomic sequence database of S. cerevisiae has shown that a second gene may encode EF3 (K. Chakraburtty, personal communication). This second gene would code for a protein with high homology but no identity to EF3. EF3 has a molecular mass of 115.7 kDa.

EF3 was determined to be a ribosome-dependent ATPase, using yeast ribosomes (30, 127). Analysis of the amino acid sequence of EF3 revealed an ATP-/GTP-binding motif, GX₄GK(S/T) (3, 41, 103). A second motif present in EF3 is found only among ATP-binding proteins (198). Together, the two sequences constitute what is called the ATP-binding cassette (ABC) (3). ABC proteins make up the largest known orthologous group of proteins, many of which are membrane transporters.

EF3-homologous proteins have been found in the fungi Candida albicans and Pneumocystis carinii (42, 131, 204). Immunoblot analysis of extracts from higher eukaryotic species using anti-EF3 antibody have not revealed any immunologically cross-reacting proteins. However, ribosomes of higher eukaryotes show significant ATPase activity, which may play a similar role in protein synthesis to that of EF3 (44, 109, 110).

The role of EF3 in protein synthesis seems to be to facilitate the binding of aminoacyl-tRNA to the A site of the ribosome. In yeast, EF1 is the homologue of the eubacterial EFTu. When EF1 is present at catalytic amounts, EF3 stimulates the EF1-mediated binding of aminoacyl-tRNA to the A site (30, 103, 195). It also influences the binding of deacyl-tRNA to the E site and stimulates the exchange of labeled deacyl-tRNA bound to the E site of yeast ribosomes (29) independent of EF1 (EFTu).

Reconstitution of synthesis using all of the homogeneous initiation, elongation, and termination factors has revealed that they do not suffice to promote the synthesis of peptides