![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
Department of Molecular Biology, Aarhus University, Aarhus, Denmark
SUMMARY INTRODUCTION BACTERIAL TRANSLATION INITIATION COMPONENTS INVOLVED IN TRANSLATION INITIATION Ribosome Stabilization of the ribosomal structure. Small ribosomal subunit Large ribosomal subunit. mRNA Initiator tRNA Translation Initiation Factors Initiation factor IF1. Initiation factor IF2. Initiation factor IF3. REGULATION OF TRANSLATION INITIATION CONCLUDING REMARKS ACKNOWLEDGMENTS REFERENCES
| SUMMARY |
|---|
 Top NextReferences |
|---|
| INTRODUCTION |
|---|
|
TopPreviousNextReferences |
|---|
The ribosome is composed of a large and a small subunit, which are assembled on the translation initiation region (TIR) of the mRNA during the initiation phase of translation. In the following elongation phase, the mRNA is decoded as it slides through the ribosome and a polypeptide chain is synthesized. Elongation continues until the ribosome encounters a stop codon on the mRNA and the process enters the termination phase of protein synthesis. Newly synthesized protein is released from the ribosome. In the final ribosome recycling phase, the ribosomal subunits dissociate and the mRNA is released. Each phase is regulated by a number of different factors. Reviews of the phases are available (52, 208).
Although the main events of the translation process are universally conserved, major differences in the detailed mechanism of each phase exist. Bacterial translation involves relatively few factors, in contrast to the more complex process in eukaryotes (164). Here we focus on translation initiation in bacteria. Although parallels are drawn to the archaeal and eukaryotic systems where relevant, everything described throughout the rest of this review concerns the bacterial system unless otherwise stated. Archaeal and eukaryotic processes of translation initiation are reviewed elsewhere (7, 44, 177).
| BACTERIAL TRANSLATION INITIATION |
|---|
|
TopPreviousNextReferences |
|---|
The rate at which ribosomes assemble on the mRNA is on the order of seconds, although it is specific for each mRNA. The ribosomes subsequently translate the mRNA at a rate of approximately 12 amino acids per second (89). The ribosome, the aminoacylated and formylated initiator tRNA (fMet-tRNAfMet), mRNA, and the three protein factors, initiation factor IF1, initiation factor IF2, and initiation factor IF3, are involved in the translation initiation phase (Fig. 1).
|
Following subunit dissociation, IF2, mRNA, and fMet-tRNAfMet associate with the 30S ribosomal subunit in an unknown and possibly random order. The Shine-Dalgamo (SD) sequence of canonical mRNAs interacts with the anti-SD sequence of the 16S rRNA (258), and the initiation codon is adjusted in the P-site of the ribosome. The initiation factors (especially IF3) seem to be responsible for this adjustment (101). The initiator tRNA is positioned in the P-site of the 30S ribosomal subunit in three steps that are designated codon-independent binding, codon-dependent binding, and fMet-tRNAfMet adjustment (reference 231 and references cited therein). All three steps are probably promoted by IF2, which interacts with fMet-tRNAfMet on the ribosome. Furthermore, IF3 stabilizes the binding of fMet-tRNAfMet to the ribosomal P-site and confers proofreading capability by destabilization of a mismatched codon-anticodon interaction (60).
The 30S preinitiation complex consists of the 30S ribosomal subunit, the three initiation factors, and mRNA in a standby position where fMet-tRNAfMet is bound in a codon-independent manner. This relatively unstable complex undergoes a rate-limiting conformational change that promotes the codon-anticodon interaction and forms the more stable 30S initiation complex (60, 174). Initiation factors IF1 and IF3 are ejected, while IF2 stimulates association of the 50S ribosomal subunit to the complex. Initiator fMet-tRNAfMet is adjusted to the correct position in the P-site, and IF2 is released from the complex. During this process, GTP bound to IF2 is hydrolyzed to GDP and Pi. The newly formed 70S initiation complex holding fMet-tRNAfMet as a substrate for the peptidyltransferase center of the 50S ribosomal subunit is ready to enter the elongation phase of translation.
Reviews of different aspects of bacterial translation initiation can be found in references 12, 61, 63, 208, and 210.
| COMPONENTS INVOLVED IN TRANSLATION INITIATION |
|---|
|
TopPreviousNextReferences |
|---|
The first visualizations of ribosomal structures were done by electron microscopy and identified a particle subdivided into two subunits of unequal sizes (82). The first determination of shapes came in the early 1970s (99). Today the resolution of structures of ribosomal particles made by cryoelectron microscopy has increased to 7 Å for the best reconstitutions (49, 247). Atomic resolution structures of ribosomes can, however, be obtained only by X-ray crystallography. High-resolution structures of the intact 70S ribosome (239, 256), the 50S ribosomal subunit (4, 72), and the 30S ribosomal subunit (195, 249) exist. Moreover, the structures of several ribosomal complexes, including different factors, RNAs, and antibiotics, have recently been solved. Only those relevant for translation initiation are discussed here. Reviews describing other complexes may be found elsewhere (5, 180, 219, 253, 259).
RNA-protein interactions occur mainly via the sugar-phosphate backbone of the RNA. Thus, the ribosomal proteins recognize the unique shape of the rRNA rather than the bases, and the interactions are therefore sequence unspecific (146). Many of the ribosomal proteins have nonglobular extensions that are highly conserved in sequence. These tails penetrate into the ribosome and fill the gaps between RNA helices. In isolation, these protein tails, which contain approximately 26% arginine and lysine residues, look like random coils that probably only assume the conformation they have on the ribosome when bound (4, 219).
Prior to peptide bond formation, an aminoacyl-tRNA is bound in the ribosomal A-site, a peptidyl-tRNA is bound in the P-site, and a deacylated tRNA, which is ready for ejection from the ribosome, is bound to the E-site. Translation moves the tRNA from the A-site through the P- and E-sites before they exit the ribosome again, with the exception of the initiator tRNA, which binds directly to the P-site. The small ribosomal subunit contains the decoding center, where the triplet codons of the mRNA are base-paired with the anticodons of the cognate tRNA, and hence determines the sequence of amino acids to be incorporated in the synthesized protein. The large subunit contains the peptidyltransferase center and is thus the catalytic unit.
|
E. coli has 41 different tRNA species with different anticodons. The ribosome must select the tRNA with an anticodon complementary to the codon of the mRNA. This is termed the cognate tRNA. The error rate of tRNA selection in the decoding process is 10–3 to 10–4. Pre-steady-state kinetics showed that in addition to having lower dissociation rates from the ribosome, cognate tRNAs have higher rates of elongation factor EF1A GTPase activation and accommodation (movement of the aminoacyl end of tRNA into the A-site of the 50S ribosomal subunit) than do near-cognate tRNAs. Based on this result, it was proposed that binding of cognate tRNA induces a conformational change of the ribosome (162). Crystal structures of the 30S subunit complexed with mRNA and cognate tRNA in the A-site reveal an induced fit mechanism. Bases A1492 and A1493 of the 16S rRNA flip out of helix 44 and interact with the correctly base-paired codon-anticodon helix in an A-minor motif type interaction. A1492 and A1493 interact with the minor groove of a correctly paired codon-anticodon but not with incorrectly paired codons and anticodons. Binding of cognate tRNA also causes a flip of G530 of the 16S rRNA from syn to anti conformation (156). Bases A1492 and A1493 interact with the first and second base pairs of the codon-anticodon helix, respectively. G530 interacts with the second position of the anticodon as well as the third position of the codon. The result is a strictly monitored codon-anticodon interaction in the first two positions, whereas the ribosome is able to tolerate noncanonical base pairs at the third position (156). During decoding, the flipping of the 30S subunit bases A1492, A1493, and G530 translates to other parts of the subunit and leaves it in a closed conformation in which the shoulder and head domains are rotated toward the subunit center, compared to the more open structure when the A-site is unoccupied. The transition to the more closed state is unfavorable for near-cognate tRNAs (158). However, X-ray crystal structures of the intact 70S E. coli ribosome reveal that the closing of the head domain of the 30S subunit is connected to formation of the intact 70S ribosome and not to decoding. These structures do, however, indicate a movement of the small-subunit body connected with decoding (239). Thus, ribosomes play an active role in tRNA selection by direct recognition of the codon-anticodon base pairing. The extent to which conformational changes occur needs further investigation. An extensive review of decoding is available (157).
During the peptidyl transfer reaction, the 
-amino group of A-site tRNA attacks the carbonyl group of the P-site peptidyl group, which is linked to the tRNA via an ester bond. The reaction proceeds via a tetrahedral intermediate to form a peptidyl bond. The reaction occurs in the PTC, where the amino acid of the A-site tRNA has been properly positioned relative to the nascent peptide chain bound to the P-site tRNA. Peptide bond formation is then catalyzed. The PTC was identified by soaking crystals of Haloarcula marismortui 50S subunits with a transition state analogue, the so-called Yarus inhibitor. Surprisingly, the subunit is completely devoid of protein within 18 Å of the PTC, and the ribosome is thus a ribozyme (145). It is beyond the scope of this review to go into detail about the mechanism of the peptidyl transferase reaction. Reviews can be found elsewhere (55, 219, 248).
After the peptidyltransferase reaction has occurred, a deacylated tRNA is left in the P-site and the A-site tRNA is covalently bound to a peptide chain extended by one residue. For elongation to proceed, the P-site tRNA has to move into the E-site ready for ejection from the ribosome and the A-site peptidyl-tRNA has to move to the P-site. The E-site is specific for deacylated tRNAs (196). The movement of tRNAs must be accompanied by a precise movement of the mRNA to preserve the reading frame. A review of the translocation process can be found in reference 150.
In the following sections, the binding of mRNA, tRNA, and the translation initiation factors to the ribosome is described, along with the properties of each individual component.
TIRs on mRNAs are not only characterized by the presence of a putative initiation codon. Additional elements are necessary to promote correct initiation and avoid initiation from, for instance, AUG codons encoding internal methionines of a protein. Upstream from the initiation codon is the 5' untranslated region (5' UTR). This region contains the SD sequence, which can undergo base-pairing to the 3' of the 16S rRNA of the 30S ribosomal subunit (207). A direct consequence of the SD interaction is the adjustment of the initiation codon to the ribosomal P-site, where it interacts with fMet-tRNAfMet. E. coli mRNAs typically have the SD sequence GGAGG located 7 ± 2 nucleotides upstream from the initiation codon, which can be AUG, GUG or UUG (123). The exceptional AUU initiation codon has been observed in infC (encoding IF3) and pcnB [encoding E. coli poly(A) polymerase] (10). Initiation codons in E. coli occur at a frequency of 90, 8, and 1% for AUG, GUG, and UUG, respectively (198).
Ribosomal protein S1 interacts with a pyrimidine-rich region 5' to the SD region on mRNAs. This pyrimidine-rich region acts as a ribosome recognition site (15). A direct interaction has been confirmed by cryoelectron microscopy (EM) studies of S1 on the 30S ribosomal subunit with a bound mRNA (200).
A region downstream from the initiation codon of several mRNAs was found to show complementarity to bases 1469 to 1483 within helix 44 of the 16S rRNA. This region was named the downstream box (DB), and there appeared to be a correlation between the degree of complementarity to the 16S rRNA and the translational efficiency of the mRNA. A mechanism similar to the SD-ASD interaction was proposed (212). The presence of a DB-anti-DB (ADB) interaction has been a matter of debate, and a recent review concludes that there is no biochemical or genetic evidence in support of the proposed role of the DB-ADB interaction in ribosomal recruitment of mRNA (133, 134). This is supported by the crystal structure of the T. thermophilus 30S ribosomal subunit, which reveals that the shoulder of the subunit is located between the putative DB of the mRNA and the proposed anti-DB of the 16S rRNA (249).
Bacterial mRNAs are either canonical or leaderless, although the latter is rare, with no more than 
40 identified cases in bacteria (133). Canonical mRNAs contain the 5' UTR elements described above, whereas leaderless mRNAs start at, or a few nucleotides 5' upstream of, the initiation codon. A clear mechanism for binding of canonical mRNAs and the order in which mRNA and fMet-tRNAfMet enter the 30S ribosomal subunit has not been established (Fig. 3). Initiation factors do not affect the SD-ASD interaction or the association between the 30S ribosomal subunit and canonical mRNA (61). However, site-directed cross-linking experiments have shown that mRNA is partially relocated on the 30S ribosomal subunit from a "standby site" to a site closer to the P-site in a process influenced by IF1, IF2, and especially IF3 (101).
|
Biochemical experiments and immuno- as well as cryo-EM studies establish a model in which the mRNA wraps around the neck of the 30S ribosomal subunit, with its 5' end on the platform side and its 3' end near the shoulder (50, 206). Structural data for the interaction between mRNA and the ribosome are now available from X-ray crystallographic studies (156, 258). The new data confirm the general features of the previous models. Interaction between the ASD and SD sequences is located at a large cleft between the head and the back of the platform on the 30S ribosomal subunit (Fig. 4). The mRNA wraps around the 30S ribosomal subunit while it passes through the up- and downstream tunnels (38). A latch-like closure between the head and body on activation of the subunit forms the tunnels (195). Early studies indicated that binding of mRNA to the ribosome through the SD interaction melts the mRNA secondary structure in the TIR of the mRNA (161). The mRNA is probably unwound by mRNA helicases before entering the downstream tunnel, since an RNA helix is too large to pass.
|
Two genes in the E. coli genome encode tRNAfMet (83). The major fraction of cellular initiator tRNA (tRNAf1Met) is encoded by the metZ gene. Three identical copies of the gene occur in tandem repeats within the operon known as the metZ operon (90). A relatively small fraction of tRNAfMet (tRNAf2Met) is encoded by the metY gene, located at the beginning of the nusA/infB operon (83). The presence of adenosine instead of 7-methylguanosine at position 46 is the only difference between tRNAf2Met and tRNAf1Met (84).
Initiator tRNAs bind directly to the P-site of the small subunit of the ribosome, whereas elongator tRNAs enter the ribosome at the A-site and subsequently translocate to the P-site. Binding of tRNAs to the P-site and the A-site is controlled by the initiation and elongation factors, respectively. Therefore, the initiator tRNAs have structural features that are recognized by initiation factors and discriminated against by elongation factors. The initiator tRNA determinants are located in the anticodon stem, the acceptor stem, and the dihydrouridine (D)-stem (Fig. 5). The significant features include (i) absence of a Watson-Crick base pair between positions 1 and 72 in the acceptor stem, (ii) three conserved consecutive GC base pairs in the anticodon stem, and (iii) the presence of a purine-11-pyrimidine-24 in contrast to the pyrimidine-11-purine-24 base pair found in other tRNAs (235). The GC pairs make the anticodon loop less flexible compared to the anticodon loop in elongator tRNAs and are important for targeting the initiator tRNA to the ribosomal P-site (199, 201).
|
|
-amino group of the methionine of Met-tRNAfMet. The most important determinant for formylation of the initiator tRNA is the absence of a 1:72 base pair. This provides the tRNA with a 5-nucleotide single-stranded 3' end of the acceptor arm just long enough to reach the active site of MTF (197). Both the initiator tRNA and MTF undergo structural changes in an induced-fit mechanism upon binding (122, 181). The regions on Met-tRNAfMet that interact with MTF are shown in Fig. 6. Peptidyl-tRNA hydrolase (PTH) is a 21-kDa monomeric enzyme that recycles all N-blocked aminoacyl-tRNA molecules accumulating from abortive translation. Initiator fMet-RNAfMet is not a substrate for PTH. The presence of a 5'-terminal phosphate at the end of a fully base-paired acceptor stem is crucial for hydrolase activity (197). Therefore, the absence of a base pair between nucleotides 1 and 72 protects fMet-RNAfMet against hydrolytic cleavage by PTH. The amino acid attached to tRNAfMet is also important since fMet-tRNAfMet is completely resistant to hydrolysis by PTH whereas fGln-tRNAfMet is not (228).
In most cases, the formyl group and the methionine residue are removed posttranslationally or even as the nascent polypeptide chain emerges from the ribosomal exit tunnel (94). Formylation of Met-tRNAfMet is important for protein synthesis in E. coli. Mutant initiator tRNAs defective in formylation are extremely poor in initiation of protein synthesis, and a strain of E. coli carrying disruptions in the fmt gene encoding MTF has severe growth defects (69, 126, 236). Formylation is, however, not necessary for translation initiation in all bacteria, as exemplified by Pseudosomonas aeruginosa (144).
Formylation favors selection of the fMet-tRNAfMet by IF2 (223), and blocks binding to the elongation factor EF1A and thus the function as elongator tRNA (71, 147, 202). The nature of the amino acid attached to the tRNA is less important for IF2 binding than is formylation. Hence, IF2 is able to bind to fVal-tRNA and fGln-tRNA but not to the unformylated tRNAs (251). However, moderate overexpression of IF2 leads to translation initiation without formylation of Met-tRNAfMet, and IF2 stimulates the binding of unformylated Met-tRNAfMet to 30S ribosomal subunits in vitro (68, 250).
IF2 protects the ester bond in fMet-tRNAfMet against spontaneous hydrolysis but does not protect the unformylated Met-tRNAfMet (167). Discrimination by IF2 against the unformylated Met-tRNAfMet has also been demonstrated by footprinting experiments performed with Met-tRNAfMet and fMet-tRNAfMet in the presence of IF2. The experiments indicate binding of IF2 to the acceptor stem, position 12 to 13 in the D-stem, two sites in the anticodon stem, and parts of the T-loop and the minor groove of the fMet-tRNAfMet T-stem (Fig. 6) (166, 243). Based on a similar cleavage pattern of RNase VI in the anticodon stem of Met-tRNAfMet bound to MTF, it was proposed that the interaction between fMet-tRNAfMet and IF2 induces a conformational change in the anticodon stem (122). However, site-directed mutagenesis and nuclear magnetic resonance (NMR) spectroscopy studies reveal that essentially all thermodynamic determinants governing the stability and specificity of the interaction are located within the fMet-3' ACCAC part of fMet-tRNAfMet (66).
Original proposals suggested that IF2 carries fMet-tRNAfMet to the ribosome by analogy to EF1A for aminoacyl-tRNAs and eIF2 for Met-tRNAiMet (reviewed in reference 94). A specific binary complex can be formed between fMet-tRNAfMet and IF2 in vitro, but the interaction is weak, with a Kd of 1.8 µM, and the complex dissociates readily in the presence of magnesium ions (113, 118, 119, 167, 251). Most evidence suggests that IF2 performs its interactions with fMet-tRNAfMet on the 30S ribosomal subunit (251).
IF3 appears to inspect the anticodon stem of the P-site bound tRNA (73). However, this inspection may occur through indirect and not direct interactions, as discussed in the section on IF3 (see below).
Binding of fMet-tRNAfMet to the P-site of the 30S ribosomal subunit is highly influenced by the conformation of the anticodon stem. Site-directed mutagenesis has shown that the three consecutive GC base pairs in the anticodon stem influence the unique conformation of fMet-tRNAfMet as well as P-site binding (202). The structure of the 70S ribosome in complex with P-site-bound fMet-tRNAfMet maps the interactions with 16S rRNA primarily to the anticodon stem and loop, whereas the 23S rRNA interacts with the CCA acceptor arm and part of the D-stem of fMet-tRNAfMet. The T-loop interacts with ribosomal protein L23 (256) (Fig. 6). A conformational change of the rRNA on P-site tRNA binding similar to that observed for A-site tRNA binding has not been found. However, C1400 of the 16S rRNA appears to stabilize the wobble base pair of the codon-anticodon interaction by stacking on base 34 of the tRNA (256). Thus, the ribosome itself does not seem to perform as stringent a proofreading of the codon-anticodon interaction in the P-site as in the A-site. As described in the following sections, the initiation factors play a key role in the selection and adjustment of the initiator tRNA in the P-site.
The structure of IF1 in solution has been determined by NMR spectroscopy (204). IF1 belongs to the family of oligonucleotide binding (OB) fold proteins. It consists of a five-strand beta barrel with the loop between strands 3 and 4, capping one end of the barrel (Fig. 7). Structures of the archaeal and eukaryotic IF1 homologues (aIF1A and eIF1A) have also been determined (6, 111). These structures are highly similar with respect to the OB fold (Fig. 7). The C terminus, however, contains -helical structures that are important for the archaeal and eukaryotic scanning mechanism and interactions with the small ribosomal subunit.
|
(151), and aspartyl-tRNA synthetase (187). It has been shown that the cellular defects resulting from a double deletion in the genes encoding cold shock proteins CspB and CspC in Bacillus subtilis can be complemented by heterologous expression of E. coli IF1 (244). This confirms the structural resemblance and functionality between IF1 and the cold shock proteins. The binding of IF1 to the 30S ribosomal subunit has been extensively mapped. IF1 binds in a cleft between the 530 loop and helix 44 of 16S rRNA and ribosomal protein S12 (Fig. 8). Cleavage of 16S rRNA with cloacin DF13 between A1493 and A1494, two positions located in the 30S ribosomal A-site, specifically disrupts the function of IF1 (2a). Binding of IF1 to the 30S ribosomal subunit protects A1492 and A1493 from modification by dimethyl sulfate and protects G530 from attack by kethoxal (132). These positions are protected by A-site-bound tRNA, strongly supporting the notion that IF1 is located at an overlapping binding site. Mutational analysis demonstrated that the C1408-G1494 base pair and the three adenosines A1408, A1492, and A1493 are required for optimal IF1 binding, and it appeared that the internal loop of helix 44 is more important for IF1 binding than the identity of the nucleotide present at a certain position in the interacting part of helix 44 (41).
|
Several functions have been attributed to bacterial IF1. It enhances the dissociation and association rate for 70S ribosomes (46, 59), primarily through the stimulating effect on the activity of IF2 and IF3 (174). Interaction between IF2 and the 30S ribosomal subunit is favored when IF1 is bound, and the release of IF2 is indirectly promoted when IF1 is ejected (27, 136, 222). IF1 cooperates with IF2 to ensure that only the initiator tRNA binds to the P-site and that it interacts with the initiation codon of the mRNA (24, 73, 127, 251). IF1 occlude tRNAs from the A-site until the 70S initiation complex has formed. Ejection of IF1 consequently opens the A-site for incoming aminoacyl-tRNAs. In vivo studies have shown that IF1-depleted cells have low growth rates and short polysomes (39). These data demonstrate that IF1 is essential for cell viability and suggest that one or more of its functions are crucial. However, no clear function has been assigned to the initiation factor yet (37).
Three isoforms of the initiation factor, named IF2-1 (97.3 kDa), IF2-2 (79.7 kDa), and IF2-3 (78.8 kDa), exist in E. coli and other members of the family Enterobacteriaceae (107, 154). Bacillus subtilis is the only organism that does not belong to the Enterobacteriaceae where more than one isoform of IF2 has been experimentally demonstrated (81). IF2-1, IF2-2, and IF2-3 are translated from three independent but in-frame translational start sites of the infB mRNA. This feature has been referred to as tandem translation. Hence, IF2-2 and IF2-3 differ from IF2-1 only by the absence of the first 157 and 164 amino acid residues, respectively (Fig. 9) (139). The presence of both the large and smaller isoforms is required for optimal growth of E. coli. The cellular content of IF2-2 and IF2-3 is close to the level of IF2-1 at optimal growth conditions (79, 191), but the ratio of IF2-2 and IF2-3 to IF2-1 increases as a response to cold shock (53). An open single-stranded structure is present in the intracistronic TIR of the infB mRNA. We suggest that this structure is required for the translation of IF2-2 and IF2-3 (107).
|
The factor can be divided into a conserved C-terminal region consisting of domains IV to VI and a less highly conserved N-terminal region corresponding to domains I to III (209, 217). Homologues of IF2 have been found in archaeabacteria and eukaryotes, where the factor is referred to as aIF5B and eIF5B, respectively (98). Remarkable interspecies homology in the C-terminal region is present among the homologues (209). The homologues have functions similar to those of bacterial IF2, including GTPase activity, promotion of ribosomal subunit association, and probably interaction with the initiator tRNA (30, 165).
No direct tertiary-structure information is available for domains IV to VI of E. coli IF2, but the structure of the homologous protein aIF5B from the archaeon Methanobacterium thermoautotrophicum has been solved (Fig. 9) (185). Amino acid sequence homology predicts a similar structure for domains IV to VI of bacterial IF2, although there are two additional helical segments at the C-terminus of the archaeal factor (Fig. 9).
The conserved C-terminal region of the protein begins with the domain responsible for GTP binding: the G-domain (E. coli domain IV). Overall homology among bacterial, eukaryotic, and archaeabacterial IF2, eIF5B, and aIF5B is highest in the G-domain area of the factor (Fig. 9). Significant sequence homology to other proteins is found only for this domain. The GTP binding motif is shared with at least four other proteins involved in translation, namely, EF1A, EF2, RF3, and SelB (184, 238). Following the G-domain is an EF1A-like ß-barrel (domain V) and a novel ß sandwich (domain VI-I) connected via a long -helix to the C-terminal domain (domain VI-2). The structure of the C-terminal domain of Bacillus stearothermophilus IF2 has been solved by NMR spectroscopy methods (128). It is similar to domain VI-2 from M. thermoautotrophicum with the exception of the two C-terminal helices found in the M. thermoautotrophicum factor. These helical segments are not present in any of the bacterial sequences (Fig. 9).
In contrast to the C-terminal region, the N-terminal region of IF2 is highly variable in both primary structure and length. The region can be divided into three separate domains in E. coli IF2 based on sequence and biochemical data (Fig. 9) (140). Domain I contains a small subdomain of approximately 50 residues, found in all bacterial and some plastid IF2s. We solved the structure of the domain by NMR spectroscopy methods (105). The subdomain is now designated IF2N in the protein families database (PFAM). IF2N has homology to the stem contact fold domains of the methionyl- and glutaminyl-tRNA synthetases and the B5 domain of the phenylalanine-tRNA synthetase. However, no specific function has been assigned to the IF2N domain (105). NMR spectroscopy of the full-length E. coli IF2-1 revealed that the IF2N domain is connected to domain IV by a highly flexible linker region (104). Domain I is extremely soluble and has been applied as a solubility-enhancing fusion partner for the expression of proteins prone to aggregate in the E. coli cytoplasm (211).
Macromolecular interactions of the domains in the N-terminal region of IF2 has been demonstrated only in E. coli, where a fragment consisting of the combined domains I and II, but not a fragment of isolated domain I, binds to the 30S ribosomal subunit (136, 137). Furthermore, we have identified domains I and II of E. coli IF2 as an interaction partner for the infB mRNA (107).
B. stearothermophilus and T. thermophilus IF2 possess a single domain in the region N-terminal to the G-domain, whereas myxobacterial IF2, which has the longest N-terminal regions characterized in bacteria, is composed of several domains with a highly unusual amino acid composition. The latter is a general feature of the N-terminal regions of IF2 (16, 64, 225, 230). Regions N-terminal to the G-domain of eukaryotic IF2 homologues are generally long, up to 700 amino acids, whereas the regions of the archaeal IF2 homologues are generally short. Conclusively, the functional importance of the N-terminal region of IF2 and its homologues remains unresolved.
The role of the GTPase activity of IF2 has been a matter of debate for decades. Available structures of aIF5B in the nucleotide free form, the inactive form with GDP bound, and the active form with GTP bound reveal that binding of Mg2+-GTP induces movements in domains V and VI-2 over a distance of more than 90 Å (185, 186).
Elongation factor EF1A associates approximately 100-fold more strongly with GDP than with GTP, whereas IF2 associates only 10-fold more strongly with GDP (163, 175). A guanine nucleotide exchange factor may therefore not be required for IF2 as in the case of EF1A, where nucleotide exchange involves the factor EF1B. Hydrolysis of GTP by IF2 depends on the presence of ribosomes, and the factor has no intrinsic GTPase activity by itself. However, B. stearothermophilus IF2 can hydrolyze GTP in the absence of ribosomes when 20% ethanol is included in the reaction mixture (64). GTP hydrolysis in translation initiation has been suggested to be important for the release of IF2 from the 70S initiation complex (108, 114), and for the adjustment of initiator tRNA in the ribosomal P-site (103). The crucial importance of GTP hydrolysis in translation initiation and its direct relation to cell viability have been confirmed by several studies of G-domain mutants of IF2 (98a, 100, 106, 115).
The G-domains of IF2 and EF1A are thought to have overlapping binding sites. Fluorescence stopped-flow experiments showed that binding of EF1A to ribosomes probed with IF2 was independent of the bound nucleotide. It was thus concluded that neither the ejection of IF2 from the ribosome nor its recycling requires GTP hydrolysis (102, 231). A recent study of initiation complex formation by using stopped-flow experiments with light scattering gave contradictory results. The conclusions from these experiments are that the GTP-bound form of IF2 accelerates association of the ribosomal subunits and that GTP hydrolysis accelerates ejection of IF2 from the 70S ribosome (2). Further studies are needed to achieve a detailed understanding of the role of GTP hydrolysis in the translation initiation event.
Early cross-linking experiments show that parts of IF1 and IF2 are in close proximity on the ribosome (13). The interaction between the two factors maps to a region between domains III and V of E. coli IF2 (136). Interactions between the eukaryotic homologues eIF5B and eIF1A have been mapped by using the yeast two-hybrid system, coimmunoprecipitation, in vitro binding assays, and NMR spectroscopy (31, 120, 159). The C-terminal unstructured region of eIF1A, which is not present in bacterial IF1, interacts with the C terminus of eIF5B. Domain V of bacterial IF2 was suggested to interact with IF1 on the ribosome (120, 136).
As mentioned above, the interactions between fMet-tRNAfMet and IF2 have been studied extensively. Formation of the binary complex is strongly dependent on the formylation of Met-tRNAfMet (223) but independent of GTP (175). The C-terminal domain VI-2 of IF2 has been suggested to contain the entire binding site for fMet-tRNAfMet (215). The interaction of the domain with fMet-tRNAfMet has been studied using mutagenesis as well as Raman and NMR spectroscopy (66, 96, 130, 225). Functionally essential residues of the B. stearothermophilus domain are C668, K699, R700, Y701, K702, E713, C714, and G715 (Fig. 10). Cross-linking experiments indicate interactions between E. coli residues N611-R645 (belonging to domain V) and the T-stem of fMet-tRNAfMet as well as residues W215-R237 (domain II) and the anticodon stem of fMet-tRNAfMet (243, 257). Finally, fMet-tRNAfMet protects a position in domain IV and weakly in domain V of B. stearothermophilus IF2 against digestion by trypsin (205). A stable interaction between the archaeal and eukaryotic IF2 homologues and Met-tRNAiMet has not been observed in vitro, but overexpression of the gene encoding tRNAiMet partially suppresses the severe slow-growth phenotype of yeast strains lacking the IF2 homologue (30).
|
The GTPases involved in translation probably occupy partly overlapping binding sites on the ribosome. A site located on the 50S ribosomal subunit, termed the factor binding site, is composed of the -sarcin loop, the L11-binding region, and the L7-L12 stalk. EF1A, EF2, and IF2 all interact with the ribosome at the factor binding site (23, 131, 182). An additional constraint to place IF2 on the ribosome is the interaction with fMet-tRNAfMet, which places domain VI-2 of IF2 in proximity of the fMet-CCA end of the P-site-bound initiator tRNA.
IF2 has been cross-linked to S13, L7-L12, IF1, and IF3 (13), as well as S1, S2, S11, S12, and S19 on the ribosome (14). Chemical probing experiments with 23S rRNA indicated that IF2 protects A2476 and A2478 in helix 89 of domain V as well as G2655, A2660, G2661, and A2665 of the sarcin-ricin domain positioned in domain VI (Fig. 10) (102). These footprints were generated regardless of the presence of GTP, IF1, mRNA, and fMet-tRNAfMet. Unfortunately, the results are unclear with respect to the 30S ribosomal subunit, since IF2 affects the reactivity of residues spread all over the subunit. This is consistent with an observed rearrangement of the subunit induced by IF2 (242). Recently, a model of IF2 binding to the ribosome was presented, based on cleavage of the rRNA by chemical nucleases tethered to cysteine residues introduced at specific sites of IF2 (121). No cleavage of the 16S rRNA was observed when IF2 was bound to 30S ribosomal subunits or to the complete 30S initiation complex. However, cleavage of the 16S rRNA was observed when IF2 was bound to the 70S initiation complex (Fig. 10). These data indicate that domain V of IF2 is localized toward the 30S subunit in the 70S initiation complex. As described above, cross-linking data of the 30S complex and footprinting data on the binary fMet-tRNAfMet-IF2 complex place domain V of IF2 in proximity to the elbow of the P-site-bound fMet-tRNAfMet. The distance between the 16S rRNA and the elbow of the fMet-tRNAfMet appears to be too far for domain V of IF2 to establish contact with both simultaneously. Conclusively, IF2 changes localization during the transition from the 30S to the 70S initiation complex (121). The cleavage experiments were performed in the presence of excess GTP. Large domain movements take place in IF2 during GTP hydrolysis (185), and the cleavage patterns in the rRNA might be dependent on whether IF2 is in the GTP- or GDP-bound form. To fully understand the function of IF2 during translation initiation, detailed atomic resolution structures of both the 30S and 70S initiation complexes as well as a better understanding of the timing and not least the consequences of GTP hydrolysis are needed.
Besides the function as a translation factor, IF2 has the properties of a chaperone. It promotes functional folding of proteins and forms stable complexes with unfolded proteins (22). Furthermore, the expression of IF2 is upregulated during the cold shock response (3), and the factor is important for the translation of leaderless transcripts (57). Finally, we have introduced the use of IF2 sequence data for the classification of organisms of closely related organisms (75-77, 152, 209, 226).
IF3 is composed of two structural domains of approximately equal size (Fig. 11) (48, 97). The two domains, called the IF3N and IF3C, are separated by a 45-Å lysine-rich flexible linker (80, 135). The IF3N domain consists of a globular /ß-fold, with helix 1 packed against a mixed five-strand ß-sheet (Fig. 11). This fold is followed by helix 2, which connects IF3N to IF3C. The length of 2 was found to be different in the structures derived from NMR spectroscopy (51) and X-ray diffraction (11) experiments and has been the subject of debate (reviewed in reference 12). The linker is essential for IF3 function, but variation of its length and composition does not considerably change the activity (43).
|
/ß sandwich fold composed of a four-strand mixed ß-sheet packed against two parallel -helices (3 and 4), leading to a ßßßß topology (Fig. 11). The structure is similar to U1A (RNA binding protein involved in RNA splicing) (51) and YppH (a protein involved in cell division) (88). IF3 perform several different functions. (i) It prevents association of the ribosomal subunits by binding to the 30S subunit, thereby blocking binding of the 50S subunit (59, 188). (ii) It monitors the codon-anticodon interaction by promoting the dissociation of fMet-tRNAfMet from initiation complexes formed at the 5' initiation codon of leaderless mRNAs (227). Likewise, initiation complexes with an incorrectly bound aminoacyl-tRNA (noninitiator tRNA) (73, 74) and complexes with triplets other than AUG, GUG, and UUG in the P-site are dissociated by IF3 (70, 127, 224). (iii) It stimulates the rapid formation of codon-anticodon interaction at the ribosomal P-site (60, 250). (iv) It is involved in the adjustment of the mRNA from the standby site to the decoding P-site of the 30S ribosomal subunit (101). Finally, a role for IF3 in recycling of subunits has been proposed. It was observed to enhance the dissociation of deacylated tRNAs from posttermination complexes and to dissociate 70S ribosomes into subunits (78, 87).
All functions of the native IF3 can be accomplished by the isolated IF3C domain in vitro, while the IF3N domain probably serves the purpose of modulating the thermodynamic stability of the IF3-30S complexes (169). Site-directed mutagenesis of the eight arginine residues in the IF3C domain has been used to map the active sites (168). The arginines at positions 99, 112, 116, 147, and 168 are important for the binding to the 30S ribosomal subunit (Fig. 11). The ability of IF3 to dissociate the ribosome into subunits was affected mainly by mutations of R112 and R147 (and less extensively by mutations of R99 and R116). The stimulation of the pseudoinitiation complex dissociation (with a noninitiator tRNA bound) was affected by mutations of R99 and R112 (and less extensively of the arginine residues at positions 116, 129, 133, and 147). Dissociation of noncanonical initiation complexes (initiation codons other than AUG, GUG, and UUG) was not affected in any of the mutants. Stimulation of translation was affected by mutations of R116 and R129 (and less extensively of the arginine residues at positions 99, 112, and 131), whereas inhibition of noncanonical mRNA translation was affected by mutations of R99, R112, and R168 (and less extensively of the arginine residues at positions 116, 129, and 131). Finally, the repositioning of the mRNA from the standby site to the P-decoding site was weakly affected by mutations of the arginine residues at positions 129, 131, 133, 147, and 168. The data indicate that IF3C contains at least two active surfaces, one embedded in the 30S subunit and the other facing the mRNA (Fig. 11) (168).
Both IF3 domains are RNA binding and interact independently with the 30S ribosomal subunit. IF3C interacts with the highest affinity through a large surface of symmetrically distributed residues in loops and -helices, whereas IF3N interacts mainly via a small number of asymmetrically distributed residues (203). Results of mapping of IF3 residues implicated in binding to the 30S ribosomal subunit by NMR spectroscopy, site-directed mutagenesis, and other chemical methods are in excellent agreement (203) (Fig. 11).
The localization of IF3 on the 30S ribosomal subunit has been studied by various methods, with conflicting results. Immuno-EM located the factor at the cleft of the 30S ribosomal subunit (220). The ribosomal proteins S7, S11, S12, S13, S18, S19, and S21 have been cross-linked to IF3 but are spread over a wide area of the 30S subunit (13, 33, 34, 117). Helices 26 (central domain) and 45 (3' minor domain) of the 16S rRNA have been cross-linked to IF3 (47). Chemical probing revealed IF3 contacts to helices 23 and 24 in the central domain of the 16S rRNA (132, 141), and NMR spectroscopy indicated that IF3 interacts with the 3' end of the 16S rRNA (246). Cryo-EM located the IF3C domain to the interface side of the small ribosomal subunit (124). However, X-ray diffraction of 30S ribosomal subunit crystals soaked with IF3C places the domain at the solvent side of the platform (170).
A model based on hydroxyl radical footprinting and directed probing from Fe(II)-derivatized IF3 has been presented (42). This model is in agreement with the cryo-EM data, and the results are summarized in Fig. 12. It was suggested that the observations in the crystallographic studies represent binding to a secondary site in the crystal-soaking experiments as a result of blockage of the primary binding site by crystal contacts. IF3C is located in the same area as helix 69 of the 23S rRNA in the 70S ribosome, which explains why IF3 blocks subunit association.
|
| REGULATION OF TRANSLATION INITIATION |
|---|
|
Top |
|---|
