An extended Shine–Dalgarno sequence in mRNA functionally bypasses a vital defect in initiator tRNA

Significance

Initiation, a regulatory step in mRNA translation is characterized by initiator tRNA (tRNA^fMet) binding to 30S ribosome together with initiation factors (IFs) to form 30S pre-initiation complex (PIC). This step is followed by binding of 50S ribosome and release of IFs to form elongation competent 70S complex. tRNA^fMet is special in possessing a vital feature of three consecutive G-C (3GC) base pairs in its anticodon stem. However, the role of this feature has remained unclear. We show that the 3GC base pairs facilitate tRNA^fMet retention in the ribosome during the transitions that mark conversion of 30S PIC into 70S complex. Furthermore, we show that translation of mRNAs having an extended Shine–Dalgarno sequence bypasses the requirement of the 3GC pairs in tRNA^fMet.

Abstract

Initiator tRNAs are special in their direct binding to the ribosomal P-site due to the hallmark occurrence of the three consecutive G-C base pairs (3GC pairs) in their anticodon stems. How the 3GC pairs function in this role, has remained unsolved. We show that mutations in either the mRNA or 16S rRNA leading to extended interaction between the Shine–Dalgarno (SD) and anti-SD sequences compensate for the vital need of the 3GC pairs in tRNA^fMet for its function in Escherichia coli. In vivo, the 3GC mutant tRNA^fMet occurred less abundantly in 70S ribosomes but normally on 30S subunits. However, the extended SD:anti-SD interaction increased its occurrence in 70S ribosomes. We propose that the 3GC pairs play a critical role in tRNA^fMet retention in ribosome during the conformational changes that mark the transition of 30S preinitiation complex into elongation competent 70S complex. Furthermore, treating cells with kasugamycin, decreasing ribosome recycling factor (RRF) activity or increasing initiation factor 2 (IF2) levels enhanced initiation with the 3GC mutant tRNA^fMet, suggesting that the 70S mode of initiation is less dependent on the 3GC pairs in tRNA^fMet.

Initiation of protein synthesis, assisted by initiation factors, is a highly regulated process in all life forms. In eubacteria, binding of both the initiator tRNA (tRNA^fMet) and mRNA to the small ribosomal subunit (30S) leads to the formation of a 30S preinitiation complex primarily with the help of the three initiation factors (IF1, IF2, and IF3). This stage is then followed by docking of the large ribosomal subunit (50S) to ultimately produce an elongation competent 70S complex upon the departure of all of the three initiation factors (1). The localization of mRNA onto the 30S subunit is facilitated by a purine rich sequence (Shine–Dalgarno, SD sequence), located upstream of the start codon, by its pairing with a complementary sequence (anti-SD sequence) at the 3′-terminus of the 16S rRNA (1, 2). The tRNA^fMet binding to ribosome is aided by the unique features it possesses. A virtually universal feature of all of the initiator tRNAs, the presence of three consecutive G-C base pairs (G₂₉G₃₀G₃₁:C₃₉C₄₀C₄₁, referred to as 3GC pairs) in the anticodon stem is known to be important for their preferential binding in the ribosomal P-site (3, 4). Mutations in the 3GC pairs result in poor binding of tRNA^fMet to the ribosomal P-site (3, 4).

However, the mechanism of how the 3GC pairs help in binding of tRNA^fMet into the ribosome has remained unclear. In the crystal structure of the initiator tRNA bound 70S ribosome, it was seen that the universally conserved A1339 and G1338 residues of 16S rRNA establish A-minor interactions with the first GC (G₂₉-C₄₁) and middle GC (G₃₀-C₄₀) pairs, respectively (5). Although these interactions were seen as suboptimal, the IF3 induced conformational changes may optimize these interactions (6). Another study showed that a major role of IF3 is to uniformly increase the rate of dissociation of all tRNAs (including tRNA^fMet) from the 30S preinitiation complexes (7). These observations suggest that the role of 3GC pairs in conferring specificity for the ribosomal P-site binding is an open question.

Formation of elongation competent 70S complex is a multistep process wherein both the tRNA^fMet and IF2 assist docking of 50S subunit onto the 30S preinitiation complex. The 70S complex so formed is in a ratcheted state with tRNA^fMet bound in the P/I state wherein the tRNA anticodon stem is placed in the P-site and the acceptor stem is placed between the P-, and E-, sites. For such a 70S complex to enter into the elongation phase, the ratcheted state must unratchet to localize the tRNA^fMet in the classical P/P site and make the A/A site available for the entry of an elongator tRNA. The unratcheting is mediated by the hydrolysis of the IF2 bound GTP (8). What specific interactions between the ribosome and tRNA^fMet facilitate retention of the latter into the 70S complex during these conformational transitions is not known. Whether the 3GC pairs in the anticodon stem of tRNA^fMet play any specific roles during these transitions is also not known.

To further our understanding of the role of the 3GC pairs in the initiation process, we have used a genetic approach to obtain suppressor strains of Escherichia coli that allow initiation to occur with the tRNA^fMet having mutations in the 3GC pairs (9). In this study, characterization of one of the isolates, B21, showed that C1536-to-T1536 mutation in the 3′ terminal region of the 16S rDNA, resulting in its extended interaction with the SD region of the reporter mRNA, allowed initiation to occur with the mutant tRNA^fMet lacking the highly conserved feature of the 3GC base pairs in the anticodon stem.

Results

In Vivo Assay and Characterization of the B21 Mutation Enabling Initiation with the 3GC Mutant tRNA^fMet in E. coli.

The plasmid, pCAT_am1 (Amp^R) encodes chloramphenicol acetyltransferase (CAT) reporter mRNA with UAG start codon (as opposed to AUG), which is not translated under normal physiological conditions. Thus, its (pCAT_am1) presence does not confer chloramphenicol (Cm) resistance to E. coli (Fig. 1 B, ii, sector 1). However, a simultaneous presence of the mutant metY gene, metY_CUA, which encodes tRNA^fMet with CUA anticodon (in place of CAU) in pCAT_am1metY_CUA, results in initiation from the UAG start codon of the reporter mRNA conferring Cm resistance (Cm^R) to the host (Fig. 1 A, i and B, ii, sector 2) (10). Not unexpectedly, when the metY_CUA is further mutated to replace the highly conserved 3GC pairs in the anticodon stem with U₂₉:A₄₁, C₃₀:G₄₀, A₃₁:Ψ₃₉, the new construct (pCAT_am1metY_CUA/3GC) fails to confer Cm^R because the encoded tRNA^fMet (called the 3GC tRNA hereafter) fails to successfully participate in initiation (Fig. 1 A, ii, and B, ii, sector 3) despite its efficient aminoacylation and formylation (3, 10, 11).

Fig. 1.

Assay system and characterization of B21 suppressor strain. (A, i) The plasmid pCAT_am1metY_CUA harbors CAT_am1 and metY_CUA genes that encode CAT_am1 mRNA with UAG initiation codon (am1) and tRNA^fMet having CUA anticodon, respectively. The metY_CUA encoded tRNA initiates from the CAT_am1 mRNA to produce CAT protein and confers Cm^R to the host. The relevant sequences of anticodon stem loop (box with dashed line) are as shown. (ii) The plasmid pCAT_am1metY_CUA/3GC is derived from pCAT_am1metY_CUA wherein the metY_CUA gene has been further mutated in the three consecutive G-C base pairs. The encoded tRNA (3GC tRNA) fails to initiate from CAT_am1 mRNA rendering the host Cm^S. (B) Growth of E. coli KL16 parent strain (sectors 1–3) and the derived suppressor B21 (sectors 4–6) harboring either pCAT_am1, pCAT_am1 metY_CUA or pCAT_am1 metY_CUA/3GC on LB-agar plates containing, Amp (i) and Amp plus Cm (100 and 30 μg/mL, respectively) (ii). Overnight cultures were streaked and incubated at 37 °C for ∼18 h. (C) Interaction of CAT_am1 SD sequence with the wild-type (i) or the B21 mutant (ii) anti-SD sequence of the 16S rRNA (rrsC).

In our earlier work (9), we isolated E. coli B21 strain wherein a chromosomal mutation rescues initiation defect of the 3GC tRNA to confer Cm^R (Fig. 1 B, ii, sector 6). As a control, the B21 harboring pCAT_am1 remains Cm sensitive (Cm^S), and retains Cm^R with pCAT_am1metY_CUA (Fig. 1 B, ii, sectors 4 and 5). As yet another control, all transformants grow on Amp alone plate (Fig. 1 B, i). Genetic mapping using P1 phage mediated transductions (SI Results) localized the site of mutation in B21 at 84.5 min in the E. coli chromosome (Fig. S1A). At this location, the rrsC gene (encoding for one of the seven 16S rRNA genes) was the most likely candidate. DNA sequence analysis revealed a novel C-to-T mutation in rrsC corresponding to a C to U change at position 1536 (termed as c1536u or the B21 mutation) in the encoded 16S rRNA (Fig. S1B) in the anti-SD (aSD) region (12), which resulted in an extended interaction with the SD region of the reporter mRNA from 6 bp to 8 bp (Fig. 1C, compare i and ii).

Impact of the Extended SD:aSD Interaction on Initiation with the 3GC Mutant and the Wild-Type tRNA^fMet.

To generate a converse system of extended SD:aSD interaction, we mutated SD sequence of the CAT reporter mRNA from UAAGGAAG to UAAGGAGG in pCAT_am1(a7g) (SD_a7g construct) to allow an extended interaction with the wild-type 16S rRNA (Fig. 2A). To check the impact of the extended SD on initiation with the 3GC tRNA, the pCAT_am1metY_CUA/3GC or pCAT_am1(a7g)metY_CUA/3GC plasmids were introduced into the parent strain, KL16, and its derivative B21. CAT assays showed that compared with the original construct (SD_wt), the a7g construct (SD_a7g) resulted in an efficient initiation with the 3GC tRNA even in the parent strain (KL16). In fact, the initiation in KL16 was higher than that seen in B21 (Fig. 2B). A plausible reason for this is that although CAT_am1 mRNA (SD_wt) could be preferably used by only a single gene (rrsC) encoded 16S rRNA in B21, the CAT_am1(a7g) mRNA (SD_a7g) could be used by 16S rRNA encoded by all of the seven genes in KL16, and by six of the seven genes in B21 (the seventh having the B21 mutation). This observation suggests that the extended SD:aSD interaction alone is sufficient to allow efficient initiation by the 3GC tRNA.

Fig. 2.

Effect of extended SD on translation initiation. (A) Schematics of interaction of SD_a7g of CAT_am1 with anti-SD (aSD) of wild-type 16S rRNA. (B) Initiation with 3GC tRNA as assayed by CAT activity in cell-free extracts of E. coli KL16 or B21 strains harboring CAT_am1 reporter in the context of SD_wt or SD_a7g. Fold differences in CAT activities with respect to 3GC tRNA in KL16 are shown. The CAT activity in KL16 for the SD_wt context was 110.4 ± 58.8 pmol of acetylated (Ac) Cm produced per μg of total protein in 20 min at 37 °C. (C) Initiation from CAT_am1 with metY_CUA (bars 1 and 2), metY_CUA/AU (bars 3 and 4), metY_CUA/GU (bars 5 and 6), and metY_CUA/3GC (bars 7 and 8) tRNAs in the context of SD_wt or SD_a7g. The fold differences in CAT activities with respect to metY_CUA in SD_wt context are shown. CAT activity of metY_CUA in SD_wt context was 5236.4 ± 1204.5 pmol Ac-Cm per μg of total protein. (D) CAT assays from cell-free extracts of E. coli KL16 harboring pCAT_am1metY_CUA/3GC along with either the empty vector, pACDH (bars 1 and 3) or another construct for CAT_am1 mRNA, pACDHCAT_am1 (bars 2 and 4), with (bars 3 and 4) or without (bars 1 and 2) 0.5 mM IPTG. Absolute CAT activity for the reaction represented by bar 1 is 116.1 ± 18.3 pmol Ac-Cm per μg total protein. (E) Assay of overexpression of CAT_am1 mRNA by semiquantitative PCR.

Similar to earlier studies (13, 14), we observed that compared with SD_wt, the extended SD (SD_a7g) caused a decrease in initiation by metY_CUA retaining wild-type sequence in its anticodon stem (Fig. 2C, compare bars 1 and 2). However, such an adverse effect of SD_a7g was not seen when, of the three consecutive G-C base pairs, we mutated only the first to A-U in metY_CUA/AU (bars 3 and 4) or the third to G-U in metY_CUA/GU (bars 5 and 6). In fact, the metY_CUA/GU construct showed an increase in initiation. As control, the metY_CUA/3GC construct showed an expected increase (bars 7 and 8). In addition, we observed that increased production of CAT_am1 mRNA (by introducing another copy of the CAT_am1 gene on another medium copy plasmid, pACDHCAT_am1, compatible with the pCAT_am1) in KL16 did not compensate for the 3GC mutations (Fig. 2 D and E) suggesting that the mechanism of rescue of the 3GC defect, in B21 was unlikely due to merely a direct impact of the extended SD on the initial binding of the CAT_am1 mRNA to the ribosome, or on an early event in initiation.

In another experiment, analysis of the ribosome bound fraction of the CAT_am1 mRNA in B21 by Northern blotting and RT-PCR, showed ∼2- to 4-fold increase (compared with KL16). Likewise, the CAT_am1(a7g) mRNA also increased ∼3.7- to 7-fold in the KL16 ribosomes (Fig. 3 A, i and B). We noted that the levels determined by Northern blotting were lower. More importantly, levels of the 3GC tRNA on ribosomes also increased ∼threefold (Fig. 3 A, ii).

Fig. 3.

Binding of mRNA and the 3GC tRNA to ribosomes. (A) Northern blot analysis for CAT_am1 mRNA (i), 3GC tRNA (ii), and 5S rRNA (iii) in the ribosomal pellets. RNA (∼20 μg) were separated on 1.2% agarose gel, transferred to nytran membrane, and probed sequentially for CAT_am1, 3GC tRNA, and 5S rRNA. Signal intensities of CAT_am1 mRNA, and 3GC tRNA were divided by those of the corresponding signals of 5S rRNA. The values so obtained for the KL16 strain harboring the pCAT_am1metY_CUA (3GC-SD_wt) were set as 1, and the remaining values were calculated relative to this reference value of 1 for both the CAT_am1 and the 3GC tRNA. (B) Fold increase in ribosome bound CAT_am1 mRNA as measured by quantitative PCR using 16S rRNA as internal control.

It may be noted that, immediately upstream of the SD sequence in the CAT_am1 reporter, yet another sequence, AGGAG, which, although suboptimally placed with respect to the start codon, could also serve as an SD sequence. However, as shown in Fig. S2, mutations in this sequence did not result in any significant effects.

Distribution of the Wild-Type tRNA^fMet and the 3GC tRNA with CAU Anticodon (3GC_CAU) in 30S and 70S Ribosomes.

Polysome preparations from KL16 strain harboring pmetY_3GC encoding 3GC_CAU tRNA were fractionated on sucrose density gradients (20–40%) (Fig. 4 A, i) and analyzed for the presence of the wild-type tRNA^fMet and the 3GC_CAU tRNAs across the sedimentation profile by dot blot hybridizations (Fig. 4 A, ii). As shown in Fig. 4 A, iii, in the fractional distribution of the tRNAs across the gradient fractions 6–17, the free tRNA levels, and those bound to the 30S ribosomes were represented by nearly the same levels (Fig. 4 A, iii, F6-F7, and F8-F10). However, in 70S fractions (F13–F17), the wild-type tRNA^fMet bound more prominently than the 3GC_CAU tRNA. Such a fractional distribution of tRNAs shows that the 3GC_CAU tRNA is not defective in binding to 30S, and suggests that the primary role of the 3GC pairs may be during the transit of tRNA^fMet from 30S to 70S complex. To validate these results, in yet another experiment (Fig. 4B), we collected the 30S, 50S, and 70S ribosome fractions separately (Fig. 4 B, i) and analyzed them by Northern blotting (Fig. 4 B, ii). In this analysis, whereas the wild-type tRNA^fMet was mostly found in the 70S complex, only a small fraction of the 3GC_CAU tRNA located in 70S. Ratios of 70S to 30S bound fractions (Fig. 4 B, iii) also revealed the same. Interestingly, unlike the case with tRNA^fMet, we observed that a significant fraction of the 3GC_CAU tRNA was also found migrating with the 50S. This migration is most likely due to its poorer association with 70S resulting in dissociation of a part of it during centrifugation. It may also be noted that, in E. coli K, wild-type tRNA^fMet encoded by metZWV (∼75%) and metY (∼25%) separates into tRNA^fMet1 and tRNA^fMet2 bands (doublet) when analyzed on the native gel shown in Fig. 4 B, ii. Expectedly, both tRNA^fMet species reveal similar distribution.

Fig. 4.

Binding of 3GC_CAU tRNA (wild-type anticodon) to 30S and 70S ribosomes. (A) Analysis of 3GC_CAU tRNA across the ribosomal profile. The ribosomal subunits and polysomes were separated on 20–40% sucrose gradient using SW55 rotor (48000 rpm for 3 h) (i). Dot blot analysis from fraction numbers 6–17 was carried out for 3GC_CAU tRNA and the tRNA^fMet. Dot blots for two biological replicates are shown (ii). The fraction of tRNA in each profile was measured by dividing the individual spot intensities with the total intensity in the profile and the distribution shown as bar diagram (iii), for wild-type tRNA (open bar) and 3GC_CAU tRNA (black bar). (B) The subunits and polysomes were separated on 20–40% sucrose gradient as in A, and the 30S, 50S and 70S peaks were collected separately by monitoring the ribosomal profile (i). The extracted RNA was analyzed on the native PAGE, transferred onto nytran membrane and probed for the presence of 3GC_CAU as well as tRNA^fMet (ii). Their fractional distributions between the subunits and 70S ribosome are as shown (iii).

Extended SD Facilitates the 3GC tRNA to Enter 70S Complex.

The analysis in Fig. 4 showed that one of the defects in initiation with the 3GC_CAU tRNA is its transition from the 30S to 70S ribosome. Hence, to understand the mechanism of how an extended SD rescued the 3GC tRNA defect, we carried out sucrose density gradient analysis (Fig. 5 A, i and ii) to determine the distribution of the 3GC tRNA (i.e., with CUA anticodon) and the tRNA^fMet. We found that in the case of wild-type SD (SD_wt) the 3GC tRNA bound well to the 30S but not to the 70S ribosome fractions (Fig. 5B, SD_wt). Interestingly, there was more 3GC tRNA on the 70S fractions in the sample with the extended SD context (Fig. 5B, SD_a7g). Quantitative analysis (Fig. 5C) also revealed the same. On the other hand, SD_a7g did not seem to make a change to the distribution of the wild-type tRNA^fMet (Fig. 5 B and C). These results indicate that compensation for the 3GC pair defect by the SD_a7g occurred at a step subsequent to its initial binding to the 30S subunit, most likely at the step of its transition into the 70S complex.

Fig. 5.

Effect of SD_a7g on 3GC tRNA binding. (A) Sucrose density gradients (20–40%) analysis of cell-lysates from KL16 strain harboring pCAT_am1metY_3GC/CUA with SD_wt (i) or SD_a7g (ii). (B) Northern blot analysis for 3GC tRNA and the tRNA^fMet across the profile. (C) The fractional distribution of the tRNAs across the fractions, measured by dividing the individual intensities with the total intensity in all fractions.

Extended SD Also Facilitates Initiation via 70S Pathway.

It had been shown that the extended SD leads to the formation of 70S ribosomal complexes without initiator tRNA (15). Furthermore, the 70S ribosomes are also known to initiate under various specialized conditions (16–20). Thus, although extended SD allowed more of the 3GC tRNA to enter the 70S complex in the standard pathway of 30S mode of initiation (as shown above), it could also be possible that the initiation by 70S ribosomes increased. To check for this possibility, we probed the initiation activity in the presence of kasugamycin, which inhibits the canonical process of 30S initiation but is less effective against 70S initiation (16, 21, 22). Thus, if translation of the CAT reporter mRNA by the 3GC tRNA had an advantage of the 70S mode of initiation, upon kasugamycin addition to the culture, its translation relative to the rest of the cellular mRNAs would be less impacted resulting in a relative increase in CAT. As shown in Fig. 6A, compared with the untreated cells, although there was no significant change in the CAT activity in the SD_wt background (Fig. 6 A, i), there was an increase in the CAT activity in the SD_a7g background (Fig. 6 A, ii) as a result of initiation by the 3GC tRNA. Furthermore, given that the presence of kanamycin (a general inhibitor of protein synthesis) under these conditions actually led to a small decrease in CAT activity (both in the SD_wt and SD_a7g backgrounds, Fig. 6A), an enhancing effect of kasugamycin in the SD_a7g background is significant. Such an effect of kasugamycin was not seen for initiation with the tRNA^fMet (CUA anticodon) possessing the wild-type sequence in the anticodon stem (Fig. S3A). These observations indicate that initiation from the SD_a7g mRNA particularly by the 3GC tRNA, at least in part, used the 70S mode of initiation.

Fig. 6.

Effect of SD_a7g on initiation with 70S ribosome. (A) Initiation with the 3GC tRNA in the context of SD_wt (i) and SD_a7g (ii) of the CAT_am1 reporter upon kasugamycin or kanamycin treatment. Cultures at OD₆₀₀ of 0.5, were split into three parts and CAT activities were determined at various times from the untreated controls or upon treatment with kasugamycin (500 μg/mL), or kanamycin (25 μg/mL). The experiment was carried for three biological replicates for each construct. Fold differences in CAT activity with respect to untreated control at the time of splitting the original culture into three are shown. For reference, the average CAT activities for 3GC tRNA in the context of SD_wt and SD_a7g in untreated KL16 were 92.9 ± 4.0 and 2225.1 ± 656.3 pmol Ac-Cm produced per μg of total protein, respectively. (B and C) Activity assays in MG1655 (RRF^wt) and MG1655 frr^ts (RRF^ts) in the context of SD_wt and SD_a7g CAT_am1 reporters. Cultures were grown to OD₆₀₀ of 0.5 at 30 °C and then either kept at 42 °C (B) or treated with 500 μg/mL kasugamycin (C) and incubated for 2 h before determination of CAT. Fold differences in CAT activity with respect to 3GC in MG1655 (6.2 ± 2.2 pmol Ac-Cm produced per μg total protein) are shown.

In yet another approach, we analyzed initiation activities in a strain (MG1655frr^ts) temperature sensitive (ts) because of a mutation in the ribosome recycling factor (RRF), an essential factor which splits 70S ribosomes into 30S and 50S subunits (23, 24). A deficiency in the RRF activity is known to increase the level of empty 70S complexes and thus enhance the 70S mode of initiation (23). In the MG1655frr^ts strain, increase in initiation by the 3GC tRNA for the SD_a7g construct (compared with SD_wt construct) both at the permissive (Fig. 6B, compare bar 3 with 4) and nonpermissive temperatures (Fig. 6B, compare bar 7 with 8), was higher than in the strain wild-type for RRF (Fig. 6B, bars 1 and 2 and 5 and 6, respectively). Furthermore, although there was a general decrease in translation at 42 °C, the increase for the SD_a7g construct was still higher in frr^ts strain (Fig. 6B, compare bars 6 and 8), supporting the possibility of 70S mode of initiation in extended SD context.

To check for the combined effect of RRF^ts and kasugamycin treatment, the MG1655frr^ts strain was treated with kasugamycin at 30 °C. In this experiment also, the increase in 3GC initiation was higher for the SD_a7g construct than for the SD_wt construct (Fig. 6C, compare bars 2 with 4; and 1 with 3), and it increased further upon treatment with kasugamycin in RRF^ts background (compare bars 2 with 6 and 4 with 8), whereas it had no effect in SD_wt context (compare bars 1 with 5 and 3 with 7). Given that in the RRF^ts background, the level of RRF are lower even at the permissive temperature and it accumulates higher level of 70S ribosomes (23), these observations further support that the 3GC tRNA used the 70S mode of initiation.

Impact of Initiation Factors.

The initiation factors IF1, IF2, and IF3 are involved in the selection of tRNA^fMet in the P-site (7, 25, 26). To investigate the impact of various initiation factors, we overexpressed them from their plasmid copies. For reference, initiation activities of pCAT_am1metY_CUA were taken as 100% (individually for each of the strain). All other activities were represented relative to this activity. As shown in Fig. 7A, the initiation activity from pCAT_am1metY_CUA/3GC (3GC tRNA) in the context of SD_wt or SD_a7g remained nearly unchanged upon overexpression of IF1 (compare activity blocks of IF1, bars 5–8, with those of vector, bars 1–4). Interestingly, overexpression of IF2 caused a significant increase in initiation from pCAT_am1metY_CUA/3GC in the context of SD_a7g (Fig. 7A, compare bars 2 and 10) but not in the SD_wt context (bars 1 and 9). The fact that IF2 functions as association factor between 30S and 50S and drives the reaction to form 70S, the observation further supports the view that initiation by 3GC tRNA used 70S ribosomes.

Fig. 7.

Effect of initiation factors on initiation with 3GC tRNA. (A) Initiation from CAT_am1 containing SD_wt or SD_a7g by 3GC or tRNA^fMet (metY_CUA) tRNAs upon overexpression of IF1 (pACDHinfA), IF2 (pACDHinfB), IF3 (pACDHinfC) or the vector control (pACDH, V). CAT activities are shown with reference to metY_CUA (SD_wt), which was set as 100%, individually for each strain. For reference, CAT activities for metY_CUA tRNA in the context of SD_wt were 6619.2 ± 989.1, 14459.3 ± 1892.4, 29456.6 ± 1760.1 and 7521.2 ± 2312.5 pmol Ac-Cm produced per μg total protein for the strains harboring vector control (V), IF1, IF2 or IF3 overexpression constructs, respectively. (B) Effect of compromising IF3 activity on initiation by 3GC tRNA. CAT assays in KL16 (infC) or infC135. CAT activities (%) with respect to metY_CUA in SD_wt for each strain are as shown. For reference, 100% CAT activities for metY_CUA (SD_wt) in infC and infC135 were 5236.4 ± 1204.5 and 13054.9 ± 2409.3 pmol Ac-Cm produced per μg of total protein, respectively.

Overexpression of IF3 had a small negative effect (27) on the activity from pCAT_am1metY_CUA/3GC in the SD_wt context (Fig. 7A, compare bars 1 and 13) but it had no effect in SD_a7g context (compare bars 2 and 14). However, we observed that compromising IF3 function in infC135 background resulted in increased activity from pCAT_am1metY_CUA/3GC in SD_a7g construct (Fig. 7B, compare wild-type infC with infC135, bars 2 and 6) but the fold increase is less than the SD_wt (Fig. 7B, bars 1 and 5).

Discussion

Characterization of an E. coli isolate, B21, in this study has revealed that a mutation in one of the rRNA genes (rrsC) corresponding to a C to U change at position 1536 of the mature 16S rRNA, resulting in an extended interaction with the SD sequence of the reporter mRNA, allowed initiation to occur with a tRNA^fMet deficient in the highly conserved feature of the three consecutive G-C base pairs in its anticodon stem. This observation was further validated by making a directed mutation in SD sequence of the reporter mRNA to extend its interaction with aSD sequence in the wild-type 16S rRNA. In contrast to the increased initiation by the 3GC tRNA, an extended SD:aSD interaction was suboptimal for initiation by the wild-type tRNA^fMet. Suboptimal initiation by the wild-type tRNA^fMet may be a consequence of lowered efficiency of ribosome clearance from the translation initiation region of the mRNA (14).

Polysome profiling and northern analysis of the fractions showed that the 3GC tRNA binding to the 30S subunit was not the rate-limiting step for its participation in initiation. Thus, it was not unexpected that overexpression of CAT_am1 mRNA (Fig. 2D) did not rescue the initiation defect of the 3GC tRNA. However, compared with the tRNA^fMet, the 3GC tRNA occurrence in the 70S fractions was very low (Fig. 4), suggesting that during transition of the 30S preinitiation complex to 70S complex, the 3GC tRNA was rejected. This observation suggests that the 3GC base pairs play an important role in stabilizing tRNA^fMet during the transition of the 30S preinitiation complex to the 70S complex (Fig. 8A). In yeast, the 3GC tRNAs also showed stable binding to the 40S (28, 29) but not the 80S ribosomes (28), supporting our proposal of the role of the 3GC pairs, a feature of initiator tRNAs conserved in all domains of life. In vitro studies using 70S ribosomes also showed that the 3GC pairs are required for tRNA^fMet binding to the ribosome (4).

Fig. 8.

Role of 3GC base pairs in translation initiation. (A) Translation initiation begins with the binding of initiator tRNA and the initiation factors IF1 (1), IF2 (2), and IF3 (3) to the 30S subunit to generate 30S initiation complex (30S IC) followed by binding of the 50S subunit resulting in 70S complex with tRNA^fMet bound in P/I state (shown by tilted initiator tRNA). An unratcheting movement assisted by the hydrolysis of GTP in IF2 together with the release of initiator factors brings the tRNA into classical state (i). Our results suggest that the 3GC base pairs in the anticodon stem of initiator tRNA play a major role in its retention on the ribosome during these transitions to generate an elongation competent 70S complex. The initial binding of the tRNA^fMet to 30S subunit is independent of the 3GC base pairs. Presence of the extended SD in mRNA partly compensates for this critical requirement. In the absence of the 3GC pairs in the initiator tRNA or an extended SD in mRNA the 70S complex decomposes (ii). (B) The extended SD may also allow bypass of the 3GC base pair requirement by the 70S mode of initiation.

How does an extended SD:aSD allow initiation with the 3GC tRNA? Using single-molecule studies, it was shown that the SD:aSD interaction stabilizes the 70S and it requires more energy to separate the subunits (30). The presence of extended SD allows more 3GC tRNAs to enter the 70S complex (Fig. 5), suggesting that a more stable 70S complex may compensate for the binding energy contributed by the 3GC base pairs. The SD:aSD helix comprising nucleotides 1534–1540 in 16S rRNA interacts with h28 (forms neck of 30S), h23a (forms platform of 30S) and h26. Using translation libration screw (TLS) analyses of the structure of 70S complex containing SD sequence with those lacking it, it was suggested that the presence of the SD:aSD helix reduces the mobility of the head and platform (31). Thus, positioning of the SD:aSD helix may fix the orientation of the mobile head of the 30S subunit for optimal interaction with tRNA^fMet in the 30S P site. It was also shown that in comparison with the vacant 70S ribosome, h26 and h28 move apart by >2 Å to house the SD:aSD helix (31). Such a displacement near the hinge of the neck results in ∼13 Å change in the positions of G1338 and A1339 in the head region. This change may allow 3GC tRNA accommodation in the 70S and lead to initiation. Interestingly, in another crystal structure, which used an extended SD in the mRNA, interaction of G1338 and A1339 with 3GC base pair of initiator tRNA was found to be suboptimal (5). This observation suggests that the discriminatory role of the 3GC pairs is diminished in the context of extended SD:aSD interaction.

IF3 has long been suggested to scrutinize the 3GC base pairs (6, 26, 32). However, this scrutiny may be indirect and negotiated through G1338- and A1339-mediated A-minor interactions (6). In our studies, overexpression of IF3 led to a small decrease in initiation with the 3GC tRNA in the SD_wt context, whereas it had no effect in the SD_a7g context (Fig. 7A). However, an inefficiently functioning IF3 (infC135) led to a 6- to 8-fold increase in initiation with 3GC tRNA in the context of SD_wt, and a 2- to 3-fold increase in the context of the SD_a7g (Fig. 7B). The diminution of IF3 action by the presence of SD sequence was also observed in the context of reinitiation by the 70S ribosomes (33). These observations lend further support to the view that the displacement of G1338 and A1339 (by the extended SD interaction) may mitigate IF3 mediated scrutiny of the 3GC sequence.

Kasugamycin treatment resulted in increased initiation with the 3GC tRNA in the context of SD_a7g, suggesting the possibility of 70S mode of initiation, which is relatively insensitive to kasugamycin (i.e., compared with the 30S mode). Decreasing RRF (RRF^ts) as well as combining the RRF^ts background with kasugamycin treatment further supported the 70S mode of initiation with the extended SD. The 70S mode of initiation is the predominant mode of initiation in the leaderless mRNAs. Importantly, the presence of initiator tRNA is necessary for the formation of 70S complex on the leaderless mRNAs (34). Also, it was shown that the kasugamycin-treated 70S complex had access to bind to SD sequences (23). Taken together, these observations suggest that leadered mRNAs with strong SD sequences may (in converse) facilitate initiation with the tRNAs lacking the 3GC pairs. In addition, the 70S mode of initiation was found to be temperature sensitive (17). Consistent with this observation, we noted that in the strain wild-type for RRF, an increase in the growth temperature resulted in a decrease in initiation from the SD_a7g construct by the 3GC tRNA (Fig. 6B, compare bar 2 with bar 6; see also Fig. S3B). Although maximum pairing of 12 or 13 nucleotides in the SD:aSD helix has been reported (35), most of the mRNAs consist of 4- to 6-base-long SD interactions (36). The most optimal SD:aSD interaction in E. coli (at 37 °C) is 6 nucleotides long, with shorter interactions being optimal at cold temperatures (36).

IF2 acts as association factor and it helps in docking of the 50S subunit onto the 30S preinitiation complex. Overexpression of IF2 has a synergistic effect on initiation with the 3GC tRNA in the extended SD context, most likely by increasing the population of 70S ribosomes. Overexpression of IF2 was also shown to increase the 70S mode of initiation in bicistronic constructs (33). Thus, the conditions that favor 70S formation also lead to increased initiation by the 3GC tRNA especially in the context of an extended SD. In fact, in the classical in vitro experiments designed to decipher the genetic code (37) initiation occurred even with the elongator tRNAs by increasing salt concentration to stabilize the 70S complex, suggesting yet again that 70S mode of initiation may dispense with the requirement of the 3GC pairs in the tRNA.

Thus, our results suggest that the 3GC pairs in the anticodon stem of initiator tRNA play a major role in its retention on the ribosome during transitions to generate an elongation competent 70S complex (Fig. 8 A, i). Presence of the extended SD in mRNA partly compensates for this critical requirement, possibly by stabilizing the 70S complex during these transitions and preventing rejection of the 3GC tRNA. In the absence of the 3GC pairs in the initiator tRNA and an extended SD, the 70S complexes decompose and fail to enter into the elongation step (Fig. 8 A, ii). The current study also suggests that for the 70S mode of initiation, which may do away with the transitional changes needed during the 30S mode of initiation, the requirement of the 3GC pairs is not vital (Fig. 8B). Importantly, although the two mechanisms (Fig. 8 A and B) are not mutually exclusive, they both support that the 3GC pairs play a critical role in tRNA^fMet retention in ribosome during the conformational changes that mark the transition of 30S preinitiation complex into 70S complex.

Finally, the initiation by 70S ribosomes by tRNAs lacking the 3GC pairs may be relevant from an evolutionary consideration. As this mode of initiation does not necessarily require 3GC base pairs, a primitive type of initiation may have been feasible solely by the elongator tRNAs. Initiation process must have evolved subsequent to the process of elongation to direct protein synthesis from a definite point in an mRNA with the coevolution of the 3GC pairs in the tRNA, and a subsequent specialization to initiate with the 30S ribosomes. Interestingly, some species of primitive bacteria, such as mycoplasma, alpha-proteobacteria and also the yeast mitochondria have been observed to lack the full complement of the 3GC pairs in initiator tRNAs.

Materials and Methods

Strains and Plasmids.

Strains and plasmids used in this study have been listed in Table S1. E. coli KL16 and its derivatives were grown in LB and LB–agar plates (Difco). Unless mentioned otherwise, media were supplemented with ampicillin (Amp, 100 µg/mL), chloramphenicol (Cm, 30 µg/mL), kanamycin (Kan, 25 µg/mL), or tetracycline (Tet, 7.5 µg/mL).

Isolation, Characterization, and Genetic Mapping of B21.

The isolation and preliminary characterization of E. coli B21 has been described (9). Mapping of the suppressor mutation was carried out by the methods described in ref. 9.

Site-Directed Mutagenesis.

The pCAT_am1metY_CUA/3GC plasmid was used as template to modify the upstream of CAT_am1 reporter. Using oligomers with desired mutation, inverse PCR was carried out, the amplification product was digested with DpnI and transformed into E. coli TG1, and the desired mutants were confirmed by DNA sequencing. The details of primers are provided in Table S2.

Polysome Profiling.

The total ribosomal preparations from E. coli were carried out as described (15) with few changes. Translation was inhibited by 0.3 μM tetracycline before harvesting the cells, and chilled on ice–salt mix. The buffer 1 contained 10% sucrose. Approximately 10–20 OD₂₆₀ of total RNA was used for profile analysis on 20–40% (wt/vol) (15–35% in some cases) sucrose gradients (buffered in 20 mM Hepes-KOH, pH 7.5, 50 mM NH₄Cl, 10 mM MgCl₂, 4 mM β-mercaptoethanol) were prepared using a BioComp gradient master (BioComp Instruments) in polyclear tubes (SETON, catalog no. 7022). Samples were loaded on gradients and spun in an ultracentrifuge using an SW55 rotor at 45,000 rpm for 2–3 h at 4 °C as described in the figure legends. Gradients were fractionated using a BioComp Gradient Fractionator with the flow rate set to 0.3 mm per second. Subunits were either manually collected by following the absorption trace at 254 nm using a BIO-RAD Econo UV monitor or equal fractions of 200–300 μL volume were collected using fraction collector (BIO RAD Model 2110 Fraction Collector).

RNA Analysis.

Analysis of fractions for the presence of mRNA and tRNA by dot blotting was carried as earlier (15). For analysis by native gel, the RNA from polysome fractions were extracted using hot phenol, ethanol precipitated and separated on native PAGE, transferred on Nytran membrane. The membranes were probed for 16S rRNA, wild-type tRNA^fMet, and the 3GC tRNA, as required.

Preparation of Cell-Free Extracts and CAT Assays.

The midlog phase grown E. coli strains were used for extract preparation as described (9, 38).