Is the cellular initiation of translation an exclusive property of the initiator tRNAs?

Abstract

Translation of mRNAs is the primary function of the ribosomal machinery. Although cells allow for a certain level of translational errors/mistranslation (which may well be a strategic need), maintenance of the fidelity of translation is vital for the cellular function and fitness. The P-site bound initiator tRNA selects the start codon in an mRNA and specifies the reading frame. A direct P-site binding of the initiator tRNA is a function of its special structural features, ribosomal elements, and the initiation factors. A highly conserved feature of the 3 consecutive G:C base pairs (3GC pairs) in the anticodon stem of the initiator tRNAs is vital in directing it to the P-site. Mutations in the 3GC pairs diminish/abolish initiation under normal physiological conditions. Using molecular genetics approaches, we have identified conditions that allow initiation with the mutant tRNAs in Escherichia coli. During our studies, we have uncovered a novel phenomenon of in vivo initiation by elongator tRNAs. Here, we recapitulate how the cellular abundance of the initiator tRNA, and nucleoside modifications in rRNA are connected with the tRNA selection in the P-site. We then discuss our recent finding of how a conserved feature in the mRNA, the Shine-Dalgarno sequence, influences tRNA selection in the P-site.

Keywords:

• anti-Shine-Dalgarno
• aSD
• initiation with elongator tRNAs
• proteome diversity/plasticity
• ribosomal heterogeneity
• Shine-Dalgarno
• SD
• 3GC base pairs
• 70S initiation

Introduction

Translation of an mRNA is a highly regulated process. It is the cellular mechanism to generate proteome diversity without affecting the basic repertoire of the genetic information. Prokaryotes devote more than 30% of their dry mass and ˜95% of the total RNA for the translation machinery itself to ensure accurate and efficient translation.^1,2 Translation of mRNA occurs in the steps of initiation, elongation and termination followed by ribosome recycling for another round of initiation. Initiation is thought to be the most rate-limiting step in translation.³ In eubacteria, the canonical pathway of initiation begins with the formation of a 30S pre-initiation complex (PIC) involving the small subunit of the ribosome (30S), mRNA, initiation factors (IF1, IF2 and IF3) and the initiator tRNA (tRNA^fMet). The tRNA^fMet plays an important role in selecting the start codon. Any failures at this step could lead to mRNA translation in incorrect reading frames and production of mis-translated peptides. Docking of the large subunit of the ribosome (50S) converts the 30S PIC into the 70S initiation complex (IC). In both complexes, tRNA^fMet is present in a tilted conformation between the P/E and P/P state, the P/I state.^4,5 Upon GTP hydrolysis (mediated by IF2), the ribosome undergoes transitions, which locate tRNA^fMet in the P/P state and result in release of the initiation factors (Fig. 1). At this point the 70S complex is ready to accept elongator tRNAs in its A-site.⁵ The selection of tRNA^fMet in the P-site is assisted by the ribosome bound initiation factors, the elements in the P-site and the distinct structural features in tRNA^fMet.^6-17 We have been interested in understanding the mechanism of initiation from a ‘tRNA^fMet-centric' view using an in vivo assay system we developed nearly 3 decades ago.¹⁸ In this short article, we summarize our findings toward understanding the mechanism of initiation in E. coli. To lead up to these findings, we first discuss the salient structural features of the initiator tRNA and the in vivo assay system central to our studies.

Figure 1. Translation initiation in eubacteria. The formation of 30S pre-initiation complex (PIC) involves binding of all the 3 initiation factors IF1, IF2 and IF3 along with tRNA^fMet and mRNA. The tRNA^fMet in this stage is slightly tilted away from the P-site, called P/I state. This complex recruits 50S subunit with the help of IF2 to form 70S IC with the tRNA^fMet in P/I state. This IC undergoes a rotational movement upon GTP hydrolysis to give rise to a 70S complex with the tRNA^fMet in P/P state. This rotation also leads to release of initiation factors from the 70S to convert into an elongation competent 70S.

Initiator tRNA: Structural Features and Cross Talk with the Ribosomal Elements

The prokaryotic tRNA^fMet are characterized by at least 3 special features (Fig. 2). Firstly, they possess a non-Watson-Crick pair (C1 × A72 in E. coli) at the top of the acceptor stem. This feature is an important determinant for, (a) formylation of the amino acid attached to tRNA^fMet, (b) avoidance of binding of the aminoacylated tRNA^fMet to EF-Tu, and (c) prevention of hydrolysis of the formyl-aminoacyl-tRNA^fMet by peptidyl-tRNA hydrolase (Pth), an essential enzyme which recycles tRNAs from the peptidyl-tRNAs that fall off the translating ribosomes.^17,19,20 Secondly, at position 11:24 (in DHU stem), tRNA^fMet possesses a purine:pyrimidine pair in contrast to the other tRNAs, which possess a pyrimidine:purine pair at this position. A known function of this feature is to contribute to the formylation efficiency of the tRNA.¹⁹ Thirdly, a feature that is highly conserved in initiator tRNAs from all domains of life, is the presence of 3 consecutive G:C base pairs (GC/GC/GC or 3GC pairs) in the anticodon stem at positions 29:41, 30:40 and 31:39. The primary function of this feature is to facilitate preferential binding of the initiator tRNA in the ribosomal P-site.^21-23

Figure 2. Cloverleaf structure of E. coli tRNA^fMet2 (metY). Sequences important for its special properties are shown in colored box. CxA mismatch at 1×72 position (shown in orange color) together with the base pairs at 2:71, 3:70 is a major determinant for formylation of aminoacid attached to tRNA^fMet. The 11:24 position (shown in yellow) also affects the efficiency of formylation. The 3 consecutive GC base pairs in anticodon stem (in green box) are important for the ribosomal P- site targeting of the initiator tRNA.

Of the 3 distinctive features in tRNA^fMet, 2 i. e., the presence of a non-Watson-Crick pair at the top of the acceptor stem and the 3GC pairs in the anticodon stem are the most crucial. When incorporated into an elongator tRNA, they allow it to function as an initiator.²⁴ The property of formylation of tRNA^fMet increases its affinity to IF2.^22,25 However, unlike formylation which occurs only in eubacteria, 3GC pairs are almost universally conserved in the initiator tRNAs emphasizing their functional significance.^21,26 Mutational analyses have shown that of the 3GC pairs, the middle GC pair is the most critical one. Interestingly, even though tRNA^fMet retaining just the middle GC pair (and mimicking the unusual tRNA^fMet from some of the mycoplasmas or rhizobia²⁷) functions in initiation, the naturally occurring elongator tRNAs with even 2 GC base pairs (including the middle GC pair) do not. Thus, it appears that the context of the 3GC pairs vis a vis rest of the tRNA may also be important. However, it should also be said that at least in yeast, the requirement of the 3GC pairs in the initiator tRNA seems more flexible as changing the middle GC pair to CG pair could still sustain its growth.²⁸ These observations suggest that the precise mechanistic role of the 3GC pairs in initiation is still an open question. How the 3GC pairs help tRNA^fMet in its function of P-site binding/initiation has been the focus of our studies.

The ribosome bound initiation factors,^6-8 the elements in the P-site such as the 16S rRNA nucleotides G1338 and A1339 (known to interact with the 3GC pairs)^29,30; methylated nucleotides m²G966 and m⁵C967^31,32 and the tails of the ribosomal proteins S9 and S13^31,33,34 etc. are also known to contribute to tRNA^fMet selection in the P-site. Besides, there is evidence that methylations at other positions in 16S rRNA play a role in tRNA^fMet selection.^35-37 The possibility that the rRNA methylations impact the affinity between the 50S and 30S subunits³⁸ needs further investigation. It has been suggested that a rapid rate of 50S subunit docking onto the 30S PIC may adversely impact the fidelity of tRNA^fMet selection in the initiation complex.^7,8

In vivo Assay System, Mutations in the 3GC Base Pairs in tRNA^fMet and Isolation of Suppressors

Our in vivo assay is a plasmid-based system, which employs chloramphenicol (Cm) acetyltransferase (CAT) reporter where the AUG start codon has been mutated to an amber codon (UAG). This reporter (CAT_am1) mRNA fails to confer Cm resistance to E. coli because the cellular tRNA^fMet (with CAU anticodon) does not initiate from the ‘UAG' start codon (Fig. 3). However, when the cells are supplied with the tRNA^fMet whose anticodon is mutated to CUA (from CAU), initiation from the UAG start codon produces CAT protein conferring Cm^R to the cell.^18,22,39 The tRNA^fMet with CUA anticodon is aminoacylated with Gln by GlnRS.¹⁸ When the 3GC pairs in the anticodon stem of the tRNA^fMet are mutated to those found in the elongator tRNA^Met (UA/CG/AΨ, called 3GC mutant tRNA^fMet), the mutant tRNA loses its competence to initiate even though it is efficiently aminoacylated and formylated, rendering the host Cm sensitive (Cm^S). We used this phenotype of Cm^S of the host in a suppressor analysis to characterize mutations in the E. coli chromosome [introduced by N-methyl-N'-nitro-N-nitrosoguanidine, (MNNG) treatment] that conferred Cm^R to the host by enabling the 3GC mutant tRNA^fMet to initiate from the CAT_am1 reporter.³⁶ Identification of the chromosomal mutations in the suppressor strains (Fig. 3) discussed below resulted in characterization of mechanisms that contribute to tRNA^fMet selection in the P-site, and in the fidelity of initiation.

Figure 3. In vivo assay system to investigate the role of 3GC pairs in tRNA^fMet. The plasmid based assay system employs CAT_am1 reporter with amber start codon together with a tRNA^fMet containing CUA anticodon (derived from metY_CUA gene). The metY_CUA encoded tRNA is aminoacylated with Gln and then formylated by 10-formyltetrahydrofolate:L-methionyl-tRNA^fMetN-formyltransferase (Fmt)¹⁸ to produce fGln-tRNA^fMet,CUA. The UAG start codon of CAT_am1 is recognized by metY_CUA encoded tRNA to produce CAT protein, which confers chloramphenicol resistance (Cm^R) to the cell (A). When the 3GC base pairs in the metY_CUA encoded tRNA are changed to UA/CG/AΨ pairs (3GC mutation), the mutant tRNA does not function in initiation (even though it is efficiently aminoacylated and formylated) rendering the cell Cm^S (B). Extragenic mutations that rescue the initiation defect of the 3GC mutant tRNA allow initiation to occur from the UAG start codon of CAT_am1 mRNA to produce CT and confer Cm^R (C).

Contribution of Cellular Initiator tRNA Levels

In E. coli, tRNA^fMet is encoded by 2 loci, metZWV at 63.5 min and metY at 71.5 min. The metZWV locus possesses 3 genes in an operon encoding tRNA^fMet1 and the metY locus possesses a single tRNA^fMet gene (as a first gene of the metY-nusA-infB operon) encoding tRNA^fMet2.⁴⁰ In E. coli B strains, both the loci encode identical tRNA^fMet.⁴¹ However, in E. coli K strains, the metY encoded tRNA^fMet differs in that it possesses an A (instead of m⁷G) at position 46⁴¹ with no apparent functional differences. The metZWV encoded tRNA^fMet contributes to about 75% of the total tRNA^fMet in E. coli. In an earlier work, one class of the suppressor strains which fostered initiation with the 3GC mutant tRNA^fMet, we observed mutations in the metZWV promoter which led to a severe down-regulation of the chromosomally encoded tRNA^fMet.⁴² Further, deletion of the metZWV locus also allowed initiation with the 3GC mutant tRNA^fMet suggesting that 3GC pairs offer a competitive advantage to tRNA^fMet to bind the P-site. In the ΔmetZWV strain we observed that not only the unusual tRNA^fMet of the mycoplasma and rhizobia origin (lacking either the first, the third or both the first and the third GC pairs) but also the elongator tRNAs initiated protein synthesis.^42,43 In fact, initiation by the unusual tRNA^fMet sustained growth of E. coli in the absence of all 4 of the tRNA^fMet genes.²⁷ Normally these unusual tRNAs fail to initiate in E. coli to any significant level. Thus, although a higher number of initiator tRNA genes has been attributed to overcome the rate limiting step of initiation, our studies suggest that higher cellular abundance of tRNA^fMet makes a definite contribution to the fidelity of initiation by preventing initiation with elongator tRNAs. The relative levels of tRNA^fMet and elongator tRNAs and their aminoacylated forms are known to vary under stress.^44-47 At least in E. coli, ppGpp as well as cAMP-CAP regulate transcription of tRNA^fMet genes.^48,44 Thus, it is possible that under the stressful conditions, initiation (from noncanonical positions) by elongator tRNAs generates novel polypeptides by translation in alternate or the original reading frames. Physiological relevance of any such novel polypeptides is unknown. However, the potential to initiate with elongator tRNAs may serve as a cellular strategy to generate proteome diversity which in turn may rescue cells from stress. Interesting as the observation of in vivo initiation with elongator tRNAs is, it raises several questions. How are these tRNAs recruited to the ribosome? Also, as initiation with elongator tRNAs is competed out by tRNA^fMet, the elongators must initiate from the P-site. If so, is the relative affinity of tRNAs for EF-Tu and IF2 somehow influenced? Could an elongator tRNA (in its EF-Tu bound form) reposition itself into the P-site for initiation? Given that E. coli can grow in the absence of formylation⁴⁹ and that an EF-Tu bound aminoacyl-tRNA may be re-sampled⁵⁰ by other proteins in cell, answers to these questions may not be improbable.

Contribution of Shine-Dalgarno (SD): anti-SD (aSD) Interaction

Recently, we observed that in yet another suppressor strain, a mutation (C1536T) in the aSD sequence of a 16S rRNA gene which resulted in an extended pairing of 8 (from the pre-existing 6) base pairs with the SD sequence of the CAT_am1 mRNA enabled initiation with the 3GC mutant tRNA^fMet.⁵¹ Similarly, a mutation in the SD sequence of the CAT_am1 reporter mRNA resulting in its extended interaction of 8 base pairs in the same region of aSD of the wild-type 16S rRNA also resulted in initiation with the 3GC mutant tRNA^fMet. While the exact mechanism remains to be investigated, we observed that the extended SD:aSD interaction resulted in an increased occupancy of the 3GC mutant tRNA^fMet onto the elongation competent 70S ribosomes suggesting that at least one of the roles of the 3GC pairs in the native tRNA^fMet is to facilitate its transition from 30S PIC to the 70S complex. The 3GC pairs may be ‘licensing' the tRNA^fMet to pass through the multistep process of the scrutiny by initiation factors (e. g. IF3^6,7,29) and/or the conformational changes the ribosome undergoes (e. g. upon GTP hydrolysis⁵) to yield an elongation competent 70S (Fig. 1). A systematic mutational analysis of the 3GC base pairs in tRNA^fMet might offer further insights into these processes. For example, in our earlier studies, we showed that overproduction of the 3GC mutant tRNA^fMet could also lead to a minor increase in initiation, an observation consistent with the role of 3GC base pairs in the direct binding of the tRNA^fMet to the 30S ribosome.^42,43 It is also known that an extended SD stabilizes the 70S complex.^52,53 Consistent with this, we observed that kasugamycin treatment (which is known to allow 70S mode of initiation in leaderless mRNAs^54–56) offered a selective advantage to initiation with the 3GC mutant tRNA^fMet from the CAT_am1 mRNA with the extended SD sequence. Other conditions such as increased IF2 levels or decreased RRF activity or decreased IF3 activity (conditions that increase 70S population in cell) also facilitate initiation with the 3GC mutant tRNA^fMet. These observations suggest that the requirement of the 3GC pairs is more acute for the canonical mode of initiation from 30S ribosome. The wild-type tRNA^fMet is known to facilitate recruitment of a leaderless mRNA for translation in the 70S mode of initiation.⁵⁷ Conversely, could a stably bound mRNA in 70S ribosome facilitate P-site recruitment of a tRNA via codon:anticodon interaction?

G1338 and A1339, 2 of the highly conserved residues of 16S rRNA, have been shown to form type II and type I A-minor interactions with the first and the middle GC base pairs of the 3 GC pairs, respectively.³³ However, as the absence of any of the GC pairs affects the efficiency of initiation, it may well be that during the various stages (transitions) of the initiation process, G1338/A1339 make dynamic interactions with all the 3 of the GC base pairs to offer discrimination between tRNA^fMet and the elongator tRNAs.^29,58,59 Thus, any displacements in the locations of G1338 and A1339 may impact the selection/retention of a tRNA in the P-site. In this context, it has been suggested⁶⁰ that the h26 and h28 in 30S move apart by ∼2Å upon interaction between SD and aSD sequences resulting in a displacement of G1338 and A1339 by ˜13 Å compared with their location in the empty ribosomes (without mRNA or tRNA). In the absence of availability of ribosome structures that directly address these changes, it is unclear how such differences might impact the scrutiny of the 3GC pairs. However, among the 3 initiation factors, IF3 plays an important role in selection of 3GC pairs^6,61 through G1338 and A1339.²⁹ A displacement of these residues due to an extended SD:aSD interaction might attenuate the IF3 mediated scrutiny of the 3GC pairs, and allow initiation with 3GC mutant tRNAs. At least, from the perspective of the genetic analyses, an extended SD:aSD interaction seems to compensate for the conformational changes that might be brought about by the interaction of the G1338 and A1339 with the 3GC pairs of the tRNA^fMet to allow initiation to proceed even in the absence of the 3GC pairs.

Outlook

Using 3GC mutant tRNA^fMet in our genetic analyses, we have explored cellular mechanisms that contribute to the maintenance of fidelity of translation initiation, especially in the selection of tRNA^fMet in the P-site. Interestingly, these studies have also uncovered cellular strategies that generate heterogeneity in the ribosomal pool^36,38 (for example, a combinatorial effect of lesser than quantitative modifications of the known nucleoside positions would generate an enormous diversity/heterogeneity of arrangements of rRNA modifications available in the individual ribosomes), which may allow selective/preferential translation of mRNAs in cell to generate proteome diversity/plasticity. Earlier studies have shown heterogeneity in the ribosomal pool that arose as a consequence of various environmental cues or changes in the toxin-antitoxin systems.^62,63 Cellular abundance of initiator tRNA has been thought to be important in regulating the rate-limiting step of initiation. Revelation that the potential of elongator tRNAs to initiate is unleashed by decreasing pools of initiator tRNA, and given that relative levels of initiator/elongator tRNAs in the cell do change,⁴⁸ it seems that alternative sites of initiation determined by the elongator tRNAs may generate novel polypeptides to create another level of proteome diversity/plasticity in the cell. Such diversity may be crucial for the cell to re-program cellular physiology to tide over the stressful conditions. There are circumstances where initiator tRNA levels are transcriptionally down-regulated^44,48 or diminished by ribonucleases such as VapC.⁶⁴ Furthermore, although not yet experimentally tested, our recent finding that extended SD:aSD interaction allows for initiation with an initiator tRNA lacking the highly conserved feature of the 3GC pairs suggests that the natural occurrences of SD sequences in translation initiation regions or at internal positions could also impact the extent and/or sites of initiation by elongator tRNAs. It is known that there are various mRNAs with extended SD sequences in the upstream or within the coding regions which lead to pausing of ribosomes.⁶⁵ Preference for SD sequence uses has already been shown to vary with temperature.⁶⁶ It is also of interest to note that the 70S ribosomes from E. coli with low levels of tRNA^fMet contained 15% less S1 protein.⁴³ Earlier studies have shown that S1 depleted ribosomes use 70S mode of initiation in leaderless mRNAs.^55,57 Thus, it seems that cells may exploit a number of strategies available to it to generate proteome diversity. However, a challenge that these observations pose is to design methods to detect and identify minor populations of peptides/proteins, which are to likely arise by initiation from the alternative sites. Clearly, more sensitive genetic and biochemical assays would need to be developed to investigate into the possibility of the impact of the alternative means of initiation by elongator tRNAs.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Funding

The work in the investigator's laboratory is supported by funds from the Department of Science and Technology (DST), Department of Biotechnology (DBT), and the Council of Scientific and Industrial Research (CSIR), New Delhi. U.V. is a J.C. Bose fellow of DST. S.S. is supported by a Shyama Prasad Mukherjee SRF of CSIR. S.B. was supported by an SRF of CSIR.