An Alternative Role of RluD in the Fidelity of Translation Initiation in Escherichia coli

The fidelity of initiator tRNA (i-tRNA) selection in the ribosomal P-site is a key step in translation initiation. The highly conserved three consecutive G:C base pairs (3GC pairs) in the i-tRNA anticodon stem play a crucial role in its selective binding in the P-site. Mutations in the 3GC pairs (3GC mutant) render the i-tRNA inactive in initiation. Here, we show that a mutation (E265K) in the unique C-terminal tail domain of RluD, a large ribosomal subunit pseudouridine synthase, results in compromised fidelity of initiation and allows initiation with the 3GC mutant i-tRNA. RluD modifies the uridine residues in H69 to pseudouridines. However, the role of its C-terminal tail domain remained unknown. The E265K mutation does not diminish the pseudouridine synthase activity of RluD, or the growth phenotype of Escherichia coli, or cause any detectable defects in the ribosomal assembly in our assays. However, in our in vivo analyses, we observed that the E265K mutation resulted in increased retention of the ribosome binding factor A (RbfA) on 30S suggesting a new role of RluD in contributing to RbfA release, a function which may be attributed to its (RluD) C-terminal tail domain. The studies also reveal that deficiency of RbfA release from 30S compromises the fidelity of i-tRNA selection in the ribosomal P-site.     

Functional Interaction Between Release Factor One and P-site Peptidyl-tRNA on the Ribosome

Translation termination at UAG is influenced by the nature of the 5′ flanking codon inEscherichia coli. Readthrough of the stop codon is always higher in a strain with mutant (prfA1) as compared to wild-type (prfA⁺) release factor one (RF1). Isocodons, which differ in the last base and are decoded by the same tRNA species, affect termination at UAG differently in strains with mutant or wild-type RF1. No general preference of the last codon base to favour readthrough or termination can be found. The data suggest that RF1 is sensitive to the nature of the wobble base anticodon-codon interaction at the ribosomal peptidyl-tRNA binding site (P-site). For some isoaccepting P-site tRNAs (tRNA₃^ProversustRNA₂^Pro, tRNA₄^ThrversustRNA_1,3^Thr) the effect is different on mutant and wild-type RF1, suggesting an interaction between RF1 at the aminoacyl-tRNA acceptor site (A-site) and the P-site tRNA itself. The glycine codons GGA (tRNA₂^Gly) and GGG (tRNA_2,3^Gly) at the ribosomal P-site are associated with an almost threefold higher readthrough of UAG than any of the other 42 codons tested, including the glycine codons GGU/C, in a strain with wild-type RF1. This differential response to the glycine codons is lost in the strain with the mutant form of RF1 since readthrough is increased to a similar high level for all four glycine codons. High α-helix propensity of the last amino acid residue at the C-terminal end of the nascent peptide is correlated with an increased termination at UAG. The effect is stronger on mutant compared to wild-type RF1. The data suggest that RF1-mediated termination at UAG is sensitive to the nature of the codon-anticodon interaction of the wobble base, the last amino acid residue of the nascent peptide chain, and the tRNA at the ribosomal P-site.     

The ribosome‐bound initiation factor 2 recruits initiator tRNA to the 30S initiation complex

Bacterial translation initiation factor 2 (IF2) is a GTPase that promotes the binding of the initiator fMet‐tRNA^fMet to the 30S ribosomal subunit. It is often assumed that IF2 delivers fMet‐tRNA^fMet to the ribosome in a ternary complex, IF2·GTP·fMet‐tRNA^fMet. By using rapid kinetic techniques, we show here that binding of IF2·GTP to the 30S ribosomal subunit precedes and is independent of fMet‐tRNA^fMet binding. The ternary complex formed in solution by IF2·GTP and fMet‐tRNA is unstable and dissociates before IF2·GTP and, subsequently, fMet‐tRNA^fMet bind to the 30S subunit. Ribosome‐bound IF2 might accelerate the recruitment of fMet‐tRNA^fMet to the 30S initiation complex by providing anchoring interactions or inducing a favourable ribosome conformation. The mechanism of action of IF2 seems to be different from that of tRNA carriers such as EF‐Tu, SelB and eukaryotic initiation factor 2 (eIF2), instead resembling that of eIF5B, the eukaryotic subunit association factor.     

Conformational change of Escherichia coli initiator methionyl-tRNA(fMet) upon binding to methionyl-tRNA formyl transferase

The specific formylation of initiator methionyl-tRNA (Met-tRNA) by methionyl-tRNA formyltransferase (MTF) is important for the initiation of protein synthesis in Escherichia coli. The determinants for formylation are located in the acceptor stem and in the dihydrouridine (D) stem of the initiator tRNA (tRNA^fMet). Here, we have used ethylation interference analysis to study the interactions between the Met-tRNA^fMet and MTF in solution. We have identified three clusters of phosphates in the tRNA that, when ethylated, interfere with binding of MTF. Interference due to ethylation of phosphates in the acceptor stem and in the D stem is most likely due to the close proximity of the protein as seen in the crystal structure of the MTF.fMet-tRNA^fMet complex. The third cluster of phosphates, whose ethylation interferes with binding of MTF, is dispersed along the anticodon stem, which is distal to the sites of tRNA protein contacts. Interestingly, these latter positions correspond to sites of increased cleavages by RNase V1 in RNA footprinting experiments. Together, these results suggest that in addition to the protein, which binds to the substrate tRNA in an induced fit mechanism, the tRNA also undergoes induced structural changes during its binding to MTF.     

Escherichia coli formylmethionine tRNA: methylation of specific guanine and adenine residues catalyzed by HeLa cells tRNA methylases and the effect of these methylations on its biological properties

Purified HeLa cell tRNA methylases have been used for site-specific methylations of Escherichia coli formylmethionine transfer ribonucleic acid (tRNA^fMet). Guanine-N²-methylase catalyzed the methylation of a specific guanine residue (G₂₇) and adenine-1-methylase that of a specific adenine residue (A₅₉). The combined action of both of these enzymes leads to a total incorporation of two methyl groups and results in the methylation of both G₂₇ and A₅₉.The effect of introducing additional methyl groups on the function of tRNA has been studied by a comparison in vitro of the biological properties of tRNA^fMet and enzymically methylated tRNA^fMet. It was found that none of the following properties of E. coli tRNA^fMet are altered to any significant extent by methylation: (a) rate, extent, and specificity of aminoacylation, (b) ability of methionyl-tRNA to be enzymically formylated, and (c) ability of formylmethionyl-tRNA to initiate protein synthesis in cell-free extracts of E. coli in the presence of f2 RNA as messenger. Also, the temperature versus absorbance profile of the doubly methylated tRNA^fmet was virtually identical to that of the E. coli tRNA^fMet, and enzymically methylated tRNA^fmet resembled tRNA^fMet in that both were resistant to deacylation by E. coli, N-acylaminoacyl-tRNA hydrolase.     

tRNA methylases from HeLa cells: purification and properties of an adenine-1-methylase and a guanine-N2-methylase

Two enzymes (methylases) that catalyze the transfer of methyl groups from S-adenosyl-l-methionine to tRNA (prepared from Escherichia coli) have been partially purified from extracts of HeLa cells. One catalyzes the methylation of adenine residues of the tRNA to give 1-methyladenine units and the other is responsible for the conversion of guanine residues to N²-methylguanine and N²,N²-dimethylguanine (and may be a mixture of two enzymes). Activities of these relatively unstable enzymes could be maintained by storage at ?20 °C in the presence of 50% glycerol. Substrate specificity studies have revealed that bacterial tRNA (E. coli, Bacillus subtilis) can be used as substrate, whereas tRNA of animal origin (HeLa cells, rat liver) cannot be used. Of the specific tRNA's tested, E. coli tRNA^fMet was used as substrate by both enzymes. E. coli tRNA^Tyr was used by the adenine-1-methylase but not by the guanine-N²-methylase. The adenine-1-methylase catalyzed the transfer of approximately one methyl group per mole of either tRNA^fMet or tRNA^Tyr offered as substrate; in the presence of the guanine-N²-methylase 1 mole of E. coli tRNA^fMet accepted 1 mole of methyl. Studies with the use of both enzymes established that enzymic methylation of the guanine site of E. coli tRNA^fMet did not interfere with subsequent methylation of an adenine residue and neither did prior methylation of adenine interfere with the subsequent methylation of a guanine residue. In the presence of both enzymes, approximately 2 moles of methyl groups were accepted by 1 mole of the E. coli tRNA^fMet.     

Role of 16S ribosomal RNA methylations in translation initiation in Escherichia coli

Translation initiation from the ribosomal P-site is the specialty of the initiator tRNAs (tRNA(fMet)). Presence of the three consecutive G-C base pairs (G29-C41, G30-C40 and G31-C39) in their anticodon stems, a highly conserved feature of the initiator tRNAs across the three kingdoms of life, has been implicated in their preferential binding to the P-site. How this feature is exploited by ribosomes has remained unclear. Using a genetic screen, we have isolated an Escherichia coli strain, carrying a G122D mutation in folD, which allows initiation with the tRNA(fMet) containing mutations in one, two or all the three G-C base pairs. The strain shows a severe deficiency of methionine and S-adenosylmethionine, and lacks nucleoside methylations in rRNA. Targeted mutations in the methyltransferase genes have revealed a connection between the rRNA modifications and the fundamental process of the initiator tRNA selection by the ribosome.     

Contribution of ribosomal residues to P-site tRNA binding

Structural studies have revealed multiple contacts between the ribosomal P site and tRNA, but how these contacts contribute to P-tRNA binding remains unclear. In this study, the effects of ribosomal mutations on the dissociation rate (k_off) of various tRNAs from the P site were measured. Mutation of the 30S P site destabilized tRNAs to various degrees, depending on the mutation and the species of tRNA. These data support the idea that ribosome-tRNA interactions are idiosyncratically tuned to ensure stable binding of all tRNA species. Unlike deacylated elongator tRNAs, N-acetyl-aminoacyl-tRNAs and tRNA^fMet dissociated from the P site at a similar low rate, even in the presence of various P-site mutations. These data provide evidence for a stability threshold for P-tRNA binding and suggest that ribosome-tRNA^fMet interactions are uniquely tuned for tight binding. The effects of 16S rRNA mutation G1338U were suppressed by 50S E-site mutation C2394A, suggesting that G1338 is particularly important for stabilizing tRNA in the P/E site. Finally, mutation C2394A or the presence of an N-acetyl-aminoacyl group slowed the association rate (k_on) of tRNA dramatically, suggesting that deacylated tRNA binds the P site of the ribosome via the E site.     

Understanding the Sequence Specificity of tRNA Binding to Elongation Factor Tu using tRNA Mutagenesis

Measuring the binding affinities of 42 single-base-pair mutants in the acceptor and TΨC stems of Saccharomyces cerevisiae tRNA^Phe to Thermus thermophilus elongation factor Tu (EF-Tu) revealed that much of the specificity for tRNA occurs at the 49-65, 50-64, and 51-63 base pairs. Introducing the same mutations at the three positions into Escherichia coli tRNA_CAG^Leu resulted in similar changes in binding affinity. Swapping the three pairs from several E. coli tRNAs into yeast tRNA^Phe resulted in chimeras with EF-Tu binding affinities similar to those for the donor tRNA. Finally, analysis of double- and triple-base-pair mutants of tRNA^Phe showed that the thermodynamic contributions at the three sites are additive, permitting reasonably accurate prediction of the EF-Tu binding affinity for all E. coli tRNAs. Thus, it appears that the thermodynamic contributions of three base pairs in the TΨC stem primarily account for tRNA binding specificity to EF-Tu.     

The human large subunit ribosomal protein L36A-like contacts the CCA end of P-site bound tRNA

Periodate-oxidized tRNA (tRNAox), the 2′,3′-dialdehyde derivative of tRNA, was used as a zero-length active site-directed affinity labeling reagent, to covalently label proteins at the binding site for the 3′-end of tRNA on human 80S ribosomes. When human 80S ribosomes were reacted with tRNA^Aspox positioned at the P-site, in the presence of an appropriate 12 mer mRNA, a set of two tRNAox-labeled ribosomal proteins (rPs) was observed. The majorily labeled protein was identified as the large subunit rP L36a-like (RPL36AL) by means of mass spectrometry. Intact tRNA^Asp competed with tRNA^Aspox for the binding to the P-site, by preventing tRNA-protein cross-linking with RPL36AL. Altogether, the data presented in this report are consistent with the presence of RPL36AL at or near the binding site for the CCA end of the tRNA substrate positioned at the P-site of human 80S ribosomes. It is the first time that a ribosomal protein is found in an intimate contact (i.e. at a zero-distance) with a nucleotide of the conserved CCA terminus of P-site tRNA which is the substrate of peptidyl transferase reaction. RPL36AL which is strongly conserved in eukaryotes belongs to the L44e family of rPs, a representative of which is Haloarcula marismortui RPL44e.     

RNA modifications stabilize the tertiary structure of tRNAfMet by locally increasing conformational dynamics

A plethora of modified nucleotides extends the chemical and conformational space for natural occurring RNAs. tRNAs constitute the class of RNAs with the highest modification rate. The extensive modification modulates their overall stability, the fidelity and efficiency of translation. However, the impact of nucleotide modifications on the local structural dynamics is not well characterized. Here we show that the incorporation of the modified nucleotides in tRNA^fMet from Escherichia coli leads to an increase in the local conformational dynamics, ultimately resulting in the stabilization of the overall tertiary structure. Through analysis of the local dynamics by NMR spectroscopic methods we find that, although the overall thermal stability of the tRNA is higher for the modified molecule, the conformational fluctuations on the local level are increased in comparison to an unmodified tRNA. In consequence, the melting of individual base pairs in the unmodified tRNA is determined by high entropic penalties compared to the modified. Further, we find that the modifications lead to a stabilization of long-range interactions harmonizing the stability of the tRNA’s secondary and tertiary structure. Our results demonstrate that the increase in chemical space through introduction of modifications enables the population of otherwise inaccessible conformational substates.     

Investigation of the thermal unfolding of secondary and tertiary structure in E. coli tRNAfMet by high-resolution nmr

The high-resolution (300 MHz) proton nmr spectrum of E. coli tRNA^fMet has been examined in 0.17M NaCl, with and without Mg²⁺, and at various temperatures. In light of recent studies of other E. coli tRNA and fragments of tRNA^fMet, some low field (11–15 ppm) resonances previously assigned to secondary structure base pairs are reassigned to a tertiary structure A₁₄–S⁴U₈ base pair and a protected uridine residue in the anticodon loop. These two resonances and other low field resonances which are assigned to secondary structure base pairs are used to monitor the thermal unfolding of the molecule. In the absence of Mg²⁺ the tertiary structure base pair is present only to ～45°C, but in the presence of Mg²⁺ it remains until at least 70°C. Analysis of the temperature dependence of other low field resonances indicates that the melting of the dihydrouridine stem occurs more or less simultaneously with the loss of tertiary structure. The observation of the resonance from the A₁₄–S⁴U₈ base pair proves that tertiary structure is present in this molecule below 40°C, even in the absence of Mg²⁺.     

Yeast initiator tRNA identity elements cooperate to influence multiple steps of translation initiation

All three kingdoms of life employ two methionine tRNAs, one for translation initiation and the other for insertion of methionines at internal positions within growing polypeptide chains. We have used a reconstituted yeast translation initiation system to explore the interactions of the initiator tRNA with the translation initiation machinery. Our data indicate that in addition to its previously characterized role in binding of the initiator tRNA to eukaryotic initiation factor 2 (eIF2), the initiator-specific A1:U72 base pair at the top of the acceptor stem is important for the binding of the eIF2.GTP.Met-tRNA(i) ternary complex to the 40S ribosomal subunit. We have also shown that the initiator-specific G:C base pairs in the anticodon stem of the initiator tRNA are required for the strong thermodynamic coupling between binding of the ternary complex and mRNA to the ribosome. This coupling reflects interactions that occur within the complex upon recognition of the start codon, suggesting that these initiator-specific G:C pairs influence this step. The effect of these anticodon stem identity elements is influenced by bases in the T loop of the tRNA, suggesting that conformational coupling between the D-loop-T-loop substructure and the anticodon stem of the initiator tRNA may occur during AUG codon selection in the ribosomal P-site, similar to the conformational coupling that occurs in A-site tRNAs engaged in mRNA decoding during the elongation phase of protein synthesis.     

Control of translation initiation involves a factor-induced rearrangement of helix 44 of 16S ribosomal RNA

Initiation of translation involves recognition of the start codon by the initiator tRNA in the 30S subunit. To investigate the role of ribosomal RNA (rRNA) in this process, we isolated a number of 16S rRNA mutations that increase translation from the non-canonical start codon AUC. These mutations cluster to distinct regions that overlap remarkably well with previously identified class III protection sites and implicate both IF1 and IF3 in start codon selection. Two mutations map to the 790 loop and presumably act by inhibiting IF3 binding. Another cluster of mutations surrounds the conserved A1413∘G1487 base pair of helix 44 in a region known to be distorted by IF1 and IF3. Site-directed mutagenesis in this region confirmed that this factor-induced rearrangement of helix 44 helps regulate initiation fidelity. A third cluster of mutations maps to the neck of the 30S subunit, suggesting that the dynamics of the head domain influences translation initiation. In addition to identifying mutations that decrease fidelity, we found that many P-site mutations increase the stringency of start codon selection. These data provide evidence that the interaction between the initiator tRNA and the 30S P site is tuned to balance efficiency and accuracy during initiation.     

Synthesis and functional activity of tRNAs labeled with fluorescent hydrazides in the D-loop

We describe an optimized procedure for replacing the dihydrouridine residues of charged tRNAs with Cy3 and Cy5 dyes linked to a hydrazide group, and demonstrate that the labeled molecules are functional in ribosomal activities including 30S initiation complex formation, EF–Tu-dependent binding to the ribosome, translocation, and polypeptide synthesis. This procedure should be straightforwardly generalizable to the incorporation of other hydrazide-linked fluorophores into tRNA or other dihydrouridine-containing RNAs. In addition, we use a rapid turnover FRET experiment, measuring energy transfer between Cy5-labeled tRNA^fMet and Cy3-labeled fMetPhe-tRNA^Phe, to obtain direct evidence supporting the hypothesis that the early steps of translocation involve movements of the flexible 3′-single-stranded regions of the tRNAs, with the considerable increase in the distance separating the two tRNA tertiary cores occurring later in the process.     

A new kinetic model reveals the synergistic effect of E-, P- and A-sites on +1 ribosomal frameshifting

Programmed ribosomal frameshifting (PRF) is a process by which ribosomes produce two different polypeptides from the same mRNA. In this study, we propose three different kinetic models of +1 PRF, incorporating the effects of the ribosomal E-, P- and A-sites toward promoting efficient +1 frameshifting in Escherichia coli. Specifically, the timing of E-site tRNA dissociation is discussed within the context of the kinetic proofreading mechanism of aminoacylated tRNA (aa-tRNA) selection. Mathematical modeling using previously determined kinetic rate constants reveals that destabilization of deacylated tRNA in the E-site, rearrangement of peptidyl-tRNA in the P-site, and availability of cognate aa-tRNA corresponding to the A-site act synergistically to promote efficient +1 PRF. The effect of E-site codon:anticodon interactions on +1 PRF was also experimentally examined with a dual fluorescence reporter construct. The combination of predictive modeling and empirical testing allowed the rate constant for P-site tRNA slippage (k_s) to be estimated as k_s ≈1.9 s⁻¹ for the release factor 2 (RF2) frameshifting sequence. These analyses suggest that P-site tRNA slippage is the driving force for +1 ribosomal frameshifting while the presence of a ‘hungry codon’ in the A-site and destabilization in the E-site further enhance +1 PRF in E. coli.     

Role of the three consecutive G:C base pairs conserved in the anticodon stem of initiator tRNAs in initiation of protein synthesis in Escherichia coli.

The three consecutive G:C base pairs, G29:C41, G30:C40, and G31:C39, are conserved in the anticodon stem of virtually all initiator tRNAs from eubacteria, eukaryotes, and archaebacteria. We show that these G:C base pairs are important for function of the tRNA in initiation of protein synthesis in vivo. We changed these base pairs individually and in combinations and analyzed the activities of the mutant Escherichia coli initiator tRNAs in initiation in vivo. For assessment of activity of the mutant tRNAs in vivo, mutations in the G:C base pairs were coupled to mutation in the anticodon sequence from CAU to CUA. Mutations in each of the G:C base pairs reduced activity of the mutant tRNA in initiation, with mutation in the second G:C base pair having the most severe effect. The greatly reduced activity of this C30:G40 mutant tRNA is not due to defects in aminoacylation or formulation of the tRNA or defects in base modification of the A37, next to the anticodon, which we had previously shown to be important for activity of the mutant tRNAs in initiation. The anticodon stem mutants are most likely affected specifically at the step of binding to the ribosomal P site. The pattern of cleavages in the anticodon loop of mutant tRNAs by S1 nuclease indicate that the G:C base pairs may be involved directly in interactions of the tRNA with components of the P site on the ribosome rather than indirectly by inducing a particular conformation of the anticodon loop critical for function of the tRNA in initiation.     

Recycling of ribosomal complexes stalled at the step of elongation in Escherichia coli

Translating ribosomes often stall during elongation. The stalled ribosomes are known to be recycled by tmRNA (SsrA)-mediated trans-translation. Another process that recycles the stalled ribosomes is characterized by peptidyl-tRNA release. However, the mechanism of peptidyl-tRNA release from the stalled ribosomes is not well understood. We used a defined system of an AGA-minigene containing a small open reading frame (ATG AGA AGA). Translation of the AGA-minigene mRNA is toxic to Escherichia coli because it stalls ribosomes during elongation and sequesters tRNA^Arg4 as a short-chain peptidyl-tRNA^Arg4 in the ribosomal P-site. We show that a ribosome recycling factor (RRF)-mediated process rescues the host from the AGA-minigene toxicity by releasing the peptidyl-tRNA^Arg4 from the ribosomes. The growth phenotypes of E. coli strains harboring mutant alleles of RRF and initiation factor 3 (IF3) genes and their consequences on λimmP22 phage replication upon AGA-minigene expression reveal that IF3 facilitates the RRF-mediated processing of the stalled ribosomes. Additionally, we have designed a uracil DNA glycosylase gene construct, ung-stopless, whose expression is toxic to E. coli. We show that the RRF-mediated process also alleviates the ung-stopless construct-mediated toxicity to the host by releasing the ung mRNA from the ribosomes harboring long-chain peptidyl-tRNAs.     

The Ribosomal Grip of the Peptidyl-tRNA is Critical for Reading Frame Maintenance

If a ribosome shifts to an alternative reading frame during translation, the information in the message is usually lost. We have selected mutants of Salmonella typhimurium with alterations in tRNA_cmo5UGG^Pro that cause increased frameshifting when present in the ribosomal P-site. In 108 such mutants, two parts of the tRNA molecule are altered: the anticodon stem and the D-arm, including its tertiary interactions with the variable arm. Some of these alterations in tRNA_cmo5UGG^Pro are in close proximity to ribosomal components in the P-site. The crystal structure of the 30S subunit suggests that the C-terminal end of ribosomal protein S9 contacts nucleotides 32-34 of peptidyl-tRNA. We have isolated mutants with defects in the C-terminus of S9 that induce + 1 frameshifting. Combinations of changes in tRNA_cmo5UGG^Pro and S9 suggest that an interaction occurs between position 32 of the peptidyl-tRNA and the C-terminal end of S9. Together, our results suggest that the cause of frameshifting is an aberrant interaction between the peptidyl-tRNA and the P-site environment. We suggest that the “ribosomal grip” of the peptidyl-tRNA is pivotal for maintaining the reading frame.     

The Role of L1 Stalk-tRNA Interaction in the Ribosome Elongation Cycle

The ribosomal L1 stalk is a mobile structure implicated in directing tRNA movement during translocation through the ribosome. This article investigates three aspects of L1 stalk-tRNA interaction. First, by combining data from cryo electron microscopy, X-ray crystallography, and molecular dynamics simulations through the molecular dynamics flexible fitting method, we obtained atomic models of different tRNAs occupying the hybrid P/E state interacting with the L1 stalk. These models confirm the assignment of fluorescence resonance energy transfer states from previous single-molecule investigations of L1 stalk dynamics. Second, the models reconcile how initiator tRNA^fMet interacts less strongly with the L1 stalk compared to elongator tRNA^Phe, as seen in previous single-molecule experiments. Third, results from a simulation of the entire ribosome in which the L1 stalk is moved from a half-closed conformation to its open conformation are found to support the hypothesis that L1 stalk opening is involved in tRNA release from the ribosome.