R213I mutation in release factor 2 (RF2) is one step forward for engineering an omnipotent release factor in bacteria Escherichia coli
2017; Elsevier BV; Volume: 292; Issue: 36 Linguagem: Inglês
10.1074/jbc.m117.785238
ISSN1083-351X
AutoresGürkan Korkmaz, Suparna Sanyal,
Tópico(s)Antimicrobial Resistance in Staphylococcus
ResumoThe current understanding of the specificity of the bacterial class I release factors (RFs) in decoding stop codons has evolved beyond a simple tripeptide anticodon model. A recent molecular dynamics study for deciphering the principles for specific stop codon recognition by RFs identified Arg-213 as a crucial residue on Escherichia coli RF2 for discriminating guanine in the third position (G3). Interestingly, Arg-213 is highly conserved in RF2 and substituted by Ile-196 in the corresponding position in RF1. Another similar pair is Leu-126 in RF1 and Asp-143 in RF2, which are also conserved within their respective groups. With the hypothesis that replacement of Arg-213 and Asp-143 with the corresponding RF1 residues will reduce G3 discrimination by RF2, we swapped these residues between E. coli RF1 and RF2 by site-directed mutagenesis and characterized their preference for different codons using a competitive peptide release assay. Among these, the R213I mutant of RF2 showed 5-fold improved reading of the RF1-specific UAG codon relative to UAA, the universal stop codon, compared with the wild type (WT). In-depth fast kinetic studies revealed that the gain in UAG reading by RF2 R213I is associated with a reduced efficiency of termination on the cognate UAA codon. Our work highlights the notion that stop codon recognition involves complex interactions with multiple residues beyond the PXT/SPF motifs. We propose that the R213I mutation in RF2 brings us one step forward toward engineering an omnipotent RF in bacteria, capable of reading all three stop codons. The current understanding of the specificity of the bacterial class I release factors (RFs) in decoding stop codons has evolved beyond a simple tripeptide anticodon model. A recent molecular dynamics study for deciphering the principles for specific stop codon recognition by RFs identified Arg-213 as a crucial residue on Escherichia coli RF2 for discriminating guanine in the third position (G3). Interestingly, Arg-213 is highly conserved in RF2 and substituted by Ile-196 in the corresponding position in RF1. Another similar pair is Leu-126 in RF1 and Asp-143 in RF2, which are also conserved within their respective groups. With the hypothesis that replacement of Arg-213 and Asp-143 with the corresponding RF1 residues will reduce G3 discrimination by RF2, we swapped these residues between E. coli RF1 and RF2 by site-directed mutagenesis and characterized their preference for different codons using a competitive peptide release assay. Among these, the R213I mutant of RF2 showed 5-fold improved reading of the RF1-specific UAG codon relative to UAA, the universal stop codon, compared with the wild type (WT). In-depth fast kinetic studies revealed that the gain in UAG reading by RF2 R213I is associated with a reduced efficiency of termination on the cognate UAA codon. Our work highlights the notion that stop codon recognition involves complex interactions with multiple residues beyond the PXT/SPF motifs. We propose that the R213I mutation in RF2 brings us one step forward toward engineering an omnipotent RF in bacteria, capable of reading all three stop codons. Peptide release during translation termination is the end of protein synthesis on the ribosome (for review see Ref. 1.Youngman E.M. McDonald M.E. Green R. Peptide release on the ribosome: mechanism and implications for translational control.Annu. Rev. Microbiol. 2008; 62: 353-373Crossref PubMed Scopus (76) Google Scholar). Translation termination begins when one of the three stop codons (UAA, UAG, or UGA) is translocated into the ribosomal A site, and the peptidyl tRNA is translocated into the P site. The ester bond between the P site tRNA and polypeptide chain must be hydrolyzed for the release of the nascent peptide. In bacteria, class I release factors RF1 3The abbreviations used are: RF, release factor; RC, release complex; DHFR, dihydrofolate reductase; IF, initiation factor. and RF2 recognize overlapping sets of three different termination signals (stop codons). Both factors recognize UAA, whereas UAG is read specifically by RF1 and UGA by RF2 (2.Scolnick E. Tompkins R. Caskey T. Nirenberg M. Release factors differing in specificity for terminator codons.Proc. Natl. Acad. Sci. U.S.A. 1968; 61: 768-774Crossref PubMed Scopus (270) Google Scholar). In eukaryotic protein synthesis, eRF1 is the single class I RF, facilitating peptide release at all three stop codons. It is an open question why bacteria have two RFs acting in a semispecific manner, whereas eukaryotes have only one RF. The accuracy with which RFs discriminate against the other 61 (sense) codons in bacteria is remarkably high. The reported error rate is 1 in 104 in vivo (3.Jørgensen F. Adamski F.M. Tate W.P. Kurland C.G. Release factor-dependent false stops are infrequent in Escherichia coli.J. Mol. Biol. 1993; 230: 41-50Crossref PubMed Scopus (55) Google Scholar); the frequency of error for all single mismatches relative to stop codons ranges from 1 in 103 to 106 in vitro (4.Freistroffer D.V. Kwiatkowski M. Buckingham R.H. Ehrenberg M. The accuracy of codon recognition by polypeptide release factors.Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 2046-2051Crossref PubMed Scopus (134) Google Scholar). According to the "tripeptide anticodon" hypothesis, conserved amino acid motifs, PXT in RF1 and SPF in RF2, mimic the tRNA triplet anticodon and determine codon specificity of the RFs. This simple mimicry model was supported by low, and later high, resolution crystal structures of various ribosomal release complexes (RCs) with different stop codons and RFs bound to the A site of the ribosome (5.Korostelev A. Asahara H. Lancaster L. Laurberg M. Hirschi A. Zhu J. Trakhanov S. Scott W.G. Noller H.F. Crystal structure of a translation termination complex formed with release factor RF2.Proc. Natl. Acad. Sci. 2008; 105: 19684-19689Crossref PubMed Scopus (196) Google Scholar6.Korostelev A. Zhu J. Asahara H. Noller H.F. Recognition of the amber UAG stop codon by release factor RF1.EMBO J. 2010; 29: 2577-2585Crossref PubMed Scopus (86) Google Scholar, 7.Laurberg M. Asahara H. Korostelev A. Zhu J. Trakhanov S. Noller H.F. Structural basis for translation termination on the 70S ribosome.Nature. 2008; 454: 852-857Crossref PubMed Scopus (283) Google Scholar, 8.Petry S. Brodersen D.E. Murphy 4th, F.V. Dunham C.M. Selmer M. Tarry M.J. Kelley A.C. Ramakrishnan V. Crystal structures of the ribosome in complex with release factors RF1 and RF2 bound to a cognate stop codon.Cell. 2005; 123: 1255-1266Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar9.Weixlbaumer A. Jin H. Neubauer C. Voorhees R.M. Petry S. Kelley A.C. Ramakrishnan V. Insights into translational termination from the structure of RF2 bound to the ribosome.Science. 2008; 322: 953-956Crossref PubMed Scopus (238) Google Scholar). In these structures the conserved amino acids of the tripeptide anticodon motif form a recognition loop that surrounds the cognate codons. The crystal structures were later used in MD-free energy perturbation simulations to demonstrate the mechanism of stop codon reading, first in bacteria (10.Sund J. Andér M. Aqvist J. Principles of stop-codon reading on the ribosome.Nature. 2010; 465: 947-950Crossref PubMed Scopus (49) Google Scholar) and later in mitochondria (11.Lind C. Sund J. Aqvist J. Codon-reading specificities of mitochondrial release factors and translation termination at non-standard stop codons.Nat. Commun. 2013; 4: 2940Crossref PubMed Scopus (39) Google Scholar). This approach, although primarily aimed to clarify the role of the key residues of the tripeptide anticodon motif, identified additional side chains on RFs, possibly involved in specific stop codon recognition. Sund et al. (10.Sund J. Andér M. Aqvist J. Principles of stop-codon reading on the ribosome.Nature. 2010; 465: 947-950Crossref PubMed Scopus (49) Google Scholar) proposed that the following residues are involved: Gly-120, Glu-123, Leu-126, Gln-185, and Ile-196 on RF1 and Gly-137, Glu-140, Asp-143, Val-192, and Arg-213 on RF2 (Escherichia coli numbering, used throughout in this work) in specific reading of the second and third positions of the stop codon. Differential reading of the third position of a stop codon by RFs attracts considerable interest because it underpins the specificity of RF2 for the UGA codon. RF1 accepts both an adenine and a guanine at the third position (A3 and G3, respectively), whereas RF2 has to discriminate against G3 reading, which would otherwise lead to misreading of the tryptophan codon (UGG) as a stop codon. Recent MD-free energy perturbation simulation (using the available structures of termination complexes) revealed that a water molecule is possibly introduced into the decoding site during RF1 binding to the UAG and UAA stop codons, which facilitates further hydrogen-bonding between G3 of the stop codon and U531 of rRNA, essential for G3 reading (10.Sund J. Andér M. Aqvist J. Principles of stop-codon reading on the ribosome.Nature. 2010; 465: 947-950Crossref PubMed Scopus (49) Google Scholar). In the case of RF2, however, a conserved residue, Arg-213, prevents the water molecule from entering the codon recognition site due to its long side chain, thereby preventing G3 reading by RF2 (10.Sund J. Andér M. Aqvist J. Principles of stop-codon reading on the ribosome.Nature. 2010; 465: 947-950Crossref PubMed Scopus (49) Google Scholar). When RF1 and RF2 amino acid sequences from various bacteria are aligned together focusing mainly on the stop codon reading elements, a high degree of homology was observed between the two groups with some exceptions, which are moderately well conserved within the group. These include the PXT motif on RF1 and the SPF motif on RF2. Similarly, Ile-196 on RF1 and Arg-213 on RF2 were found in the corresponding positions (Fig. 1). Because Arg-213 was proposed to play a crucial role for RF2's discrimination for G3 from MD simulation (10.Sund J. Andér M. Aqvist J. Principles of stop-codon reading on the ribosome.Nature. 2010; 465: 947-950Crossref PubMed Scopus (49) Google Scholar), we hypothesized that exchanging Arg-213 with isoleucine (Ile) as in RF1 or with alanine (Ala), an even smaller side chain might allow RF2 to read G3 better than the WT because the water-blocking effect of Arg-213 would be removed. Alternatively, changing Ile-196 to Arg in RF1 might increase its discrimination against G3, whereas replacing Ile-196 with Ala would be less discriminative of G3 compared with the WT RF1. Other than the residues discussed above, Leu-126 in RF1 and Asp-143 in RF2 were also found in the corresponding positions, which are highly conserved in their respective groups (Fig. 1A). Interestingly, these residues are located in the α-helix, supporting the stop codon recognition loop (Fig. 1, B and C). Even though these residues may not be directly involved in stop codon recognition, they play supporting roles for the major residues. Thus, we reasoned that exchanging Asp-143 with Leu will also reduce RF2's discrimination for G3 and changing Leu-126 in RF1 to Asp would increase discrimination for G3. Mutational analyses of the key residues on RF1/RF2, although crucial for complementing the available structural data, are limited. Mutations of the PXT and SPF motifs of RF1 and RF2 first illustrated the crucial role of these tripeptide motifs in specific reading of the stop codons (12.Uno M. Ito K. Nakamura Y. Polypeptide release at sense and non-cognate stop codons by localized charge-exchange alterations in translational release factors.Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 1819-1824Crossref PubMed Scopus (31) Google Scholar). Besides those, biochemical data are available for the alanine mutants of few conserved residues within the decoding region of RF1 (Gln-185, Arg-186, Thr-190, His-197, and Thr-198) (13.Field A. Hetrick B. Mathew M. Joseph S. Histidine 197 in release factor 1 is essential for A Site binding and peptide release.Biochemistry. 2010; 49: 9385-9390Crossref PubMed Scopus (11) Google Scholar, 14.Trappl K. Mathew M.A. Joseph S. Thermodynamic and kinetic insights into stop codon recognition by Release Factor 1.PLoS ONE. 2014; 9: e94058Crossref PubMed Scopus (7) Google Scholar). These mutations showed a modest decrease in the maximal rate of peptide release (kcat) but resulted in a large 100-fold increase in the dissociation constant (kD) when bound to the ribosome with a cognate stop codon in the A site. However, similar mutational analysis of RF2 is not available to date. Based on a fitness compensatory mutation (E167K) in the RF1 knock-out background, Glu-167 was suggested to play a crucial role in UAG reading by RF2 (12.Uno M. Ito K. Nakamura Y. Polypeptide release at sense and non-cognate stop codons by localized charge-exchange alterations in translational release factors.Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 1819-1824Crossref PubMed Scopus (31) Google Scholar, 15.Ito K. Uno M. Nakamura Y. Single amino acid substitution in prokaryote polypeptide release factor 2 permits it to terminate translation at all three stop codons.Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 8165-8169Crossref PubMed Scopus (71) Google Scholar). However, the structural basis of improved UAG reading by this RF2 mutant is not understood. Two earlier studies (12.Uno M. Ito K. Nakamura Y. Polypeptide release at sense and non-cognate stop codons by localized charge-exchange alterations in translational release factors.Proc. Natl. Acad. Sci. U.S.A. 2002; 99: 1819-1824Crossref PubMed Scopus (31) Google Scholar, 16.Ito K. Ebihara K. Uno M. Nakamura Y. Conserved motifs in prokaryotic and eukaryotic polypeptide release factors: tRNA-protein mimicry hypothesis.Proc. Natl. Acad. Sci. U.S.A. 1996; 93: 5443-5448Crossref PubMed Scopus (130) Google Scholar) claimed that the R213I mutation is toxic for overexpression, and it gives a dominant lethal phenotype in the bacteria, but the actual reason for such behavior may not be this mutation alone (17.Dinçbas-Renqvist V. Engström A. Mora L. Heurgué-Hamard V. Buckingham R. Ehrenberg M. A post-translational modification in the GGQ motif of RF2 from Escherichia coli stimulates termination of translation.EMBO J. 2000; 19: 6900-6907Crossref PubMed Scopus (125) Google Scholar, 18.Uno M. Ito K. Nakamura Y. Functional specificity of amino acid at position 246 in the tRNA mimicry domain of bacterial release factor 2.Biochimie. 1996; 78: 935-943Crossref PubMed Scopus (60) Google Scholar). Also, no mutational studies are available for Asp-143 in RF2. Here, using site-directed mutagenesis, we generated several E. coli RF1/RF2 variants (RF1 I196A, RF1 I196R, RF1 L126D, RF2 R213A, RF2 R213I, and RF2 D143L) and characterized those in a competition assay for single-round peptide release (19.Bouakaz L. Bouakaz E. Murgola E.J. Ehrenberg M. Sanyal S. The role of ribosomal protein L11 in class I release factor-mediated translation termination and translational accuracy.J. Biol. Chem. 2006; 281: 4548-4556Abstract Full Text Full Text PDF PubMed Scopus (23) Google Scholar) from ribosomal RCs containing major stop codon UAA (RCUAA) versus RCXXX carrying various codons (symbolized as XXX) in the A site. These include UAG and UGA stop codons and UGG (Trp), UCA (Ser), and AAA (Lys) codons. Among all the mutants tested, the R213I mutant RF2 showed the highest degree of improvement (5-fold) in UAG reading relative to UAA during translation termination while maintaining discrimination against UCA and UGG codons. To understand the basis of the altered sensitivity of the R213I mutant, we characterized it further with fast kinetic analysis for reading UAA and UAG codons and also in full-length protein synthesis assay using a reconstituted transcription-translation system. Ribosomal RCs were prepared with fMet-tRNAfMet in the P site and various codons in the A site (see the list of codons in Table 1). The fMet in the RCUAA was radioactively labeled with 3H. Other RCs (RCXXX) contained one of the five different codons (UAG, UGA, UGG, UCA, or AAA) in the A site, and [35S]fMet-tRNAfMet in the P site. All RF proteins RF1 and RF2 WT and mutants RF1 I196A, RF1 I196R, RF1 L126D, RF2 R213A, RF2 R213I, and RF2 D143L were prepared using standard laboratory protocols.Table 1A values (as defined in Equation 1) determined from the competition assays using [35S]fMet-RCUAA versus [3H]fMet-RCXXX, where XXX represents the codon specified in the first row of the tableUAGUGAUCAUGGAAARF1 WT0.929.529.532.5–*Beyond the detection limit.RF1 I196A127.768.2135–*Beyond the detection limit.RF1 I196R1.468.936.831.3–*Beyond the detection limit.RF1 L126D1.39.216.112.1–*Beyond the detection limit.RF2 WT33136.733.9–*Beyond the detection limit.RF2 R213A22.41.15966.3–*Beyond the detection limit.RF2 R213I6.51.461.520.2–*Beyond the detection limit.RF2 D143L12.21.110.352.2–*Beyond the detection limit.* Beyond the detection limit. Open table in a new tab In the competition experiments, equal amounts of RCUAA carrying [3H]fMet-tRNAfMet and RCXXX carrying [35S]fMet-tRNAfMet were mixed together. To this mix, RF variants were added in increasing concentrations, and the amounts of [3H]fMet and [35S]fMet released in a single round reaction were measured (Fig. 2). From these data, a discrimination parameter A, reflecting the relative preference of the RFs for UAA over the other codon was determined as described under "Experimental procedures." The higher the value of A, the higher is the discrimination against the competing codon with respect to UAA. Thus, decrease in the A value for a particular codon due to any mutation in a RF will indicate relatively improved reading of that codon by the mutant RF. The A values are listed in Table 1. All RF mutants recognized UAA and the respective cognate codons of their WT counterparts (UAG for RF1 or UGA for RF2 mutants) with similar efficiency (A values ranging from 0.9 to 1.4), thereby demonstrating no discrimination between UAA and the specific cognate codon (Fig. 2, A and B, Table 1). In contrast, no peptide release was detected with RCs with a sense codon AAA coding for lysine; consequently, the slope of the discrimination curve was infinite, and the A value could not be determined (Table 1). None of the RF1 variants displayed an improved reading of UGA respective to UAA while maintaining substantial levels of discrimination on other non-cognate codons tested here. The RF1 I196A produced a WT-like A value for the non-cognate UGA codon but showed 4- and 2-fold higher discriminations against UGG and UCA codons, respectively. The I196R mutant, on the other hand, showed a 2-fold increased discrimination against UGA but WT-like A values for UCA and UGG codons. Interestingly, the L126D mutation showed an ∼2-fold improved reading of UGA, but at the same time it also lost discrimination against UGG and UCA codons. Based on these results, we excluded all RF1 mutants from further investigation, as none of those showed improvement in omnipotent reading of the stop codons. For RF2 mutants the same screening process was applied; in contrast to RF1, all RF2 variants showed a moderate to significant decrease in discrimination against RF1-specific UAG codon. The R213A mutation showed a 1.5× smaller A value for the UAG codon but ∼2-fold higher A values for both UCA and UGG codons. The D143L mutant showed a 3-fold smaller A value for the UAG codon, but it also lost discrimination against the UCA codon to the same extent, whereas its discrimination against UGG codon increased 1.5×. The most interesting mutant was RF2 R213I, as it displayed 5× less discrimination against the UAG codon than the RF2 WT (Fig. 2C) while having an almost 2-fold higher discrimination against UCA and a moderate (1.5×) loss of discrimination against UGG codon. Thus, our competition assay identified RF2 R213I as a promising candidate toward making an omnipotent release factor. Therefore, RF2 R213I was selected for further in-depth kinetic analysis. The competition assays were limited only to three sense codons. Thus, to test the ability of RF2 R213I to discriminate against a variety of sense codons in a cellular scenario, we performed an in vitro full-length protein synthesis experiment using a fully reconstituted transcription (T7 based)-translation system with individually purified components from E. coli (20.Mandava C.S. Peisker K. Ederth J. Kumar R. Ge X. Szaflarski W. Sanyal S. Bacterial ribosome requires multiple L12 dimers for efficient initiation and elongation of protein synthesis involving IF2 and EF-G.Nucleic Acids Res. 2012; 40: 2054-2064Crossref PubMed Scopus (34) Google Scholar). Recognition of any sense codon by RF2 R213I would lead to peptide release and, thus, would generate truncated products due to premature translation termination. Alternatively, a full-length protein band of expected size would signify its capacity in recognizing the stop codon accurately. E. coli dihydrofolate reductase (DHFR) was used as the reporter protein as it contains a broad range of sense codons (all except ACG, TCC, AGG) and UAA as the stop codon. As shown in Fig. 3, a single clear band of the full-length DHFR protein was seen with RF1 WT and RF2 WT as well as RF2 R213I (Fig. 3, lanes A, B, and C, respectively). The negative control reaction without RFs showed only a faint spot in the same place (Fig. 3, lane D). In addition, we evaluated the amount of synthesized protein by measuring radioactive count. The same counts per mm2 were noted for RF1 WT and RF2 WT samples (8600 and 8700 counts/mm2, respectively), whereas it was somewhat lower for RF2 R213I samples (7700 counts/mm2). The value for the negative control was ∼600 counts/mm2. The experiment was done in duplicates with similar outcomes. Because we did not detect any truncated products of DHFR with either RF2 WT or R213I mutant, we conclude that similar to RF2 WT, the RF2 R213I mutant does not induce a discrete premature peptide release or translation termination on any sense codon within the limits of this assay. It also means that RF2 R213I retains the specificity against the sense codons at the same level as the RF2 WT. To investigate the kinetic properties of the RF2 R213I, we performed a single turnover fMet (peptide) release assay using fast kinetics (Fig. 4). RCUAA and RCUAG presenting UAA and UAG codons in the A site, respectively, were produced by binding [3H]fMet to AUG-UAA/UAG mRNA-programmed 70S ribosomes. RF variants at different concentrations were rapidly mixed with the RCs in the quench-flow, and the rate of [3H]fMet release was measured by plotting the amount of released [3H]fMet against time. The rates, when plotted against RF concentration, generated hyperbolic curves, from which Michaelis-Menten parameters, namely kcat (the maximal rate of peptide release) and Km (the RF concentration at half-maximal rate), were estimated (Fig. 4). At UAA codons, the RF2 WT released [3H]fMet with a kcat of 12.1 ± 1.1 s−1 and a Km of 123 ± 29 nm (Fig. 4A), leading to a translational efficiency (kcat/Km) of 98.2 × 10−6 μm−1 s−1, in good agreement with published results (4.Freistroffer D.V. Kwiatkowski M. Buckingham R.H. Ehrenberg M. The accuracy of codon recognition by polypeptide release factors.Proc. Natl. Acad. Sci. U.S.A. 2000; 97: 2046-2051Crossref PubMed Scopus (134) Google Scholar, 21.Indrisiunaite G. Pavlov M.Y. Heurgué-Hamard V. Ehrenberg M. On the pH dependence of class-1 RF-dependent termination of mRNA translation.J. Mol. Biol. 2015; 427: 1848-1860Crossref PubMed Scopus (33) Google Scholar). In comparison, RF2 R213I showed a 3-fold smaller kcat (3.8 ± 0.03 s−1) than WT, whereas the Km was similar to the WT (102 ± 3.5 nm); this resulted in a translational efficiency of 37.2 × 10−6 μm−1 s−1. At the non-cognate UAG stop codon, the RF2 WT showed a kcat equal to 1.49 ± 0.16 s−1 and a Km of 1045 ± 342 nm, resulting in kcat/Km values of 1.4 × 10−6 μm−1 s−1, almost 70× less than the UAA codon. For UAG reading by RF2 R213I, kcat was 0.79 ± 0.13 s−1, and Km was 875 ± 282 nm; thus, kcat/Km was 0.9 × 10−6 μm−1 s−1, ∼40 times less than the cognate UAA codon. When compared with the WT, the translation termination efficiency (kcat/Km) of the RF2 R213I mutant was found to be 2.6× less at the cognate UAA codon but quite similar for the non-cognate UAG codon. Thus, the relatively improved UAG reading by RF2 R213I with respect to UAA in the competition assay (Fig. 2C) is essentially not due to its increased efficiency of UAG reading but, rather, due to its reduced efficiency in UAA reading. We also note that the reduced efficiency of RF2 R213I in UAA reading originated from the 3-fold decrease in the kcat value compared with the WT. However, on non-cognate UAG codon, a 2-fold decrease in the kcat value for RF2 R213I relative to the WT was partially compensated by a decrease in Km, thereby leading to termination efficiency at UAG codon comparable with the WT. During the last decade, it was shown that stop codon recognition by the class I release factors is far more complex than the simple tripeptide anticodon theory (22.Ito K. Uno M. Nakamura Y. A tripeptide "anticodon" deciphers stop codons in messenger RNA.Nature. 2000; 403: 680-684Crossref PubMed Scopus (39) Google Scholar). The tRNA mimicry hypothesis is certainly valid from the structural viewpoint, as the RFs span from the decoding center to the peptidyl transferase center, like the tRNAs, and the tripeptide motifs PXT in RF1 and SPF in RF2 appear in the similar positions as the tRNA anticodons, but it oversimplifies the mechanism of stop codon recognition. Computational, biochemical, and structural studies unraveled a complex network (including, but not limited to the tripeptide anticodon) of hydrogen bonds between the RFs, the stop codon, and the ribosome, which are now believed to be essential for accurate recognition of the stop codons (5.Korostelev A. Asahara H. Lancaster L. Laurberg M. Hirschi A. Zhu J. Trakhanov S. Scott W.G. Noller H.F. Crystal structure of a translation termination complex formed with release factor RF2.Proc. Natl. Acad. Sci. 2008; 105: 19684-19689Crossref PubMed Scopus (196) Google Scholar6.Korostelev A. Zhu J. Asahara H. Noller H.F. Recognition of the amber UAG stop codon by release factor RF1.EMBO J. 2010; 29: 2577-2585Crossref PubMed Scopus (86) Google Scholar, 7.Laurberg M. Asahara H. Korostelev A. Zhu J. Trakhanov S. Noller H.F. Structural basis for translation termination on the 70S ribosome.Nature. 2008; 454: 852-857Crossref PubMed Scopus (283) Google Scholar, 8.Petry S. Brodersen D.E. Murphy 4th, F.V. Dunham C.M. Selmer M. Tarry M.J. Kelley A.C. Ramakrishnan V. Crystal structures of the ribosome in complex with release factors RF1 and RF2 bound to a cognate stop codon.Cell. 2005; 123: 1255-1266Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar, 9.Weixlbaumer A. Jin H. Neubauer C. Voorhees R.M. Petry S. Kelley A.C. Ramakrishnan V. Insights into translational termination from the structure of RF2 bound to the ribosome.Science. 2008; 322: 953-956Crossref PubMed Scopus (238) Google Scholar10.Sund J. Andér M. Aqvist J. Principles of stop-codon reading on the ribosome.Nature. 2010; 465: 947-950Crossref PubMed Scopus (49) Google Scholar, 13.Field A. Hetrick B. Mathew M. Joseph S. Histidine 197 in release factor 1 is essential for A Site binding and peptide release.Biochemistry. 2010; 49: 9385-9390Crossref PubMed Scopus (11) Google Scholar, 14.Trappl K. Mathew M.A. Joseph S. Thermodynamic and kinetic insights into stop codon recognition by Release Factor 1.PLoS ONE. 2014; 9: e94058Crossref PubMed Scopus (7) Google Scholar, 23.He S.L. Green R. Visualization of codon-dependent conformational rearrangements during translation termination.Nat. Struct. Mol. Biol. 2010; 17: 465-470Crossref PubMed Scopus (23) Google Scholar). In this context, an early observation that a single point mutation E167K in RF2, acquired at the prfA-deleted background, enables it to read all three stop codons is worth mentioning (15.Ito K. Uno M. Nakamura Y. Single amino acid substitution in prokaryote polypeptide release factor 2 permits it to terminate translation at all three stop codons.Proc. Natl. Acad. Sci. U.S.A. 1998; 95: 8165-8169Crossref PubMed Scopus (71) Google Scholar). However, the basis of RF2 E167K to be an omnipotent RF in vivo is not well understood. This is not only because Glu-167 is located far from the decoding center but also because it showed significant loss of its activity for reading the cognate UAA codon. Thus, there could be additional fitness compensatory mutations (within the ribosome or elsewhere) in the RF2 E167K strain, which were not checked or characterized. Thus, in the current study we decided to try the reverse approach by incorporating mutations in the RFs based on structural and computational studies, which would direct us toward rational design of an omnipotent release factor. The first step of designing a RF that would read both UAG and UGA codons other than UAA codon is either to reduce the discrimination for G3 by RF2 or for G2 by RF1. In the current study we focused mainly on modifying RF2 for improved G3 reading; for that we closely inspected the analysis of the third position reading by Sund et al. (10.Sund J. Andér M. Aqvist J. Principles of stop-codon reading on the ribosome.Nature. 2010; 465: 947-950Crossref PubMed Scopus (49) Google Scholar) using MD simulation based on high-resolution crystal structures of the RFs on the ribosome (7.Laurberg M. Asahara H. Korostelev A. Zhu J. Trakhanov S. Noller H.F. Structural basis for translation termination on the 70S ribosome.Nature. 2008; 454: 852-857Crossref PubMed Scopus (283) Google Scholar, 9.Weixlbaumer A. Jin H. Neubauer C. Voorhees R.M. Petry S. Kelley A.C. Ramakrishnan V. Insights into translational termination from the structure of RF2 bound to
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