GGQ methylation enhances both speed and accuracy of stop codon recognition by bacterial class-I release factors
2021; Elsevier BV; Volume: 296; Linguagem: Inglês
10.1016/j.jbc.2021.100681
ISSN1083-351X
AutoresShreya Pundir, Xueliang Ge, Suparna Sanyal,
Tópico(s)Genomics and Phylogenetic Studies
ResumoAccurate translation termination in bacteria requires correct recognition of the stop codons by the class-I release factors (RFs) RF1 and RF2, which release the nascent peptide from the peptidyl tRNA after undergoing a "compact to open" conformational transition. These RFs possess a conserved Gly-Gly-Gln (GGQ) peptide release motif, of which the Q residue is posttranslationally methylated. GGQ-methylated RFs have been shown to be faster in peptide release than the unmethylated ones, but it was unknown whether this modification had additional roles. Using a fluorescence-based real-time in vitro translation termination assay in a stopped-flow instrument, we demonstrate that methylated RF1 and RF2 are two- to four-fold more accurate in the cognate stop codon recognition than their unmethylated variants. Using pH titration, we show that the lack of GGQ methylation facilitates the "compact to open" transition, which results in compromised accuracy of the unmethylated RFs. Furthermore, thermal melting studies using circular dichroism and SYPRO-orange fluorescence demonstrate that GGQ methylation increases overall stability of the RF proteins. This increased stability, we suspect, is the basis for the more controlled conformational change of the methylated RFs upon codon recognition, which enhances both their speed and accuracy. This GGQ methylation-based modulation of the accuracy of RFs can be a tool for regulating translational termination in vivo. Accurate translation termination in bacteria requires correct recognition of the stop codons by the class-I release factors (RFs) RF1 and RF2, which release the nascent peptide from the peptidyl tRNA after undergoing a "compact to open" conformational transition. These RFs possess a conserved Gly-Gly-Gln (GGQ) peptide release motif, of which the Q residue is posttranslationally methylated. GGQ-methylated RFs have been shown to be faster in peptide release than the unmethylated ones, but it was unknown whether this modification had additional roles. Using a fluorescence-based real-time in vitro translation termination assay in a stopped-flow instrument, we demonstrate that methylated RF1 and RF2 are two- to four-fold more accurate in the cognate stop codon recognition than their unmethylated variants. Using pH titration, we show that the lack of GGQ methylation facilitates the "compact to open" transition, which results in compromised accuracy of the unmethylated RFs. Furthermore, thermal melting studies using circular dichroism and SYPRO-orange fluorescence demonstrate that GGQ methylation increases overall stability of the RF proteins. This increased stability, we suspect, is the basis for the more controlled conformational change of the methylated RFs upon codon recognition, which enhances both their speed and accuracy. This GGQ methylation-based modulation of the accuracy of RFs can be a tool for regulating translational termination in vivo. Termination is an important step of translation during which the nascent peptides are released from the ribosome. It should be efficient for allowing fast turnover of the translation machinery, and at the same time, accurate to avoid accumulation of the potentially inactive and toxic, truncated, or overlong proteins. Protein synthesis terminates when a translating ribosome encounters one of the three stop codons (UAA, UAG, and UGA) on the mRNA at the ribosomal A site. These codons are recognized in a semispecific manner by the class-I release factors (referred hereafter as RFs) in bacteria, namely release factor 1 (RF1) and release factor 2 (RF2). UAA codon is read by both RF1 and RF2. But, UAG is read specifically by RF1 and UGA by RF2 (1Scolnick 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 (250) Google Scholar). In eukaryotes, all three stop codons are read by a common class-I RF named eRF1 (2Konecki D.S. Aune K.C. Tate W. Caskey C.T. Characterization of reticulocyte release factor.J. Biol. Chem. 1977; 252: 4514-4520Abstract Full Text PDF PubMed Google Scholar). The class-I RFs in bacteria depend on the class-II RF RF3 for their fast dissociation from the ribosome after peptide release (3Freistroffer D.V. Pavlov M.Y. MacDougall J. Buckingham R.H. Ehrenberg M. Release factor RF3 in E. coli accelerates the dissociation of release factors RF1 and RF2 from the ribosome in a GTP-dependent manner.EMBO J. 1997; 16: 4126-4133Crossref PubMed Scopus (238) Google Scholar). RF1 and RF2 possess a common universally conserved Glycine–Glycine–Glutamine (GGQ) motif (4Frolova L.Y. Tsivkovskii R.Y. Sivolobova G.F. Oparina N.Y. Serpinsky O.I. Blinov V.M. Tatkov S.I. Kisselev L.L. Mutations in the highly conserved GGQ motif of class I polypeptide release factors abolish ability of human eRF1 to trigger peptidyl-tRNA hydrolysis.RNA. 1999; 5: 1014-1020Crossref PubMed Scopus (278) Google Scholar) in the tip of domain III, which is involved in release of the nascent peptide by ester bond hydrolysis from the peptidyl tRNA at the peptidyl transferase center (PTC) of the ribosome (5Rawat U.B.S. Zavialov A.V. Sengupta J. Valle M. Grassucci R.A. Linde J. Vestergaard B. Ehrenberg M. Frank J. A cryo-electron microscopic study of ribosome-bound termination factor RF2.Nature. 2003; 421: 87-90Crossref PubMed Scopus (202) Google Scholar, 6Klaholz B.P. Pape T. Zavialov A.V. Myasnikov A.G. Orlova E.V. Vestergaard B. Ehrenberg M. Van Heel M. Structure of the Escherichia coli ribosomal termination complex with release factor 2.Nature. 2003; 421: 90-94Crossref PubMed Scopus (170) Google Scholar, 7Petry S. Brodersen D.E. Murphy IV, F.V. Dunham C.M. Selmer M. Tarry M.J. Kelley A.C. 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Crystal structure of a translation termination complex formed with release factor RF2.Proc. Natl. Acad. Sci. U. S. A. 2008; 105: 19684-19689Crossref PubMed Scopus (175) Google Scholar). Mutations of the GGQ motif render RF1 and RF2 inactive in peptide release (8Shaw J.J. Green R. Two distinct components of release factor function uncovered by nucleophile partitioning analysis.Mol. Cell. 2007; 28: 458-467Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 12Zavialov A.V. Mora L. Buckingham R.H. Ehrenberg M. Release of peptide promoted by the GGQ motif of class 1 release factors regulates the GTPase activity of RF3.Mol. Cell. 2002; 10: 789-798Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar, 13Mora L. Heurgué-Hamard V. Champ S. Ehrenberg M. Kisselev L.L. Buckingham R.H. The essential role of the invariant GGQ motif in the function and stability in vivo of bacterial release factors RF1 and RF2.Mol. Microbiol. 2003; 47: 267-275Crossref PubMed Scopus (78) Google Scholar). In both prokaryotes and eukaryotes, the amide group of the Gln (Q) residue in the GGQ motif is posttranslationally methylated at the N5 position by a methyltransferase enzyme encoded by prmC (also known as HemK) (for review see (14Dinçbas-Renqvist V. Engström Å. 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 (115) Google Scholar, 15Graille M. Figaro S. Kervestin S. Buckingham R.H. Liger D. Heurgué-Hamard V. Methylation of class I translation termination factors: Structural and functional aspects.Biochimie. 2012; 94: 1533-1543Crossref PubMed Scopus (12) Google Scholar, 16Heurgu V. Champ S. The hemK gene in Escherichia coli encodes the N5-glutamine methyltransferase that modifies peptide release factors.EMBO J. 2002; 21: 769-778Crossref PubMed Scopus (105) Google Scholar, 17Graille M. Heurgué-Hamard V. Champ S. Mora L. Scrima N. Ulryck N. Van Tilbeurgh H. Buckingham R.H. Molecular basis for bacterial class I release factor methylation by PrmC.Mol. Cell. 2005; 20: 917-927Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar)). Knocking out prmC gene is not lethal but results in slow phenotypic growth (18Nakahigashi K. Kubo N. Narita S.-I. Shimaoka T. Goto S. Oshima T. Mori H. Maeda M. Wada C. Inokuchi H. HemK, a class of protein methyl transferase with similarity to DNA methyl transferases, methylates polypeptide chain release factors, and hemK knockout induces defects in translational termination.Proc. Natl. Acad. Sci. U. S. A. 2002; 5: 1473-1478Crossref Scopus (90) Google Scholar). Earlier in vitro (10Zeng F. Jin H. Conformation of methylated GGQ in the peptidyl transferase center during translation termination.Sci. Rep. 2018; 8: 2349Crossref PubMed Scopus (7) Google Scholar, 14Dinçbas-Renqvist V. Engström Å. 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 (115) Google Scholar, 19Indrisiunaite 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 (24) Google Scholar, 20Pierson W.E. Hoffer E.D. Keedy H.E. Simms C.L. Dunham C.M. Zaher H.S. Uniformity of peptide release is maintained by methylation of release factors.Cell Rep. 2016; 17: 11-18Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar) and in vivo studies (18Nakahigashi K. Kubo N. Narita S.-I. Shimaoka T. Goto S. Oshima T. Mori H. Maeda M. Wada C. Inokuchi H. HemK, a class of protein methyl transferase with similarity to DNA methyl transferases, methylates polypeptide chain release factors, and hemK knockout induces defects in translational termination.Proc. Natl. Acad. Sci. U. S. A. 2002; 5: 1473-1478Crossref Scopus (90) Google Scholar, 21Mora L. Heurgué-Hamard V. De Zamaroczy M. Kervestin S. Buckingham R.H. Methylation of bacterial release factors RF1 and RF2 is required for normal translation termination in vivo.J. Biol. Chem. 2007; 282: 35638-35645Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar) demonstrated that GGQ methylation significantly enhances the catalytic activity of the RFs. The enhancement is more pronounced for some particular amino acids (20Pierson W.E. Hoffer E.D. Keedy H.E. Simms C.L. Dunham C.M. Zaher H.S. Uniformity of peptide release is maintained by methylation of release factors.Cell Rep. 2016; 17: 11-18Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Using X-ray crystallography and cryo-electron microscopy (cryo-EM), it has been recently shown that the GGQ methylation helps this motif in acquiring a stable conformation in the PTC, which in turn facilitates efficient peptide release by the RFs (10Zeng F. Jin H. Conformation of methylated GGQ in the peptidyl transferase center during translation termination.Sci. Rep. 2018; 8: 2349Crossref PubMed Scopus (7) Google Scholar). Accuracy of translation termination depends on correct stop codon recognition by the class-I RFs. RF1 and RF2 possess different sequence motifs for recognition of the specific stop codons at the decoding center (DC) of the ribosome. Earlier studies based on mutational analysis demonstrated that RF1 uses a Proline-X-Threonine/Alanine/Valine (PXT) motif and RF2 uses a Serine–Proline–Phenylalanine (SPF) motif located in the domain-II for specific recognition of their cognate stop codons (22Uno 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 (57) Google Scholar, 23Nakamura Y. Ito K. Uno M. A tripeptide "anticodon" deciphers stop codons in messenger RNA.Nature. 2000; 403: 680-684Crossref PubMed Scopus (214) Google Scholar). Later, molecular dynamics (MD) simulations-based studies (24Sund J. Andér M. Åqvist J. Principles of stop-codon reading on the ribosome.Nature. 2010; 465: 947-950Crossref PubMed Scopus (47) Google Scholar) identified additional residues beyond the PXT and SPF motifs as crucial for correct stop codon recognition by RF1 and RF2. Among these the role of Arg213 of RF2 has been studied extensively by mutagenesis and fast kinetics (25Korkmaz G. Sanyal S. R213I mutation in release factor 2 (RF2) is one step forward for engineering an omnipotent release factor in bacteria Escherichia coli.J. Biol. Chem. 2017; 292: 15134-15142Abstract Full Text Full Text PDF PubMed Scopus (6) Google Scholar). Structural studies of the RFs with X-ray crystallography and cryo-EM have revealed that RF1 and RF2 exist in two different conformations in free state and when bound to the ribosome (5Rawat U.B.S. Zavialov A.V. Sengupta J. Valle M. Grassucci R.A. Linde J. Vestergaard B. Ehrenberg M. Frank J. A cryo-electron microscopic study of ribosome-bound termination factor RF2.Nature. 2003; 421: 87-90Crossref PubMed Scopus (202) Google Scholar, 6Klaholz B.P. Pape T. Zavialov A.V. Myasnikov A.G. Orlova E.V. Vestergaard B. Ehrenberg M. Van Heel M. Structure of the Escherichia coli ribosomal termination complex with release factor 2.Nature. 2003; 421: 90-94Crossref PubMed Scopus (170) Google Scholar, 7Petry S. Brodersen D.E. Murphy IV, 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 (187) Google Scholar). In the crystal structures, corresponding to the free state, RF1 and RF2 are seen in a compact form, with PXT/SPF motif and GGQ motif separated by a distance of 25 Å (Fig. 1A) (26Vestergaard B. Van L.B. Andersen G.R. Nyborg J. Buckingham R.H. Kjeldgaard M. Bacterial polypeptide release factor RF2 is structurally distinct from eukaryotic eRF1.Mol. Cell. 2001; 8: 1375-1382Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 27Shin D.H. Brandsen J. Jancarik J. Yokota H. Kim R. Kim S.H. Structural analyses of peptide release factor 1 from Thermotoga maritima reveal domain flexibility required for its interaction with the ribosome.J. Mol. Biol. 2004; 341: 227-239Crossref PubMed Scopus (65) Google Scholar). In contrast, the class-I RFs mimic aminoacyl-tRNAs on the ribosome and adopt an open conformation spanning ∼75 Å from the DC on the small subunit to the PTC on the large subunit (Fig. 1B) (5Rawat U.B.S. Zavialov A.V. Sengupta J. Valle M. Grassucci R.A. Linde J. Vestergaard B. Ehrenberg M. Frank J. A cryo-electron microscopic study of ribosome-bound termination factor RF2.Nature. 2003; 421: 87-90Crossref PubMed Scopus (202) Google Scholar, 7Petry S. Brodersen D.E. Murphy IV, 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 (187) Google Scholar, 11Korostelev 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. U. S. A. 2008; 105: 19684-19689Crossref PubMed Scopus (175) Google Scholar, 28Weixlbaumer 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 (220) Google Scholar, 29Korostelev 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 (74) Google Scholar, 30Laurberg 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 (260) Google Scholar, 31Jin H. Kelley A.C. Loakes D. Ramakrishnan V. Structure of the 70S ribosome bound to release factor 2 and a substrate analog provides insights into catalysis of peptide release.Proc. Natl. Acad. Sci. U. S. A. 2010; 107: 8593-8598Crossref PubMed Scopus (76) Google Scholar). It was proposed earlier that RF1 and RF2 confer the catalytically active open conformation upon correct codon recognition after binding to the ribosome (Fig. 1C) (5Rawat U.B.S. Zavialov A.V. Sengupta J. Valle M. Grassucci R.A. Linde J. Vestergaard B. Ehrenberg M. Frank J. A cryo-electron microscopic study of ribosome-bound termination factor RF2.Nature. 2003; 421: 87-90Crossref PubMed Scopus (202) Google Scholar). The visual evidence for this hypothesis, however, appeared only very recently through time-resolved cryo-EM, which demonstrated a "compact to open" transition of RF1 and RF2 on the ribosome after proper recognition of the stop codon (32Fu Z. Indrisiunaite G. Kaledhonkar S. Shah B. Sun M. Chen B. Grassucci R.A. Ehrenberg M. Frank J. The structural basis for release-factor activation during translation termination revealed by time-resolved cryogenic electron microscopy.Nat. Commun. 2019; 10: 2579Crossref PubMed Scopus (17) Google Scholar). Contemporary cryo-EM studies have also reported different intermediate conformations (between fully compact and open) of RF1 and RF2 on the ribosome (33Svidritskiy E. Korostelev A.A. Conformational control of translation termination on the 70S ribosome.Structure. 2018; 26: 821-828Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar, 34Svidritskiy E. Korostelev A.A. Mechanism of inhibition of translation termination by Blasticidin S.J. Mol. Biol. 2018; 430: 591-593Crossref PubMed Scopus (8) Google Scholar, 35Svidritskiy E. Demo G. Loveland A.B. Xu C. Korostelev A.A. Extensive ribosome and RF2 rearrangements during translation termination.Elife. 2019; 8e46850Crossref PubMed Scopus (12) Google Scholar). Moreover, kinetics of the "compact to open" transition has been followed by state-of-the-art fluorescence resonance energy transfer (FRET) (36Trappl K. Joseph S. Ribosome induces a closed to open conformational change in release factor 1.J. Mol. Biol. 2016; 428: 1333-1344Crossref PubMed Scopus (13) Google Scholar) and in silico simulations (9Trobro S. Åqvist J. A model for how ribosomal release factors induce peptidyl-tRNA cleavage in termination of protein synthesis.Mol. Cell. 2007; 27: 758-766Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Altogether these studies decipher a near complete mechanistic picture of translation termination by the bacterial class-I RFs, where the stop codon recognition and conformational change relate to the accuracy in termination, whereas the peptide release governs the speed of the process (Fig. 1C). The first quantitative analysis of accuracy of stop codon recognition by RF1 and RF2 was performed by Ehrenberg and colleagues. With precise biochemical experiments, they demonstrated the degree of preference of RF1 and RF2 for their cognate stop codons over the near-cognate codons for efficient peptide release (37Freistroffer 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 (128) Google Scholar). Their conclusion was that accuracy in termination depends entirely on the selective recognition of the stop codons by the RFs. However, the new finding that RF1 and RF2 open up on the ribosome upon correct stop codon recognition and thereby become catalytically active (Fig. 1) (32Fu Z. Indrisiunaite G. Kaledhonkar S. Shah B. Sun M. Chen B. Grassucci R.A. Ehrenberg M. Frank J. The structural basis for release-factor activation during translation termination revealed by time-resolved cryogenic electron microscopy.Nat. Commun. 2019; 10: 2579Crossref PubMed Scopus (17) Google Scholar), adds an additional regulatory step in the accuracy process. In addition, it raises the question whether GGQ methylation has any role in modulating this conformational transition and thus, affecting the accuracy of stop codon recognition by RF1 and RF2. Recently, Zeng et al. (10Zeng F. Jin H. Conformation of methylated GGQ in the peptidyl transferase center during translation termination.Sci. Rep. 2018; 8: 2349Crossref PubMed Scopus (7) Google Scholar) reported kinetic parameters for peptide release by the methylated and unmethylated RFs on the stop codons, but the accuracy impact has not been explored. Here, using a fluorescence-based in vitro peptide release assay in stopped-flow, we show that GGQ methylation in RF1 and RF2 enhances both catalytic speed and accuracy of stop codon recognition. Furthermore, by pH titration, we demonstrate that the lack of GGQ methylation expedites the "compact to open" transition of the RFs, thereby making the unmethylated RFs less accurate in peptide release. Finally, by biophysical characterization of the unmethylated and methylated RFs, we find that the GGQ methylation aids in overall stability of the proteins. This increased stability, we suspect, is the basis for more controlled conformational change in the methylated RF1 and RF2 upon codon recognition, which enhances their speed and accuracy. For peptide release assay, we used a ribosomal release complex (RC), which harbored in the P-site, a peptidyl tRNA carrying fluorescent BODIPY 576/589 (BOP) labeled Met-Leu-Leu tripeptide, and in the A-site, one of the three stop codons or the UGG codon specific for tryptophan (Trp), on the mRNA. The RF2 variants, methylated (mRF2) and unmethylated (RF2), were added in increasing concentration to the RC with either cognate UAA and UGA codons or near-cognate UAG and UGG codons, and the time course of peptide release was monitored by following decrease in BOP fluorescence in a stopped-flow instrument (Fig. 2, left and middle panel). The rates of peptide release were determined by fitting the fluorescence curves with exponential functions (see Experimental procedures for details). The observed rates plotted against concentration of the RFs were fitted with hyperbolic function following Michaelis–Menten equation (Fig. 2, right panel), from which the kinetic parameters kcat, KM, and kcat/KM were derived. Our kinetics data (Table 1), consistent with earlier reports (19Indrisiunaite 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 (24) Google Scholar, 20Pierson W.E. Hoffer E.D. Keedy H.E. Simms C.L. Dunham C.M. Zaher H.S. Uniformity of peptide release is maintained by methylation of release factors.Cell Rep. 2016; 17: 11-18Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 37Freistroffer 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 (128) Google Scholar, 38Pavlov M.Y. Freistroffer D.V. Dincbas V. MacDougall J. Buckingham R.H. Ehrenberg M. A direct estimation of the context effect on the efficiency of termination.J. Mol. Biol. 1998; 284: 579-590Crossref PubMed Scopus (46) Google Scholar), show that mRF2 releases peptide from cognate RCUAA and RCUGA with the maximal catalytic rate (kcat) of about 10 s−1, which is about 1.7-fold higher than that of RF2 (Fig. 2, C and F). However, the increase in kcat is accompanied by an equivalent increase in KM by mRF2, thereby resulting in similar catalytic efficiencies (kcat/KM) of mRF2 and RF2 on the cognate UAA and UGA codons (Table 1). This result, in complete agreement with the previous report (38Pavlov M.Y. Freistroffer D.V. Dincbas V. MacDougall J. Buckingham R.H. Ehrenberg M. A direct estimation of the context effect on the efficiency of termination.J. Mol. Biol. 1998; 284: 579-590Crossref PubMed Scopus (46) Google Scholar), confirms that the methylation of GGQ does not have any impact on catalytic efficiency of peptide release by RF2 on the cognate codons. However, when the same assay was performed with near-cognate RCUAG, RF2 showed about twofold higher kcat than mRF2 with almost no change in KM (Fig. 2I). Thus, catalytic efficiency of RF2 on UAG codon is about 2.5-fold higher than mRF2 (Table 1). In line with this result, RF2 shows about fourfold higher catalytic efficiency of peptide release than mRF2 on Trp coding UGG codon (Table 1). On RCUGG, RF2 releases peptide with 2.5-fold higher kcat than mRF2 (Fig. 2L). Interestingly, mRF2 shows 1.5-fold higher KM than RF2 on RCUGG. Thus, together these two parameters cause a bigger difference in the catalytic efficiency of RF2 versus mRF2 on UGG than on UAG codon (Table 1).Table 1The Michaelis–Menten parameters for peptide release by the methylated and unmethylated RFs on cognate and near-cognate codonsCodonA site codonRelease factorkcat (s−1)KM (μM−1)kcat/KM (s−1 μM−1)CognateUAAmRF210.4 ± 0.040.49 ± 0.00721.2 ± 0.40RF26.0 ± 0.170.30 ± 0.0120.0 ± 0.008UGAmRF210.7 ± 0.030.38 ± 0.00928.2 ± 0.36RF26.8 ± 0.190.28 ± 0.0424.3 ± 0.007Near-cognateUAGmRF20.13 ± 0.0115.5 ± 1.760.008 ± 0.0006RF20.28 ± 0.00414.7 ± 0.100.019 ± 0.0004UGGmRF20.10 ± 0.0422 ± 1.40.0045 ± 0.0001RF20.25 ± 0.0314.4 ± 0.110.017 ± 0.0006CognateUAAmRF18.3 ± 0.060.06 ± 0.005138.3 ± 0.003RF12.0 ± 0.040.03 ± 0.0366.7 ± 0.005Near-cognateUGAmRF10.16 ± 0.0042.7 ± 0.030.059 ± 0.004RF10.12 ± 0.0032.6 ± 0.260.046 ± 0.004The values are obtained from M-M plots (Figs. 2 and 3, right panels) with SEM (Standard Error of Mean) estimated from at least three independent sets of experiments. Open table in a new tab The values are obtained from M-M plots (Figs. 2 and 3, right panels) with SEM (Standard Error of Mean) estimated from at least three independent sets of experiments. As expected, mRF2 and RF2 show similar kcat and KM values on the two cognate codons UAA and UGA (Table 1). However, both mRF2 and RF2 show slightly higher catalytic efficiency (kcat/KM) for UGA (28.2 ± 0.36 and 24.3 ± 0.007) than UAA (21.2 ± 0.4 and 20 ± 0.008). Our results clearly demonstrate that mRF2 has higher catalytic activity, but similar catalytic efficiency of peptide release on cognate codons. However, on near-cognate codons, RF2 shows higher catalytic activity as well as catalytic efficiency than mRF2. We compared mRF1 and RF1 for their catalytic efficiency in peptide release from the RCs containing cognate (UAA) and near-cognate (UGA) codon using the stopped-flow-based Bop-Met-Leu-Leu tripeptide release assay (Fig. 3, left and middle panel). The observed rates estimated from the time course of the peptide release are plotted against mRF1 and RF1 concentration and fitted with hyperbolic function to obtain the Michaelis–Menten parameters (kcat, KM, and kcat/KM) (Fig. 3, right panel). Our kinetic data show that mRF1 possesses 4.2-fold higher catalytic activity than RF1 for peptide release from the cognate RCUAA, with kcat 8.3 s−1 (Fig. 3C, Table 1). Along with the increase in kcat, mRF1 also shows a twofold increase in KM, thereby resulting in twofold higher catalytic efficiency (kcat/KM) than RF1 for peptide release on the cognate UAA codon (Table 1). This result confirms that mRF1 has higher catalytic efficiency than RF1 for peptide release on the cognate UAA codon. However, on the near-cognate RCUGA, mRF1 and RF1 show similar catalytic activity with kcat 0.16 s−1 and 0.12 s−1 respectively, with no change in KM (Fig. 3F). Thus, mRF1 and RF1 show similar catalytic efficiency of peptide release on UGA codon (Table 1). Thus, our results demonstrate that methylation of RF1 results in significantly higher catalytic activity and catalytic efficiency of peptide release on the cognate codon (Table 1). To determine the accuracy of the RFs, we introduce a parameter called "A value," which is the ratio of their catalytic efficiencies of peptide release (kcat/KM) on cognate versus near-cognate codons (37Freistroffer 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 (128) Google Scholar). In other words, the A value describes the relative preference of the RFs for the cognate codon (reference codon) over the near-cognate codon (or essentially any other codon). Here, we have used the major stop codon UAA as the reference codon. With regard to the two cognate codons UAA and UGA, both mRF2 and RF2 show slightly higher preference for UGA with the A values 0.74 ± 0.017 and 0.83 ± 0.0004, respectively. However, significantly larger A values are obtained when catalytic efficiencies are compared between UAA and near-cognate UAG and UGG codons. The A values summarized in Table 2 show that mRF2 favors peptide release on UAA over UAG by a factor of 2625 ± 203 and over UGG by a factor of 4200 ± 116. These values are in close agreement with the values previously reported by Freistroffer et al. (37Freistroffer D.V. Kwiatkowski M. Buckingham R.H. Ehrenber
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