Ribosomal protein S18 acetyltransferase RimI is responsible for the acetylation of elongation factor Tu

Abstract

N-terminal acetylation is widespread in the eukaryotic proteome but in bacteria is restricted to a small number of proteins mainly involved in translation. It was long known that elongation factor Tu (EF-Tu) is N-terminally acetylated, whereas the enzyme responsible for this process was unclear. Here, we report that RimI acetyltransferase, known to modify ribosomal protein S18, is likewise responsible for N-acetylation of the EF-Tu. With the help of inducible tufA expression plasmid, we demonstrated that the acetylation does not alter the stability of EF-Tu. Binding of aminoacyl tRNA to the recombinant EF-Tu in vitro was found to be unaffected by the acetylation. At the same time, with the help of fast kinetics methods, we demonstrate that an acetylated variant of EF-Tu more efficiently accelerates A-site occupation by aminoacyl-tRNA, thus increasing the efficiency of in vitro translation. Finally, we show that a strain devoid of RimI has a reduced growth rate, expanded to an evolutionary timescale, and might potentially promote conservation of the acetylation mechanism of S18 and EF-Tu. This study increased our understanding of the modification of bacterial translation apparatus. N-terminal acetylation is widespread in the eukaryotic proteome but in bacteria is restricted to a small number of proteins mainly involved in translation. It was long known that elongation factor Tu (EF-Tu) is N-terminally acetylated, whereas the enzyme responsible for this process was unclear. Here, we report that RimI acetyltransferase, known to modify ribosomal protein S18, is likewise responsible for N-acetylation of the EF-Tu. With the help of inducible tufA expression plasmid, we demonstrated that the acetylation does not alter the stability of EF-Tu. Binding of aminoacyl tRNA to the recombinant EF-Tu in vitro was found to be unaffected by the acetylation. At the same time, with the help of fast kinetics methods, we demonstrate that an acetylated variant of EF-Tu more efficiently accelerates A-site occupation by aminoacyl-tRNA, thus increasing the efficiency of in vitro translation. Finally, we show that a strain devoid of RimI has a reduced growth rate, expanded to an evolutionary timescale, and might potentially promote conservation of the acetylation mechanism of S18 and EF-Tu. This study increased our understanding of the modification of bacterial translation apparatus. Post-translational modifications are paramount for the regulation of protein activity, stability, and localization. Acetylation is among the most abundant protein modifications in eukaryotes (1Verdin E. Ott M. 50 Years of protein acetylation: From gene regulation to epigenetics, metabolism and beyond.Nat. Rev. Mol. Cell Biol. 2015; 16: 258-264Crossref PubMed Scopus (470) Google Scholar), while bacteria have generally few acetylated proteins (2Favrot L. Blanchard J.S. Vergnolle O. Bacterial GCN5-related N -acetyltransferases: From resistance to regulation.Biochemistry. 2016; 55: 989-1002Crossref PubMed Scopus (100) Google Scholar). A number of Escherichia coli proteins are N-acetylated (3Nesterchuk M.V. Sergiev P.V. Dontsova O.A. Posttranslational modifications of ribosomal proteins in Escherichia coli.Acta Naturae. 2011; 3: 22-33Crossref PubMed Google Scholar), such as ribosomal proteins S5 (4Wittmann-Liebold B. Greuer B. The primary structure of protein S5 from the small subunit of the Escherichia coli ribosome.FEBS Lett. 1978; 95: 91-98Crossref PubMed Scopus (45) Google Scholar), S18 (5Yaguchi M. Primary structure of protein S18 from the small Escherichia coli ribosomal subunit.FEBS Lett. 1975; 59: 217-220Crossref PubMed Scopus (40) Google Scholar), and L12 (6Terhorst C. Möller W. Laursen R. Wittmann-Liebold B. The primary structure of an acidic protein from 50-S ribosomes of Escherichia coli which is involved in GTP hydrolysis dependent on elongation factors G and T.Eur. J. Biochem. 1973; 34: 138-152Crossref PubMed Scopus (156) Google Scholar) as well as the translation elongation factor Tu (EF-Tu) (7Laursen R.A. L'Italien J.J. Nagarkatti S. Miller D.L. The amino acid sequence of elongation factor Tu of Escherichia coli. The complete sequence.J. Biol. Chem. 1981; 256: 8102-8109Abstract Full Text PDF PubMed Google Scholar). Protein acetylation is commonly carried out by the GCN5-related (GNAT) family of enzymes (2Favrot L. Blanchard J.S. Vergnolle O. Bacterial GCN5-related N -acetyltransferases: From resistance to regulation.Biochemistry. 2016; 55: 989-1002Crossref PubMed Scopus (100) Google Scholar). A family of acetyltransferases RimJ (8Yoshikawa A. Isono S. Sheback A. Isono K. Cloning and nucleotide sequencing of the genes rimI and rimJ which encode enzymes acetylating ribosomal proteins S18 and S5 of Escherichia coli K12.Mol. Gen. Genet. 1987; 209: 481-488Crossref PubMed Scopus (98) Google Scholar), RimI (8Yoshikawa A. Isono S. Sheback A. Isono K. Cloning and nucleotide sequencing of the genes rimI and rimJ which encode enzymes acetylating ribosomal proteins S18 and S5 of Escherichia coli K12.Mol. Gen. Genet. 1987; 209: 481-488Crossref PubMed Scopus (98) Google Scholar), and RimL (9Isono S. Isono K. Ribosomal protein modification in Escherichia coli. III. Studies of mutants lacking an acetylase activity specific for protein L12.Mol. Gen. Genet. 1981; 183: 473-477Crossref PubMed Scopus (23) Google Scholar) were found to be responsible for the N-terminal acetylation of ribosomal proteins. Acetylation of S5 was found to be involved in ribosome assembly (10Roy-Chaudhuri B. Kirthi N. Kelley T. Culver G.M. Suppression of a cold-sensitive mutation in ribosomal protein S5 reveals a role for RimJ in ribosome biogenesis.Mol. Microbiol. 2008; 68: 1547-1559Crossref PubMed Scopus (38) Google Scholar, 11Cumberlidge A.G. Isono K. Ribosomal protein modification in Escherichia coli. I. A mutant lacking the N-terminal acetylation of protein S5 exhibits thermosensitivity.J. Mol. Biol. 1979; 131: 169-189Crossref PubMed Scopus (45) Google Scholar), in a way that its inactivation leads to bacterial growth cold sensitivity. RimL is known to catalyze ribosomal protein L12 acetylation (9Isono S. Isono K. Ribosomal protein modification in Escherichia coli. III. Studies of mutants lacking an acetylase activity specific for protein L12.Mol. Gen. Genet. 1981; 183: 473-477Crossref PubMed Scopus (23) Google Scholar). Recently, RimL ascribed an additional function of microcin C acetylation resulting in a ground state E. coli resistance level toward this antibiotic (12Kazakov T. Kuznedelov K. Semenova E. Mukhamedyarov D. Datsenko K.A. Metlitskaya A. Vondenhoff G.H. Tikhonov A. Agarwal V. Nair S. Van Aerschot A. Severinov K. The RimL transacetylase provides resistance to translation inhibitor microcin C.J. Bacteriol. 2014; 196: 3377-3385Crossref PubMed Scopus (16) Google Scholar). While the phenotype of rimL inactivation is moderate (9Isono S. Isono K. Ribosomal protein modification in Escherichia coli. III. Studies of mutants lacking an acetylase activity specific for protein L12.Mol. Gen. Genet. 1981; 183: 473-477Crossref PubMed Scopus (23) Google Scholar, 13Gordiyenko Y. Deroo S. Zhou M. Videler H. Robinson C.V. Acetylation of L12 increases interactions in the Escherichia coli ribosomal stalk complex.J. Mol. Biol. 2008; 380: 404-414Crossref PubMed Scopus (48) Google Scholar), rimL expression was shown to be increased at the stationary phase of bacterial culture (13Gordiyenko Y. Deroo S. Zhou M. Videler H. Robinson C.V. Acetylation of L12 increases interactions in the Escherichia coli ribosomal stalk complex.J. Mol. Biol. 2008; 380: 404-414Crossref PubMed Scopus (48) Google Scholar). RimI-dependent acetylation of the ribosomal protein S18 (8Yoshikawa A. Isono S. Sheback A. Isono K. Cloning and nucleotide sequencing of the genes rimI and rimJ which encode enzymes acetylating ribosomal proteins S18 and S5 of Escherichia coli K12.Mol. Gen. Genet. 1987; 209: 481-488Crossref PubMed Scopus (98) Google Scholar, 14Isono K. Isono S. Ribosomal protein modification in Escherichia coli. II. Studies of a mutant lacking the N-terminal acetylation of protein S18.Mol. Gen. Genet. 1980; 177: 645-651Crossref PubMed Scopus (38) Google Scholar) was characterized structurally and biochemically (15Vetting M.W. Bareich D.C. Yu M. Blanchard J.S. Crystal structure of RimI from Salmonella typhimurium LT2, the GNAT responsible for N(alpha)-acetylation of ribosomal protein S18.Protein Sci. 2008; 17: 1781-1790Crossref PubMed Scopus (64) Google Scholar). EF-Tu, the most abundant bacterial protein (16Furano A.V. Content of elongation factor Tu in Escherichia coli.Proc. Natl. Acad. Sci. U. S. A. 1975; 72: 4780-4784Crossref PubMed Scopus (140) Google Scholar), is N-terminally acetylated. Its abundance and conservation in bacteria allowed it to be recognized by the innate immunity system of plants as one of pathogen-associated molecular pattern (17Zipfel C. Kunze G. Chinchilla D. Caniard A. Jones J.D.G. Boller T. Felix G. Perception of the bacterial PAMP EF-Tu by the receptor EFR restricts Agrobacterium-mediated transformation.Cell. 2006; 125: 749-760Abstract Full Text Full Text PDF PubMed Scopus (1237) Google Scholar), and its acetylated N-terminal fragment is used as a recognition element (18Kunze G. Zipfel C. Robatzek S. Niehaus K. Boller T. Felix G. The N terminus of bacterial elongation factor Tu elicits innate immunity in Arabidopsis plants.Plant Cell. 2004; 16: 3496-3507Crossref PubMed Scopus (601) Google Scholar). While there are indications that acetylation of EF-Tu may happen at internal lysine residues and this modification is under a growth stage control in Bacillus subtilis (19Suzuki S. Kondo N. Yoshida M. Nishiyama M. Kosono S. Dynamic changes in lysine acetylation and succinylation of the elongation factor Tu in Bacillus subtilis.Microbiology (Reading). 2019; 165: 65-77Crossref PubMed Scopus (7) Google Scholar), neither regulation nor function of N-terminal acetylation of EF-Tu was studied. The enzyme responsible for acetylation of EF-Tu has eluded identification for almost 4 decades. In this work, we have demonstrated that S18-specific acetyltransferase RimI is in addition responsible for the N-terminal acetylation of EF-Tu, thus being a dual-specificity enzyme. The majority of eukaryotic protein acetyltransferases is capable to modify multiple targets, while similar enzymes of bacteria are usually considered to be single protein specific. Since amino group acetylation removes a single positive charge, it might be detected by 2D protein gel electrophoresis. We compared total proteins extracted from ΔrimI strain from Keio knockout collection (20Baba T. Ara T. Hasegawa M. Takai Y. Okumura Y. Baba M. Datsenko K.A. Tomita M. Wanner B.L. Mori H. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: The Keio collection.Mol. Syst. Biol. 2006; 2 (2006.0008)Crossref Scopus (5196) Google Scholar), labeled by Cy2 green fluorescent dye with that from the isogenic parental strain (21Datsenko K.A. Wanner B.L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products.Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6640-6645Crossref PubMed Scopus (10838) Google Scholar) labeled with the Cy5 red fluorescent dye with the help of 2D protein electrophoresis (Fig. 1). We expected that proteins that differed by the acetylation would be represented by a couple of green and red spots of the same molecular mass but different isoelectric points. We readily observed such an effect when comparing proteins from the WT and ΔrimI strains (Fig. 1). The protein spots with an isoelectric point dependent on the RimI were excised from the gel and identified by MALDI mass spectrometry (MS) following tryptic digestion. Both spots appeared to correspond to the EF-Tu protein. While intensity of several protein spots on the gel (Fig. 1) appears to depend on rimI gene functionality, they do not demonstrate the pattern that would indicate they represent the substrates of acetylation. To identify EF-Tu amino acid that is acetylated by RimI, we transformed ΔrimI strain with the plasmid carrying tufA gene with the C-terminal hexahistidine tag and used the resulting culture for EF-Tu preparation by metallochelate chromatography. WT strain transformed with the same plasmid was used for purification of the control EF-Tu sample. Tryptic hydrolysate of recombinant EF-Tu was subjected to MALDI MS analysis, which failed to reveal the modification site presumably because N-terminal end of EF-Tu is rich with basic amino acids, and digestion with trypsin results in a very short tryptic peptide. To overcome this difficulty, we applied chymotrypsin hydrolysis followed by MALDI MS analysis (Fig. 2A) and readily identified that EF-Tu from the WT, but not from ΔrimI strain, contains acetyl group attached to the N-terminal SKEKF peptide. Further fragmentation of this peptide in mass spectrometer (Fig. 2B) indicated that the N-terminal serine group is acetylated. This result corroborated earlier findings that EF-Tu is N-terminally acetylated (7Laursen R.A. L'Italien J.J. Nagarkatti S. Miller D.L. The amino acid sequence of elongation factor Tu of Escherichia coli. The complete sequence.J. Biol. Chem. 1981; 256: 8102-8109Abstract Full Text PDF PubMed Google Scholar). Thus, RimI is a dual-specificity protein acetyltransferase responsible for the N-terminal modification of two proteins, S18 and EF-Tu. To verify that rimI gene is indeed inactivated in the ΔrimI strain from Keio collection and since S18 protein is too small to be resolved by the standard 2D gel, we compared the mass of S18 protein, the known target of RimI (8Yoshikawa A. Isono S. Sheback A. Isono K. Cloning and nucleotide sequencing of the genes rimI and rimJ which encode enzymes acetylating ribosomal proteins S18 and S5 of Escherichia coli K12.Mol. Gen. Genet. 1987; 209: 481-488Crossref PubMed Scopus (98) Google Scholar), from knockout and the WT strains (Fig. 2C). As expected, the mass of S18 from the knockout strain was found to be lower by precisely the mass of the acetyl group. To check that it is inactivation of rimI gene responsible for the difference in the EF-Tu isoelectric point, but not some hypothetic secondary mutation, we transformed ΔrimI strain with the plasmid coding for RimI protein. Analysis of EF-Tu by MALDI MS confirmed that reintroduction of rimI gene restored acetylation of N-terminal serine (Fig. 2D). Thus, we demonstrated that RimI is necessary for EF-Tu N terminus acetylation. It is most likely that RimI directly acetylates EF-Tu, although we should formally acknowledge that a possibility might exist that an influence of RimI on EF-Tu acetylation could be indirect. The structure of Salmonella typhimurium RimI protein in a complex with bisubstrate inhibitor CoA-S-acetyl-ARYFRR a peptide the N-terminal peptide of S18 ribosomal protein was by Blanchard (15Vetting M.W. Bareich D.C. Yu M. Blanchard J.S. Crystal structure of RimI from Salmonella typhimurium LT2, the GNAT responsible for N(alpha)-acetylation of ribosomal protein S18.Protein Sci. 2008; 17: 1781-1790Crossref PubMed Scopus (64) Google Scholar) of RimI with S18 N-terminal peptide several such as of with of The amino acid of the peptide inhibitor to of S18 with of RimI, whereas Van with and of a of the N-terminal of S18 and EF-Tu is the that proteins a small amino in S18 and in EF-Tu, whereas the is in S18 and in EF-Tu. Since protein N-terminal acetylation is considered to protein stability at in eukaryotes of protein by N-terminal acetylation and the Mol. PubMed Scopus Google Scholar), we to check acetylation of EF-Tu by RimI the stability of the To this we used ΔrimI strain transformed by the plasmid coding for under the control of of was on for at the growth phase of at followed by the with lacking The of in of expression was by with (Fig. and that acetylation does not alter EF-Tu Finally, to the of EF-Tu in of protein we used factors from the WT (EF-Tu) and ΔrimI strains we an of tRNA with EF-Tu on the acetylation of the of the complex by with has by a of The changes were to single (Fig. of the of (Fig. the for EF-Tu, for were to in Since the structure of complex M. G. L. J. Crystal structure of the complex of EF-Tu, and a GTP PubMed Scopus Google Scholar), the nucleotide of tRNA is to the N terminus of EF-Tu, we that amino group might To this we the stability of and in the complex with with the help of (Fig. C and The hydrolysis were single As expected, to the from but difference in the hydrolysis in a complex with has we the role of EF-Tu N-terminal acetylation in of translation on the with coding for and in the were with the complete of translation factors Analysis of the has at of to EF-Tu from to (Fig. In was almost We not difference in for the of WT EF-Tu While the end of demonstrated on the acetylation of EF-Tu, we to of which and of to the ribosome by might depend on EF-Tu of of ribosomal with (Fig. allowed to the of the the 2 for and for were found to be the same the In we used a to of the to the ribosome with the A-site in a of (Fig. in a the following of the complex to the ribosome and and which and results in GTP hydrolysis by EF-Tu. The of the is to the of tRNA from the of EF-Tu and of the tRNA in the A site of the ribosome T. mechanism of elongation factor of to the A site of the E. coli J. 17: PubMed Scopus Google Scholar). of the allowed to the of complex and A-site for a number of of the (Fig. we have observed that acetylated of EF-Tu of to the ribosome for EF-Tu and 2 for At the same time, the of tRNA were the same for of EF-Tu for EF-Tu and 2 for Finally, to acetylation of EF-Tu that increases the of to the ribosome to bacterial protein we the growth of the WT and ΔrimI strains (Fig. of this a moderate growth of the ΔrimI While the difference is small in rich (Fig. in the growth more (Fig. In a timescale, growth by the system of S18 and EF-Tu acetylation would to the of this in bacterial To this we a growth (Fig. of the parental strain and ΔrimI strain were and for at in with At the end of the growth of the the of the was used for of the at of growth the were and on with The of WT to ΔrimI in the was but and that knockout of rimI influence of RimI activity on growth may of rimI gene in bacterial B. of protein lysine acetyltransferases in Escherichia PubMed Google Scholar). Thus, we that EF-Tu acetylation accelerates tRNA to the A-site of the ribosome that results in of bacterial growth rate, which is for the growth in when might be it should be that it is to the of EF-Tu acetylation on since RimI is responsible for modification of S18 as It is most likely that the of acetylation on S18 and EF-Tu may to the observed growth in ΔrimI E. coli strain (21Datsenko K.A. Wanner B.L. One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products.Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 6640-6645Crossref PubMed Scopus (10838) Google Scholar) used as WT and the isogenic strain (20Baba T. Ara T. Hasegawa M. Takai Y. Okumura Y. Baba M. Datsenko K.A. Tomita M. Wanner B.L. Mori H. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: The Keio collection.Mol. Syst. Biol. 2006; 2 (2006.0008)Crossref Scopus (5196) Google Scholar), lacking the rimI were by of E. coli strains were at in and with 50 the of growth was by a single of the WT ΔrimI strains and for at The culture was to of at culture of with and at for in with at The at of culture was was with A growth was using with at of parental strain and ΔrimI strain were used for of At growth the was At the same time, of the bacterial culture were a of was from the were to and with were at and the were and To EF-Tu stability time, sequence coding for under control of was for the of PubMed Scopus Google Scholar) the plasmid with the following the recombinant EF-Tu sequence coding for under control of was the plasmid with the following 2D difference gel electrophoresis analysis was as M.V. Sergiev P.V. and analysis of Bacillus subtilis single and 2015; PubMed Scopus (16) Google Scholar, E. A. S. M. V. A. I. A. M. M. Dontsova O.A. analysis of Escherichia coli ribosomal Genet. PubMed Scopus Google Scholar). E. coli were with 2D 2 50 of total protein was labeled with Cy2 Cy5 and applied for isoelectric on a The was with the recombinant EF-Tu of the WT ΔrimI strains transformed with the plasmid were used for of the with At an of at expression of EF-Tu gene was by are the of were by in the 50 inhibitor and by a EF-Tu was from by with acid and at 4 50 50 and with of to was used for acid chromatography. and with of to were used for chromatography. EF-Tu, to the gel were EF-Tu was and to by and and and were to K. D. M.V. of elongation factor nucleotide in Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, R. W. W. M.V. of GTP hydrolysis by elongation factor G on the Google Scholar, A. M.V. and in of of translation in PubMed Scopus Google Scholar, V. E. A. the ribosome with to Biol. PubMed Scopus (23) Google Scholar, and of an in Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, W. W. M.V. GTP hydrolysis by tRNA on small and ribosomal J. 2014; PubMed Scopus Google Scholar, W. of tRNA 1979; 59: PubMed Scopus Google Scholar). sequence coding for and sequence and coding for were by in vitro and used the in vitro not indicated A sequence of the for of A sequence of the for of The sequence of is shown in coding for are shown in in vitro were in 50 and at a of complex of the ribosomes were with 2 and in with GTP and 2 for at of were by of the WT EF-Tu with 2 in for at followed by addition of and and were by and at whereas was complex was with and tRNA in for at In the kinetics to of the complex and to of the complex were used of the complex were used In the on of the complex and of the complex were used MS analysis was carried out as protein of were out of with with and for in for and in the of the gel were with trypsin in for 4 at an of acid was the were on a in the and by MALDI MS using an mass spectrometer MS analysis of EF-Tu modification protein from the WT and ΔrimI strains were subjected to 2D difference gel electrophoresis. The protein spots of were identified with trypsin digestion and MS analysis of S18 modification total ribosomal proteins from the WT and ΔrimI strains were subjected to analysis directly To identify an modification site of EF-Tu, protein spots were by 50 with of and were the same as in trypsin digestion The mass were with the WT and ΔrimI were transformed with a plasmid coding for under the control of The expression of was on for at the growth phase of at followed by the with the were from the culture indicated and by 2D Protein were by and was for at using The was with and with primary 4 and with were used 2 and with and the were using the in the in control was by to with kinetics were using an was at and a were in in the were by to were using were using the same To study the kinetics of complex was with increasing of as were by to a function with a and to A is the at A function was used to the of the complex from the of To the of A-site we with the increasing of the with at were by to a function with two and and to The function was used to the of the A-site of with and EF-Tu were as with of EF-Tu the factor in EF-Tu at were by the and at the To the and kinetics of with were with and at the and for the and for the of kinetics for the the were for were with of and for at were with of acid and by The of the was by the of the To the kinetics of the were in a The was at by the addition of and were as The were using two are in the The that they have of with the of this This was by the and the A. A. B. and V. S. I. O. S. M. M. V. I. A. and A. I. O. S. V. I. A. and A. V. S. S. A. A. L. and V. S. A. O. A. A. L. and V. S. O. A. D. This was by the O. A.

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