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• W2019509622 abstract "Aminoacyl-tRNA synthetases are responsible for activating specific amino acids and transferring them onto cognate tRNA molecules. Due to the similarity in many amino acid side chains, certain synthetases misactivate non-cognate amino acids to an extent that would be detrimental to protein synthesis if left uncorrected. To ensure accurate translation of the genetic code, some synthetases therefore utilize editing mechanisms to hydrolyze non-cognate products. Previously class II Escherichia coli proline-tRNA synthetase (ProRS) was shown to exhibit pre- and post-transfer editing activity, hydrolyzing a misactivated alanine-adenylate (Ala-AMP) and a mischarged Ala-tRNAPro variant, respectively. Residues critical for the editing activity (Asp-350 and Lys-279) are found in a novel insertion domain (INS) positioned between motifs 2 and 3 of the class defining aminoacylation active site. In this work, we present further evidence that INS is responsible for editing in ProRS. We deleted the INS from wild-type E. coli ProRS to yield ΔINS-ProRS. While ΔINS-ProRS was still capable of misactivating alanine, the truncated construct was defective in hydrolyzing non-cognate Ala-AMP. When the INS domain was cloned and expressed as an independent protein, it was capable of deacylating a mischarged Ala-microhelixPro variant. Similar to full-length ProRS, post-transfer editing was abolished in a K279A mutant INS. We also show that YbaK, a protein of unknown function from Haemophilus influenzae with high sequence homology to the prokaryotic INS domain, was capable of deacylating Ala-tRNAPro and Ala-microhelixPro variants but not cognate Pro-tRNAPro. Thus, we demonstrate for the first time that an independently folded class II synthetase editing domain and a previously identified homolog can catalyze a hydrolytic editing reaction. Aminoacyl-tRNA synthetases are responsible for activating specific amino acids and transferring them onto cognate tRNA molecules. Due to the similarity in many amino acid side chains, certain synthetases misactivate non-cognate amino acids to an extent that would be detrimental to protein synthesis if left uncorrected. To ensure accurate translation of the genetic code, some synthetases therefore utilize editing mechanisms to hydrolyze non-cognate products. Previously class II Escherichia coli proline-tRNA synthetase (ProRS) was shown to exhibit pre- and post-transfer editing activity, hydrolyzing a misactivated alanine-adenylate (Ala-AMP) and a mischarged Ala-tRNAPro variant, respectively. Residues critical for the editing activity (Asp-350 and Lys-279) are found in a novel insertion domain (INS) positioned between motifs 2 and 3 of the class defining aminoacylation active site. In this work, we present further evidence that INS is responsible for editing in ProRS. We deleted the INS from wild-type E. coli ProRS to yield ΔINS-ProRS. While ΔINS-ProRS was still capable of misactivating alanine, the truncated construct was defective in hydrolyzing non-cognate Ala-AMP. When the INS domain was cloned and expressed as an independent protein, it was capable of deacylating a mischarged Ala-microhelixPro variant. Similar to full-length ProRS, post-transfer editing was abolished in a K279A mutant INS. We also show that YbaK, a protein of unknown function from Haemophilus influenzae with high sequence homology to the prokaryotic INS domain, was capable of deacylating Ala-tRNAPro and Ala-microhelixPro variants but not cognate Pro-tRNAPro. Thus, we demonstrate for the first time that an independently folded class II synthetase editing domain and a previously identified homolog can catalyze a hydrolytic editing reaction. Aminoacyl-tRNA synthetases are the family of enzymes responsible for activating cognate amino acids with ATP to form aminoacyl-adenylates and subsequently transferring the activated amino acids onto their corresponding tRNAs (1Freist W. Biochemistry. 1989; 28: 6787-6795Crossref PubMed Scopus (71) Google Scholar). The aminoacylated or charged tRNA is then delivered to the ribosome for use in protein synthesis. Each aminoacyl-tRNA synthetase must select and activate its cognate amino acid from the cellular pool of 20 different proteinaceous amino acids. High fidelity in this selection process is necessary to ensure faithful protein translation as an accumulation of mistakes in the aminoacylation process will eventually lead to cell death (2Orgel L.E. Proc. Natl. Acad. Sci. U. S. A. 1963; 49: 517-521Crossref PubMed Scopus (764) Google Scholar, 3Laughrea M. Exp. Gerontol. 1982; 17: 305-317Crossref PubMed Scopus (16) Google Scholar, 4Freist W. Sternbach H. Pardowitz I. Cramer F. J. Theor. Biol. 1998; 193: 19-38Crossref PubMed Scopus (23) Google Scholar).Some amino acids have chemical structures that closely resemble each other, making accurate discrimination by synthetases difficult. In particular, smaller non-cognate amino acids may enter into the active sites of synthetases and be misactivated and subsequently transferred onto the wrong tRNA. Some synthetases are capable of correcting mistakes in amino acid selection through hydrolysis of the misactivated aminoacyl-adenylate prior to amino acid transfer to the tRNA (pretransfer editing) and hydrolysis of the ester linkage of the misacylated tRNA (post-transfer editing) (5Jakubowski H. Goldman E. Microbiol. Rev. 1992; 56: 412-429Crossref PubMed Google Scholar).Editing has been well documented in the case of class I synthetases. For example, class I isoleucine-tRNA synthetase (IleRS) 1The abbreviations used are: IleRSisoleucine-tRNA synthetaseValRSvaline-tRNA synthetaseLeuRSleucine-tRNA synthetaseCP1connective polypeptide 1ThrRSthreonine-tRNA synthetase, ProRS, proline-tRNA synthetaseAlaRSalanine-tRNA synthetaseINSinsertion domainMBPmaltose-binding proteinHI1434H. influenzae YbaK protein.1The abbreviations used are: IleRSisoleucine-tRNA synthetaseValRSvaline-tRNA synthetaseLeuRSleucine-tRNA synthetaseCP1connective polypeptide 1ThrRSthreonine-tRNA synthetase, ProRS, proline-tRNA synthetaseAlaRSalanine-tRNA synthetaseINSinsertion domainMBPmaltose-binding proteinHI1434H. influenzae YbaK protein. and valine-tRNA synthetase (ValRS) activate and edit the non-cognate amino acids leucine and threonine, respectively (6Baldwin A.N. Berg P. J. Biol. Chem. 1966; 241: 839-845Abstract Full Text PDF PubMed Google Scholar, 7Eldred E.W. Schimmel P.R. J. Biol. Chem. 1972; 247: 2961-2964Abstract Full Text PDF PubMed Google Scholar). More recently, class I leucine-tRNA synthetase (LeuRS) has also been shown to misactivate and edit isoleucine as well as a series of non-standard amino acids (8Englisch S. Englisch U. von der Haar F. Cramer F. Nucleic Acids Res. 1986; 14: 7529-7539Crossref PubMed Scopus (78) Google Scholar, 9Chen J.-F. Guo N.-N. Li T. Wang E.-D. Wang Y.-L. Biochemistry. 2000; 39: 6726-6731Crossref PubMed Scopus (116) Google Scholar, 10Lincecum T.L. Martinis S.A. SAAS Bull. Biochem. Biotechnol. 2000; 13: 25-33Google Scholar, 11Mursinna R. Lincecum T.L. Martinis S.A. Biochemistry. 2001; 40: 5376-5381Crossref PubMed Scopus (106) Google Scholar). In all of these class I enzymes, a structural domain distinct from the aminoacylation active site known as connective polypeptide 1 (CP1) (12Starzyk R.M. Webster T.A. Schimmel P. Science. 1987; 237: 1614-1618Crossref PubMed Scopus (141) Google Scholar) is responsible for the editing activity (9Chen J.-F. Guo N.-N. Li T. Wang E.-D. Wang Y.-L. Biochemistry. 2000; 39: 6726-6731Crossref PubMed Scopus (116) Google Scholar, 11Mursinna R. Lincecum T.L. Martinis S.A. Biochemistry. 2001; 40: 5376-5381Crossref PubMed Scopus (106) Google Scholar, 13Schmidt E. Schimmel P. Biochemistry. 1995; 34: 11204-11210Crossref PubMed Scopus (78) Google Scholar, 14Lin L. Hale S.P. Schimmel P. Nature. 1996; 384: 33-34Crossref PubMed Scopus (92) Google Scholar). Biochemical and structural studies have also identified editing activities in class II synthetases, including editing of serine by threonine-tRNA synthetase (ThrRS) (15Dock-Bregeon A.C. Sankaranarayanan R. Romby P. Caillet J. Springer M. Rees B. Francklyn C.S. Ehresmann C. Moras D. Cell. 2000; 103: 877-884Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 16Sankaranarayanan R. Dock-Bregeon A.-C. Rees B. Bovee M. Caillet J. Romby P. Francklyn C.S. Moras D. Nat. Struct. Biol. 2000; 7: 461-465Crossref PubMed Scopus (137) Google Scholar), editing of alanine by proline-tRNA synthetase (ProRS) (17Beuning P.J. Musier-Forsyth K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8916-8960Crossref PubMed Scopus (121) Google Scholar, 18Wong F.C. Beuning P.J. Nagan M.C. Shiba K. Musier-Forsyth K. Biochemistry. 2002; 41: 7108-7115Crossref PubMed Scopus (64) Google Scholar), and editing of glycine and serine by alanine-tRNA synthetase (AlaRS) (19Tsui W.-C. Fersht A.R. Nucleic Acids Res. 1981; 9: 4627-4637Crossref PubMed Scopus (79) Google Scholar, 20Beebe K. Ribas de Pouplana L. Schimmel P. EMBO J. 2003; 22: 668-675Crossref PubMed Scopus (140) Google Scholar).Synthetases are modular proteins composed of domains that have distinct functional roles (21Schimmel P. Ribas de Pouplana L. Trends Biochem. Sci. 2000; 25: 207-209Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 22Alexander R.W. Schimmel P. Prog. Nucleic Acids Res. Mol. Biol. 2001; 69: 317-349Crossref PubMed Google Scholar). The core catalytic domain is responsible for amino acid activation and tRNA acceptor stem docking. It has been proposed that ancestral aminoacyl-tRNA synthetases consisted only of the core catalytic domain, while the anticodon-binding domain was recruited later to improve the binding and discrimination of tRNA substrates (21Schimmel P. Ribas de Pouplana L. Trends Biochem. Sci. 2000; 25: 207-209Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 22Alexander R.W. Schimmel P. Prog. Nucleic Acids Res. Mol. Biol. 2001; 69: 317-349Crossref PubMed Google Scholar, 23Schimmel P. Ribas de Pouplana L. Cold Spring Harbor Symp. Quant. Biol. 2001; 66: 161-166Crossref PubMed Scopus (40) Google Scholar). The catalytic domain alone of some synthetases is capable of aminoacylating tRNA or microhelix substrates that mimic the acceptor stem of full-length tRNA (24Buechter D.D. Schimmel P. Biochemistry. 1995; 34: 6014-6019Crossref PubMed Scopus (17) Google Scholar, 25Augustine J. Francklyn C.S. Biochemistry. 1997; 36: 3473-3482Crossref PubMed Scopus (32) Google Scholar), supporting such an evolutionary scenario. In some cases, an editing domain may have been recruited to improve amino acid specificity and eliminate non-cognate product formation (22Alexander R.W. Schimmel P. Prog. Nucleic Acids Res. Mol. Biol. 2001; 69: 317-349Crossref PubMed Google Scholar, 23Schimmel P. Ribas de Pouplana L. Cold Spring Harbor Symp. Quant. Biol. 2001; 66: 161-166Crossref PubMed Scopus (40) Google Scholar). “Extra” domains such as the editing domains of certain class I synthetases have been cloned and expressed as independent functional domains. In particular, the independently expressed CP1 domains from IleRS and ValRS were shown to deacylate Val-tRNAIle and Thr-tRNAVal, respectively (14Lin L. Hale S.P. Schimmel P. Nature. 1996; 384: 33-34Crossref PubMed Scopus (92) Google Scholar). Interestingly the CP1 domain of LeuRS has been shown to play a critical role in group I intron splicing (26Rho S.B. Lincecum Jr., T.L. Martinis S.A. EMBO J. 2002; 21: 6874-6881Crossref PubMed Scopus (35) Google Scholar).Editing domains found in class II aminoacyl-tRNA synthetases are significantly different from the CP1 domain responsible for editing in class I synthetases. Moreover, in the case of class II enzymes, editing domains identified to date are believed to be distinct from each other, although there is weak homology between the editing domains of ThrRS and AlaRS (15Dock-Bregeon A.C. Sankaranarayanan R. Romby P. Caillet J. Springer M. Rees B. Francklyn C.S. Ehresmann C. Moras D. Cell. 2000; 103: 877-884Abstract Full Text Full Text PDF PubMed Scopus (152) Google Scholar, 20Beebe K. Ribas de Pouplana L. Schimmel P. EMBO J. 2003; 22: 668-675Crossref PubMed Scopus (140) Google Scholar). Based on sequence alignments, class II ProRSs can be divided into two distinct groups (27Cusack S. Yaremchuk A. Krikliviy I. Tukalo M. Structure. 1998; 6: 101-108Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 28Stehlin C. Burke B. Yang F. Liu H. Shiba K. Musier-Forsyth K. Biochemistry. 1998; 37: 8605-8613Crossref PubMed Scopus (61) Google Scholar, 29Ribas de Pouplana L. Brown J.R. Schimmel P. J. Mol. Evol. 2001; 53: 261-268Crossref PubMed Scopus (20) Google Scholar). The “prokaryotic-like” group contains synthetases from bacteria and eukaryotic mitochondrial enzymes, whereas the “eukaryotic-like” group contains synthetases from Eukaryotae, Archaea, and Bacteria. In the case of Escherichia coli ProRS, a representative member of the prokaryotic-like grouping, the editing active site is a novel insertion domain (INS) located between motifs 2 and 3, which together constitute the aminoacylation active site (18Wong F.C. Beuning P.J. Nagan M.C. Shiba K. Musier-Forsyth K. Biochemistry. 2002; 41: 7108-7115Crossref PubMed Scopus (64) Google Scholar). Surprisingly this ∼200-amino acid INS is absent from eukaryotic-like ProRS. This observation suggests either that the INS domain was recruited late in evolution to enhance the performance of the prokaryotic-like enzymes (23Schimmel P. Ribas de Pouplana L. Cold Spring Harbor Symp. Quant. Biol. 2001; 66: 161-166Crossref PubMed Scopus (40) Google Scholar) or that it was present early in both groupings of ProRS and then lost from the eukaryotic-like group, which generally exhibits higher amino acid specificity (30Beuning P.J. Musier-Forsyth K. J. Biol. Chem. 2001; 276: 30779-30785Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). Whereas the three-dimensional structures of two members of the eukaryotic-like ProRS group have been solved by x-ray crystallography (27Cusack S. Yaremchuk A. Krikliviy I. Tukalo M. Structure. 1998; 6: 101-108Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar, 31Yaremchuk A. Cusack S. Tukalo M. EMBO J. 2000; 19: 4745-4758Crossref PubMed Scopus (77) Google Scholar, 32Kamtekar S. Kennedy W.D. Wang J. Stathopoulos C. Söll D. Steitz T.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 1673-1678Crossref PubMed Scopus (35) Google Scholar), the structure of a bacterial ProRS containing the INS domain is not yet known.Previously we have shown that E. coli ProRS is capable of hydrolyzing non-cognate Ala-AMP in a tRNA-independent pre-transfer editing reaction and of deacylating Ala-tRNAPro via post-transfer editing (17Beuning P.J. Musier-Forsyth K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8916-8960Crossref PubMed Scopus (121) Google Scholar). Using alanine-scanning mutagenesis, we have also identified residues within the INS domain that are critical for both pre- and post-transfer editing (18Wong F.C. Beuning P.J. Nagan M.C. Shiba K. Musier-Forsyth K. Biochemistry. 2002; 41: 7108-7115Crossref PubMed Scopus (64) Google Scholar). More recently, ProRS from the bacterium Aquifex aeolicus was shown to possess post-transfer editing activity, whereas a mutant lacking 117 residues of the insertion domain also lacked detectable deacylation activity (33Ahel I. Stathopoulos C. Ambrogelly A. Sauerwald A. Toogood H. Hartsch T. Söll D. J. Biol. Chem. 2002; 277: 34743-34748Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). Taken together with the mutagenesis data obtained with the E. coli enzyme, these data provide strong support for the role of the prokaryotic INS domain in amino acid editing by ProRS.By analogy to the class I CP1 domain, we hypothesized that the INS domain alone might be capable of hydrolytic editing activity. To test this hypothesis, we cloned and expressed the E. coli INS domain and tested its post-transfer editing capability. In addition, we deleted the INS from full-length E. coli ProRS to yield ΔINS-ProRS. We also tested the Haemophilus influenzae YbaK protein, previously reported to have high homology to the bacterial ProRS INS domain (34Wolf Y.I. Aravind L. Grishin N.V. Koonin E.V. Genome Res. 1999; 9: 689-710PubMed Google Scholar, 35Zhang H. Huang K. Li Z. Banerjei L. Fisher K.E. Grishin N.V. Eisenstein E. Herzberg O. Proteins Struct. Funct. Genet. 2000; 40: 86-97Crossref PubMed Scopus (46) Google Scholar), for editing activity. The data presented here provide further support for the functional role of the INS domain in translational editing and demonstrate for the first time that an independently folded domain derived from a class II synthetase can catalyze hydrolytic editing.EXPERIMENTAL PROCEDURESPlasmid Construction—A plasmid encoding ΔINS-ProRS was constructed from plasmid pCS-M1S, which encodes wild-type E. coli ProRS with an N-terminal histidine tag (28Stehlin C. Burke B. Yang F. Liu H. Shiba K. Musier-Forsyth K. Biochemistry. 1998; 37: 8605-8613Crossref PubMed Scopus (61) Google Scholar). This plasmid contains a single KpnI restriction site at the N terminus of the INS domain. A second KpnI restriction site was introduced at the C-terminal end of the INS domain using the QuikChange site-directed mutagenesis kit (Stratagene) and the following primers: 5′-GCGCGCTGCTGGTACCCAGGAAATG-3′ and 5′-CATTTCCTGGGTACCAGCAGCGCGC-3′. After digesting with KpnI to excise the insertion domain, the digested plasmid was then ligated to yield the ΔINS-ProRS with amino acid residues 249–418 of wild-type E. coli ProRS deleted.The plasmid containing the E. coli INS domain was prepared by PCR amplifying the INS fragment from full-length plasmid pCS-M1S using the following primers: 5′-CGCGGATCCATGGGGCTGGATTTCCGCGC-3′ and 5′-CCCAAGCTTGTTACGGCCATCTTCACCC-3′. After digesting with BamHI and HindIII, the PCR fragments were ligated into the corresponding sites of the pMAL-c2E vector (New England Biolabs) immediately following the maltose-binding protein (MBP) coding region. Following ligation, plasmids were transformed into XL-1 Blue supercompetent cells (Stratagene). Results of all cloning and deletion steps were confirmed by automated DNA sequencing (Microchemical Facility, University of Minnesota).RNA Preparation—Wild-type E. coli tRNAPro and a G1:C72/U70-tRNAPro triple mutant were prepared by in vitro transcription from BstNI-linearized plasmids using T7 RNA polymerase as described previously (28Stehlin C. Burke B. Yang F. Liu H. Shiba K. Musier-Forsyth K. Biochemistry. 1998; 37: 8605-8613Crossref PubMed Scopus (61) Google Scholar, 36Liu H. Yap L.-P. Musier-Forsyth K. J. Am. Chem. Soc. 1996; 118: 2523-2524Crossref Scopus (19) Google Scholar). The G1:C72/U70-microhelixPro variant was synthesized using automated chemical RNA synthesis as described previously (37Yap L.-P. Stehlin C. Musier-Forsyth K. Chem. Biol. 1995; 2: 661-666Abstract Full Text PDF PubMed Scopus (15) Google Scholar, 38Burke B. Yang F. Chen F. Stehlin C. Chan B. Musier-Forsyth K. Biochemistry. 2000; 39: 15540-15547Crossref PubMed Scopus (21) Google Scholar). Mischarged G1:C72/U70-tRNAPro and G1:C72/U70-microhelixPro for use in post-transfer deacylation assays were prepared as described previously (17Beuning P.J. Musier-Forsyth K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8916-8960Crossref PubMed Scopus (121) Google Scholar, 18Wong F.C. Beuning P.J. Nagan M.C. Shiba K. Musier-Forsyth K. Biochemistry. 2002; 41: 7108-7115Crossref PubMed Scopus (64) Google Scholar). All reactions contained 2 units/ml inorganic pyrophosphatase, and reactions were quenched by the addition of acetic acid to 1% final concentration. Purified charged tRNA and microhelix were quantified by scintillation counting.Protein Preparation—Expression of wild-type and truncated ΔINS-ProRS was induced with 1 mm isopropyl-β-d-thiogalactopyranoside in SG13009(pREP4) or BL21(DE3) pLysE competent cells. His-tagged proteins were then purified and prepared using a Talon cobalt affinity resin (Clontech) as described previously (39Stehlin C. Heacock D.H. Liu H. Musier-Forsyth K. Biochemistry. 1997; 36: 2932-2938Crossref PubMed Scopus (29) Google Scholar). The MBP-INS fusion protein was purified using amylose affinity chromatography according to the manufacturer's protocol (New England Biolabs). In brief, sonicated protein solution was loaded onto the amylose affinity column followed by extensive washing with 15 column volumes of column buffer (20 mm Tris-HCl, pH 7.4, 200 mm NaCl, 1 mm EDTA). The MBP-INS fusion protein was then eluted with column buffer containing 10 mm maltose. Fractions containing the fusion protein were identified via SDS-PAGE, pooled, and subjected to cleavage by 0.1% enterokinase (New England Biolabs). The latter was performed at 37 °C for 1 h followed by incubation at 25 °C overnight. Following the removal of maltose from the cleaved fusion protein by hydroxyapatite chromatography (Bio-Rad), the solution was loaded onto a second amylose affinity column. The cleaved INS domain was then eluted using column buffer, and fractions containing the purified protein were pooled and concentrated using Centricon 10 microconcentrators (Amicon). Purified enzyme concentrations were determined either by active site titration (full-length and ΔINS-ProRS) or by Bradford protein assay (INS) (BioRad). Concentrated protein was stored in column buffer with the addition of an equal volume of 80% glycerol.Plasmid pCYB2_HI1434 encoding H. influenzae YbaK protein (HI1434) was a gift from Prof. Osnat Herzberg (University of Maryland). The HI1434 is expressed as a fusion protein consisting of an N-terminal HI1434 polypeptide followed by a self-splicing intein and a chitin-binding domain. Protein purification was performed essentially as described previously (35Zhang H. Huang K. Li Z. Banerjei L. Fisher K.E. Grishin N.V. Eisenstein E. Herzberg O. Proteins Struct. Funct. Genet. 2000; 40: 86-97Crossref PubMed Scopus (46) Google Scholar) using the IMPACT™ I system (New England Biolabs). In brief, protein expression was induced in the presence of 1 mm isopropyl-β-d-thiogalactopyranoside, and cell pellets were harvested after 4 h of growth at 37 °C. Cell pellets were then lysed by sonication and clarified by centrifugation. Cell-free lysate was then passed through a chitin column. After washing with column buffer (20 mm HEPES, pH 8.0, 500 mm NaCl, 0.1 mm EDTA, and 0.1% Triton), intein-mediated cleavage was initiated by the addition of column buffer containing 50 mm dithiothreitol. Protein was then eluted with column buffer, and fractions containing the YbaK protein were pooled and concentrated using Centriprep 10 and Centricon 10 concentrators (Amicon). Concentrated protein was stored in column buffer containing an equal volume of 80% glycerol.Circular Dichroism (CD) Spectroscopy—CD spectra of proteins were obtained at room temperature using a J-710 spectropolarimeter (Jasco). Protein samples at a concentration of 0.5 mg/ml in 50 mm HEPES, pH 7.0 were analyzed using a 0.1-cm-path length cuvette, and spectra were accumulated over six scans. Subtraction of the INS domain spectrum from the wild-type ProRS spectrum was performed using Excel and Origin programs.Enzyme Assays—Active site titration was performed using the adenylate burst assay as described previously (40Fersht A.R. Ashford J.S. Bruton C.J. Jakes R. Koch G.L. Hartley B.S. Biochemistry. 1975; 14: 1-4Crossref PubMed Scopus (216) Google Scholar). Cognate tRNA aminoacylation assays were performed at room temperature using the published conditions (41Musier-Forsyth K. Scaringe S. Usman N. Schimmel P. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 209-213Crossref PubMed Scopus (68) Google Scholar, 42Liu H. Peterson R. Kessler J. Musier-Forsyth K. Nucleic Acids Res. 1995; 23: 165-169Crossref PubMed Scopus (51) Google Scholar). Mischarging assays were performed using 22.5 μm [3H]alanine, 2 μm enzyme, and 10 μm tRNA as described previously (18Wong F.C. Beuning P.J. Nagan M.C. Shiba K. Musier-Forsyth K. Biochemistry. 2002; 41: 7108-7115Crossref PubMed Scopus (64) Google Scholar). ATP-PPi exchange assays were performed at 37 °C using the published conditions (43Heacock D. Forsyth C.J. Shiba K. Musier-Forsyth K. Bioorg. Chem. 1996; 24: 273-289Crossref Scopus (107) Google Scholar). The amino acid concentrations used were 0.05–2 mm (proline) and 25–500 mm (alanine), and the final enzyme concentrations were 1 nm (with proline) and 20 nm (with alanine). Kinetic parameters were determined from Lineweaver-Burk plots and represent the average of three determinations. Editing assays were performed at room temperature using the published conditions (17Beuning P.J. Musier-Forsyth K. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 8916-8960Crossref PubMed Scopus (121) Google Scholar). In brief, ATPase assays for assessing pretransfer editing were initiated with a 1–5 μm final concentration of enzymes in the presence of 3 mm [γ-32P]ATP, 4 units/ml inorganic pyrophosphatase, 2 mm proline or 500 mm alanine. Reactions were quenched with 25 volumes of 7% HClO4, 10 mm NaPPi, and 3% charcoal, and the charcoal-bound ATP/AMP was separated from the [32P]Pi in solution by centrifugation. A 50-μl aliquot of the supernatant was quantified by liquid scintillation counting. Deacylation assays for assessing post-transfer editing were performed using 2.5–21 μm protein, as indicated in the figure legends, and 20,000–100,000 cpm mischarged RNA substrate prepared as described above. Aliquots of the reaction mixture were quenched on 5% trichloroacetic acid-soaked Whatman No. 3MM filter pads and washed extensively to remove free 3H-labeled amino acid prior to quantifying by scintillation counting.RESULTSExpression and Stability of Truncated ProRS Constructs—Fig. 1 shows a schematic illustration of the domain architecture of full-length E. coli ProRS (top). Two additional proteins derived from full-length E. coli ProRS were constructed for this work. ΔINS-ProRS, a variant with a deletion from residues 249 to 418 resulting in removal of 86% of the INS domain, is shown in Fig. 1 (bottom). The end points for this internal deletion were chosen to encompass the majority of the INS domain and also reflected convenient sites for cloning without disrupting sequences within the class II consensus motifs 2 and 3. A protein that contains the entire 183-amino acid INS domain and 65 flanking residues chosen to help stabilize the isolated domain (residues 188–436) was also constructed (Fig. 1, middle). This construct was appended to a cleavable MBP fusion protein.To evaluate the structure and folding of the truncated ΔINS-ProRS and the cloned INS domain in comparison to full-length E. coli ProRS, we used CD spectroscopy. Fig. 2 shows the CD spectra of wild-type ProRS (Fig. 2A, solid curve), ΔINS-ProRS (Fig. 2A, dashed curve), and INS (Fig. 2B, solid curve). The wild-type and ΔINS-ProRS spectra are consistent with a combination of α-helical and β-sheet secondary structural elements with minima near 208 and 220 nm. The INS domain has a single minimum at 205 nm, suggesting a higher content of β-sheet and random coil elements than the full-length protein. Subtraction of the INS domain spectrum from the wild-type ProRS spectrum yields the spectrum shown in Fig. 2A, inset (WT-INS). The close resemblance between the calculated WT-INS spectrum and the experimental spectrum for ΔINS-ProRS (Fig. 2A, dashed curve) provides strong support for the structural integrity of the engineered proteins.Fig. 2CD spectra of wild-type E. coli ProRS and truncated constructs. Protein samples were measured at a concentration of 0.5 mg/ml in 50 mm HEPES, pH 7.0 at room temperature using a 0.1-cm-path length cuvette. Spectra were accumulated over six scans. A shows full-length E. coli ProRS (solid curve) and ΔINS-ProRS (dashed curve). B shows the INS domain (solid curve) and K279A-INS (dashed curve). The inset to A shows a spectrum calculated by subtraction of the INS spectrum from that of the full-length protein (WT-INS). WT, wild type; deg, degrees.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Misactivation of Non-cognate Amino Acids—Misactivation of non-cognate alanine by wild-type E. coli ProRS and ΔINS-ProRS was tested by assaying ATP-PPi exchange. Wild-type ProRS misactivates alanine with a kcat of 3 ± 2 s–1 and Km of 300 ± 30 mm, while ΔINS-ProRS was found to misactivate alanine with a kcat of 0.4 ± 0.2 s–1 and Km of 80 ± 30 mm. Thus, relative to wild-type ProRS, the kcat/Km of the deletion mutant is only reduced by ∼2-fold. These data indicate that the deletion of the INS domain results in only a moderate decrease in the ability of the ΔINS-ProRS to activate alanine. As expected, no activation of alanine was detected with the cloned INS domain, which lacks the aminoacylation active site. We also tested the purified ΔINS-ProRS for misactivation of glycine in the ATP-PPi exchange assay as wild-type E. coli ProRS is known to weakly misactivate non-cognate glycine (30Beuning P.J. Musier-Forsyth K. J. Biol. Chem. 2001; 276: 30779-30785Abstract Full Text Full Text PDF PubMed Scopus (68) Google Scholar). However, no glycine activation was detected.To ensure that the observed alanine misactivation by ΔINS-ProRS was not due to trace contamination of E. coli AlaRS during protein purification, we tested the purified ΔINS-ProRS for its ability to charge an E. coli tRNAAla transcript with alanine. No aminoacylation activity was detected using 0.5 μm ΔINS-ProRS and 0.5 μm tRNAAla. Under the same conditions, aminoacylation activity was readily detected using 0.1 μm purified E. coli AlaRS and 0.5 μm tRNAAla.Activation of Cognate Proline, Cognate tRNA Aminoacylation, and Mischarging—Under standard reaction condit" @default.
• W2019509622 created "2016-06-24" @default.
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