Mutational Analysis of Bacteriophage T4 RNA Ligase 1

T4 RNA ligase 1 (Rnl1) exemplifies an ATP-dependent RNA ligase family that includes fungal tRNA ligase (Trl1) and a putative baculovirus RNA ligase. Rnl1 acts via a covalent enzyme-AMP intermediate generated by attack of Lys-99 Nζ on the α phosphorus of ATP. Mutation of Lys-99 abolishes ligase activity. Here we tested the effects of alanine mutations at 19 conserved positions in Rnl1 and thereby identified 9 new residues essential for ligase activity: Arg-54, Lys-75, Phe-77, Gly-102, Lys-119, Glu-227, Gly-228, Lys-240, and Lys-242. Seven of the essential residues are located within counterparts of conserved nucleotidyltransferase motifs I (99KEDG102), Ia (118SK119), IV (227EGYVA231), and V (238HFKIK242) that comprise the active sites of DNA ligases, RNA capping enzymes, and T4 RNA ligase 2. Three other essential residues, Arg-54, Lys-75 and Phe-77, are located upstream of the AMP attachment site within a conserved domain unique to the Rnl1-like ligase family. We infer a shared evolutionary history and active site architecture in Rnl1 (a tRNA repair enzyme) and Trl1 (a tRNA splicing enzyme). We determined structure-activity relationships via conservative substitutions and examined mutational effects on the isolated steps of Rnl1 adenylylation (step 1) and phosphodiester bond formation (step 3). Lys-75, Lys-240, and Lys-242 were found to be essential for step 1 and overall ligation of 5′-phosphorylated RNA but not for phosphodiester bond formation. These results suggest that the composition of the Rnl1 active site is different during steps 1 and 3. Mutations at Arg-54 and Lys-119 abolished the overall RNA ligation reaction without affecting steps 1 and 3. Arg-54 and Lys-119 are thereby implicated as specific catalysts of the RNA adenylation reaction (step 2) of the ligation pathway. T4 RNA ligase 1 (Rnl1) exemplifies an ATP-dependent RNA ligase family that includes fungal tRNA ligase (Trl1) and a putative baculovirus RNA ligase. Rnl1 acts via a covalent enzyme-AMP intermediate generated by attack of Lys-99 Nζ on the α phosphorus of ATP. Mutation of Lys-99 abolishes ligase activity. Here we tested the effects of alanine mutations at 19 conserved positions in Rnl1 and thereby identified 9 new residues essential for ligase activity: Arg-54, Lys-75, Phe-77, Gly-102, Lys-119, Glu-227, Gly-228, Lys-240, and Lys-242. Seven of the essential residues are located within counterparts of conserved nucleotidyltransferase motifs I (99KEDG102), Ia (118SK119), IV (227EGYVA231), and V (238HFKIK242) that comprise the active sites of DNA ligases, RNA capping enzymes, and T4 RNA ligase 2. Three other essential residues, Arg-54, Lys-75 and Phe-77, are located upstream of the AMP attachment site within a conserved domain unique to the Rnl1-like ligase family. We infer a shared evolutionary history and active site architecture in Rnl1 (a tRNA repair enzyme) and Trl1 (a tRNA splicing enzyme). We determined structure-activity relationships via conservative substitutions and examined mutational effects on the isolated steps of Rnl1 adenylylation (step 1) and phosphodiester bond formation (step 3). Lys-75, Lys-240, and Lys-242 were found to be essential for step 1 and overall ligation of 5′-phosphorylated RNA but not for phosphodiester bond formation. These results suggest that the composition of the Rnl1 active site is different during steps 1 and 3. Mutations at Arg-54 and Lys-119 abolished the overall RNA ligation reaction without affecting steps 1 and 3. Arg-54 and Lys-119 are thereby implicated as specific catalysts of the RNA adenylation reaction (step 2) of the ligation pathway. RNA ligases join 3′ OH and 5′ PO4 RNA termini via a series of three nucleotidyl transfer steps similar to those of DNA ligases: (i) RNA ligase reacts with ATP to form a covalent ligase-(lysyl-N)–AMP intermediate plus pyrophosphate; (ii) AMP is transferred from ligase-adenylate to the 5′ PO4 RNA end to form an RNA-adenylate intermediate (AppRNA); and (iii) ligase catalyzes attack by an RNA 3′ OH on the RNA-adenylate to seal the two ends via a phosphodiester bond and release AMP (1Silber R. Malathi V.G. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1972; 69: 3009-3013Crossref PubMed Scopus (186) Google Scholar, 2Cranston J.W. Silber R. Malathi V.G. Hurwitz J. J. Biol. Chem. 1974; 249: 7447-7456Abstract Full Text PDF PubMed Google Scholar, 3Sugino A. Snopek T.J. Cozarelli N.R. J. Biol. Chem. 1978; 252: 1732-1738Abstract Full Text PDF Google Scholar, 4Uhlenbeck O.C. Gumport R.I. Enzymes. 1982; 15: 31-58Crossref Scopus (134) Google Scholar, 5Engler M.J. Richardson C.C. Enzymes. 1982; 15: 3-29Crossref Scopus (106) Google Scholar). Bacteriophage T4 RNA ligase 1 (Rnl1) is the founding member of the RNA ligase family (1Silber R. Malathi V.G. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1972; 69: 3009-3013Crossref PubMed Scopus (186) Google Scholar). The function of Rnl1 in vivo is to repair a break in the anticodon loop of Escherichia coli tRNALys triggered by phage activation of a host-encoded anticodon nuclease (6Amitsur M. Levitz R. Kaufman G. EMBO J. 1987; 6: 2499-2503Crossref PubMed Scopus (205) Google Scholar). T4 Rnl1 is a 374-amino acid polypeptide (7Rand K.N. Gait M.J. EMBO J. 1984; 3: 397-402Crossref PubMed Scopus (20) Google Scholar). Gait and co-workers (8Thogerson H.C. Morris H.R. Rand K.N. Gait M.J. Eur. J. Biochem. 1985; 147: 325-329Crossref PubMed Scopus (33) Google Scholar, 9Heaphy S. Singh M. Gait M.J. Biochemistry. 1987; 26: 1688-1696Crossref PubMed Scopus (48) Google Scholar) mapped the site of covalent adenylation to Lys-99 and demonstrated the essential of Lys-99 in by The active site of Rnl1 is located within a conserved I in 1) that a of covalent includes DNA ligases and capping S. PubMed Scopus Google Scholar, S. Biol. Google Scholar). DNA ligases and capping a of conserved motifs and that acid for and Full Text Full Text PDF PubMed Scopus Google Scholar, S. Full Text Full Text PDF PubMed Scopus Google Scholar, M. S. 6: Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. EMBO J. PubMed Scopus Google Scholar, S. Full Text Full Text PDF PubMed Scopus Google Scholar). that DNA ligases and capping from a nucleotidyltransferase S. Biol. Google from an RNA in the and active site of Rnl1 that of DNA ligases and RNA capping is for and mutational of the We identified and a T4 RNA ligase by T4 S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). exemplifies a family of RNA ligases that motifs found in DNA ligases and capping enzymes, plus a of unique and essential S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, S. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). The ligase family includes the ligases of and and a of putative RNA ligases by and of the ligases are in three T4 Rnl1 exemplifies a of RNA ligases with a The Rnl1-like a putative RNA of A. a and the tRNA ligases of G. M. J. PubMed Scopus Google Scholar, J. J. Biol. Chem. Full Text PDF Google Scholar, J. Biochemistry. PubMed Scopus Google Scholar, J. S. R. PubMed Scopus Google Scholar). The of the Rnl1-like and ligases are by with the shared the of three of the nucleotidyltransferase motifs and that comprise the site of DNA ligases and RNA capping of the of the Rnl1-like ligases three as (i) an of counterparts of nucleotidyltransferase motifs and shared DNA ligases, capping enzymes, and (ii) the of of conserved residues upstream of the I that are not found in ligases in DNA ligases and capping and (iii) Rnl1 and the ligase in the motifs I and is not found in the tRNA ligases of the Rnl1-like to of the conserved are we a mutational of positions of T4 Rnl1 by in on motifs and the to and the motifs I and We that motifs IV and V are essential for covalent nucleotidyl transfer by We essential residues the nucleotidyltransferase that Rnl1-like of active T4 DNA the by from T4 DNA (a of with to an site at the and a site 3′ of the The with and and the to the acid mutations were the by the PubMed Scopus Google Scholar). The were with and and The of the and were to the of during and and were Escherichia coli of coli were at in the The were to and at for were by and the at were at were in of and were to of 1 1 and The were to and by for at in a The were to of that with A. The were with of the and with of and in The polypeptide of the were by to the and in the of The Rnl1 were at were determined with the as the and of Rnl1 were for at The were with and the were by The by of the and by the with a specific from the of the RNA 5′ T4 and The by a RNA ligation reaction 1 of 5′ RNA and Rnl1 as were for at The were by of The were by a in 1 The ligation were by of the and with a of of 5′ RNA-adenylate and as S. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and Rnl1 as were for at The were with and the were by T4 Rnl1 in coli as a and the from the by to and with The of Rnl1 (step 1 of the ligation by transfer from to the Rnl1 polypeptide to form a covalent The reaction at and the of at We that of the Rnl1 with The of the Rnl1 of of Rnl1 with a 5′ RNA and in the of ATP in the formation of two new The steps the to a by ligation of the and termini of the (1Silber R. Malathi V.G. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1972; 69: 3009-3013Crossref PubMed Scopus (186) Google Scholar, S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, S. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). step the to the RNA-adenylate generated by AMP transfer from to the 5′ end of the RNA A. Snopek T.J. Cozarelli N.R. J. Biol. Chem. 1978; 252: 1732-1738Abstract Full Text PDF Google Scholar, S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, S. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). of Rnl1 and V for of putative counterparts of nucleotidyltransferase motifs and V in Rnl1 the of and to RNA ligase S. Singh M. Gait M.J. Biochemistry. 1987; 26: 1688-1696Crossref PubMed Scopus (48) Google Scholar) that Lys-99 of I to abolished overall ligation and the to form the as from the of Lys-99 as the site of covalent adenylylation (8Thogerson H.C. Morris H.R. Rand K.N. Gait M.J. Eur. J. Biochem. 1985; 147: 325-329Crossref PubMed Scopus (33) Google Scholar). to is with the of at different mutations of abolished the ligation reaction but on formation of the intermediate S. Singh M. Gait M.J. Biochemistry. 1987; 26: 1688-1696Crossref PubMed Scopus (48) Google Scholar). The of a of the in I for the nucleotidyl transfer step is a shared with DNA ligase M. S. 6: Full Text Full Text PDF PubMed Scopus Google Scholar, PubMed Scopus Google Scholar, J. PubMed Scopus Google Scholar, S. 26: PubMed Scopus Google Scholar, Hurwitz J. PubMed Scopus Google capping S. J. Biol. Chem. Full Text PDF PubMed Google Scholar, S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google and S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, S. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). the that Rnl1 at for reaction steps is that is at the in tRNA ligases and the is a in S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). The of I is in RNA ligases, DNA ligases, and capping enzymes, but the of in RNA ligation not we with the of the residues in motifs IV is a and V we alanine substitutions at and in IV and and Lys-242 in We of V to and an is at the positions in ligase and S. tRNA ligase and in DNA ligases and RNA capping a we Lys-99 to The and were in and from by effects on RNA ligation are in 3. The were of in to the We found that and to form the RNA-adenylate activity. The of the to form the enzyme-AMP intermediate by the were to that of Rnl1 the in the ligase adenylylation The were that the and were of in ligase thereby to the ligation the with in We that the conserved I and the residues of motifs IV and V are essential for Rnl1 of that Rnl1 and baculovirus and fungal three of the covalent nucleotidyltransferase motifs the that (i) the active site of Rnl1-like ligases is that of DNA ligases, capping enzymes, and ligases (ii) Rnl1 active site that are unique to of the nucleotidyltransferase the we alanine at of 9 of Lys-75, Phe-77, and are conserved in the putative baculovirus RNA ligase and the fungal tRNA ligases and of and are conserved in the baculovirus We in on residues, we as for the ATP RNA residues as for a and residues as to in with the of the Full Text Full Text PDF PubMed Scopus Google Scholar, S. Full Text Full Text PDF PubMed Scopus Google Scholar, M. S. 6: Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. EMBO J. PubMed Scopus Google Scholar, S. Full Text Full Text PDF PubMed Scopus Google Scholar). The and were in and from by specific were by and to that of Rnl1 We the mutational effects as Lys-75 and at alanine to of were essential for activity. Lys-119, and at alanine in specific within a of of the Rnl1 were Three other mutations and to and of the with step 1 the and were in RNA ligation 3). and that were active in step 1 were active in the ligation The that in RNA ligation active at the ligase adenylation The in of in ligase We infer that Arg-54 and Lys-119 are essential for of the ligation to formation of the and were active in overall RNA ligation in ligase that the of in the ligation mutational effects on active in step 1 but a in by of RNA and of the intermediate 3). on at a RNA 5′ and a RNA for of step of the ligation in Rnl1 with in in the of ATP. The of Rnl1 to form a phosphodiester at the 5′ end by the of a RNA of the to and RNA by the reaction ligation (step and from of step of the ligation of RNA-adenylate a not and step with to the ligation reaction 3). The with that the I is essential for phosphodiester formation the step reaction not a covalent is with the that of Lys-99 to the step of T4 Rnl1 S. Singh M. Gait M.J. Biochemistry. 1987; 26: 1688-1696Crossref PubMed Scopus (48) Google Scholar). and were in as were in the ligase adenylation step and overall ligation 3). The step with at the adenylation step and in overall from the of of the and on the isolated step reaction with of the overall ligation reaction 3). that step 1 and were These results that different of are for of the three steps of the ligation pathway. of step function for and were of the in ligation of 3). The active in and from the other in that generated in to RNA that the in the isolated step in ligase adenylation and overall that the of in from the of the intermediate in on the at of results 9 in to the I that are essential for Rnl1 activity: Arg-54, Lys-75, Phe-77, Gly-102, Lys-119, Glu-227, Lys-240, and Lys-242. the of residues to the RNA ligase we tested the effects of conservative substitutions at of the essential positions the for alanine is to a conservative by and by and by and and by with and in to in the isolated step new Rnl1 were in coli and from by in ligation of of with a with that with the These the for a at positions and a from the to the that is by but not that are conserved at in IV of tRNA ligases and the baculovirus Rnl1 of Lys-119 by ligase to the the alanine of Arg-54 by in a of ligase function with but the the of in of Lys-75 ligation the We that the at positions and are for that the sites to Rnl1 Lys-75 and Lys-119 are by in the putative baculovirus Rnl1 and Lys-242 in V of Rnl1 were essential and not be by The in ligation and active to the of the at The and were active in as that The specific of the conservative were by and to that of Rnl1 effects on step 1 were with the on overall ligation at of the Lys-75, a essential for step 1 in the alanine we found that the to of with the of function in the as as The and were in ligase adenylation and as active as thereby for in RNA in step 1 of overall ligation activity. conservative mutations effects on step 1 overall and were active in step 1 in The and were active in step 1 of RNA ligase activity. These results the by two residues at a step to ligase in the V effects on step 1 The to of the as as that a at for ligase adenylylation not for The and step 1 and of with the alanine but to is at for in step 1 and overall of Mutations on of for the Three of the and were active in the isolated step reaction in to the overall ligation reaction in the active but the The at Lys-119 the of as the alanine of steps 1 and but of overall ligase in These Lys-119 as a specific of the RNA adenylation step (step 2) of the ligase of mutational effects on Rnl1 in a new relationships in step were at the two essential of mutations and at the not the isolated step reaction with the for the mutations and at the upstream in of the We that the of and Lys-242 in overall ligation during the ligase adenylation reaction (step is that Lys-242 in AMP transfer to as the step 1 but in Arg-54 with in a of step function with the step that the and generated a of as with the is not essential for phosphodiester a The that is in overall ligation active in steps 1 and plus the that at ligation a for a at in the reaction (step 2) of the ligase pathway. in of Lys-75 step to The and were active in step The of the in in step active in step and in overall the specific of Lys-75 during the reaction of the ligase of overall ligase for the that is the at The active in the RNA that the for in step as in step 1 and the overall ligation reaction The of the a for in a to steps 1 and an with the of ATP a in an active We found that the in the isolated step reaction of a at step but the in step in ligase adenylation and overall ligation These results that the of a at the of the intermediate in on Rnl1 conservative of IV with step the We infer that a in steps 1 and of the ligation thereby for the of function in overall ligation for the and the Rnl1 the for RNA we an mutational of residues of T4 Rnl1 that are conserved in fungal tRNA ligases the putative baculovirus Rnl1 We that conserved residues in nucleotidyltransferase motifs I and IV and and V and are essential for the ligase of Rnl1 The were to be essential for the of DNA ligases PubMed Scopus Google Scholar, J. 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Google Scholar). the results residues Lys-75, and located upstream of the I that are found in of the Rnl1-like are essential for Rnl1 ligase and counterparts in ATP-dependent DNA ligases, RNA capping enzymes, and RNA mutational of fungal tRNA ligases, we that the conserved essential positions demonstrated for Rnl1 are to be for Trl1 as that the of T4 Rnl1 in tRNA is similar to the splicing step by Trl1 (6Amitsur M. Levitz R. Kaufman G. EMBO J. 1987; 6: 2499-2503Crossref PubMed Scopus (205) Google Scholar, G. Biochem. Sci. Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google we that T4 Rnl1 and Trl1 a shared evolutionary history from an ligase to We that the baculovirus Rnl1 a similar in RNA as is of T4 RNA T4 and T4 3′ G. M. J. PubMed Scopus Google Scholar, S. PubMed Scopus Google Scholar). from to Rnl1 alanine and conservative mutational effects on overall RNA ligation and the isolated steps of ligase adenylylation and phosphodiester formation. residues in to the Lys-99 were essential for overall and for the isolated step reaction but not for overall ligation S. Singh M. Gait M.J. Biochemistry. 1987; 26: 1688-1696Crossref PubMed Scopus (48) Google the I essential for steps 1 and 3. positions found to be for of the Rnl1 ligase reaction Glu-227, and and Three residues, Lys-75, Lys-240, and were identified as essential for step 1 and overall ligation but not for phosphodiester bond formation. we that the composition of the active site is different during steps 1 and 3. Mutations at Arg-54 and Lys-119 abolished the overall ligation reaction without affecting steps 1 and 3. Arg-54 and Lys-119 are thereby implicated as specific catalysts of the RNA adenylation reaction (step 2) of the ligation pathway. of the are for we on the and the be in of the for other of the covalent nucleotidyltransferase the of an for RNA we a the essential in motifs and V of Rnl1 and the found at positions of motifs and V of DNA ligases and capping Full Text Full Text PDF PubMed Scopus Google Scholar, S. Full Text Full Text PDF PubMed Scopus Google Scholar, M. S. 6: Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. EMBO J. PubMed Scopus Google Scholar, S. Full Text Full Text PDF PubMed Scopus Google Scholar). The Rnl1 IV for Rnl1 ligation and for the steps of ligase adenylation and phosphodiester formation. were not mutational effects on step step and overall ligation were for T4 S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar, S. S. J. Biol. Chem. 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S. 6: Full Text Full Text PDF PubMed Scopus Google Scholar). is that mutations of the Lys-119 in Rnl1 to with step of the ligase of the of the and capping a conserved located of the I and S. in Scholar). The upstream of the is in the capping of capping that is essential for and S. in Scholar). The is located at a in of and capping to that of the in ATP-dependent DNA ligases S. Full Text Full Text PDF PubMed Scopus Google Scholar, S. Full Text Full Text PDF PubMed Scopus Google Scholar). the the the of the covalent the a on the to the 5′ of the RNA to be with S. Full Text Full Text PDF PubMed Scopus Google Scholar). The of the intermediate a on the located to the is to the positions of the RNA to the transfer step of the capping S. Full Text Full Text PDF PubMed Scopus Google Scholar). T4 RNA ligase and a conserved of the I S. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). mutational of that the is essential for ligation alanine and substitutions and that conservative with ligase S. S. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). on and we that the conserved of I is an essential of the active sites of ATP-dependent RNA and DNA ligases and capping we the as of the covalent nucleotidyltransferase of the Rnl1-like residues Arg-54, Lys-75, and are conserved in of the Rnl1-like ATP-dependent DNA ligases, RNA capping enzymes, and RNA ligases of is the I of Rnl1-like ligases and the of ATP-dependent DNA ligases, RNA capping enzymes, and RNA residues mapped at a upstream of I in of the of The of a unique domain in Rnl1-like ligases to with the in Rnl1 of counterparts of motifs and that are essential for the function of ATP-dependent DNA ligases, RNA and is located of and includes an essential that the of the Full Text Full Text PDF PubMed Scopus Google Scholar, S. Full Text Full Text PDF PubMed Scopus Google Scholar). is located of of the to a conserved that a on the of the and the covalent intermediate Full Text Full Text PDF PubMed Scopus Google Scholar, S. Full Text Full Text PDF PubMed Scopus Google Scholar, M. S. 6: Full Text Full Text PDF PubMed Scopus Google Scholar, J. J. EMBO J. PubMed Scopus Google Scholar, S. Full Text Full Text PDF PubMed Scopus Google Scholar). the of the domain of Rnl1 and the of Arg-54, Lys-75, and are we on the function Arg-54 is implicated in step of the Rnl1 ligation pathway. Arg-54 is a to the of the with Lys-119, as The that a function in of Arg-54 is with an with Lys-75 is for the Rnl1 adenylation step but not for phosphodiester formation. is with an Lys-75 and the of ATP in step that is of a in the in step is implicated in steps 1 and 3. is a to form of the ATP and via with the as those by the in of other covalent we effects of mutations that the of by Rnl1 but the overall ligation of step 3. the that the of of an DNA by DNA ligase the of the ligation reaction S. 26: PubMed Scopus Google Scholar). were by and J. Biol. Chem. Scholar) for coli DNA ligase. The for that of an a step in the of that not the intermediate is in at the site S. 26: PubMed Scopus Google Scholar). We a similar for the effects of the mutations on the isolated step reaction of that the of but is the RNA-adenylate is in is located within a that is conserved in the putative baculovirus Rnl1 but is of in the fungal tRNA The of of in of Rnl1-like is with that is not a of the active