Kinetic Pathway of Pyrophosphorolysis by a Retrotransposon Reverse Transcriptase

摘要: DNA and RNA polymerases use a common phosphoryl transfer mechanism for base addition that requires two or three acidic amino acid residues at their active sites. We previously showed, the reverse transcriptase (RT) encoded by yeast retrotransposon Ty1, one of conserved site aspartates (D(211)) can be substituted asparagine still retain in vitro polymerase activity, although vivo transposition is lost. Transposition partially restored second suppressor mutations RNAse H domain. The novel properties this substitution led us to express WT D(211)N mutant enzymes, study pre-steady state kinetic parameters. found k(pol) was reduced factor 223 mutant, K(d) nucleotide binding unaltered. Further, enzyme had marked preference Mn(2+) over Mg(2+). To better understand functions residue within Ty1 RT site, we have now examined RTs carrying out pyrophosphorolysis, reaction polymerization, where pyrophosphate substrate dNTPs are product. find pyrophosphorolysis efficient only when base-paired primer template region >14 bases, activity increases end blunt-ended recessed few bases. Using analysis, rate (k(pyro)) nearly 320 fold lower than enzyme, has an approximately 170 apparent pyrophosphate. These findings indicate subtle differences strongly affect enzyme's ability properly position primer-end carry pyrophosphorolysis. Further data suggests D(211) role release, which could translocation, help explain mutant's defect.