Insights into the role of an active site aspartate in Ty1 reverse transcriptase polymerization.
Long terminal repeat-containing retrotransposons encode reverse transcriptases (RTs) that replicate their RNA into integratable, double-stranded DNA. A mutant version of the RT from Saccharomyces cerevisiae retrotransposon Ty1, in which one of the three active site aspartates has been changed to asparagine (D211N), is still capable of in vitro polymerization, although it is blocked for in vivo transposition. We generated recombinant WT and D211N Ty1 RTs to study RT function and determine specific roles for the Asp(211) residue. Presteady-state kinetic analysis of the two enzymes shows that the D211N mutation has minimal effect on nucleotide binding but reduces the k(pol) by approximately 230-fold. The mutation reduces binding affinity for both Mn(2+) and Mg(2+), indicating that the Asp(211) side chain helps create a tight metal binding pocket. Although both enzymes are highly processive and tend to remain bound to their initial substrate, each shows distinctive patterns of pausing, attributable to interactions between metal ions and the active site residue. These results provide insights to specific roles for the Asp(211) residue during polymerization and indicate unusual enzymatic properties that bear on the Ty1 replication pathway.
Pandey
Manjula
M
Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, New Jersey 08854, USA.
Patel
Smita
S
Gabriel
Abram
A
D001224:Aspartic Acid / Q000378:metabolism*; D001665:Binding Sites; D002413:Cations, Divalent / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D018626:Retroelements; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic
D002413:Cations, Divalent; D011994:Recombinant Proteins; D018626:Retroelements; D029701:Saccharomyces cerevisiae Proteins; D001224:Aspartic Acid; D004247:DNA; D012194:RNA-Directed DNA Polymerase