The role of template-primer in protection of reverse transcriptase from thermal inactivation.
We compared the thermal stabilities of wild-type recombinant avian myeloblastosis virus (AMV) and Moloney murine leukemia virus (M-MLV) reverse transcriptase (RT) with those of mutants of the recombinant enzymes lacking RNase H activity. They differed in resistance to thermal inactivation at elevated temperatures in the presence of an RNA/DNA template-primer. RNase H-minus RTs retained the ability to efficiently synthesize cDNA at much higher temperatures. We show that the structure of the template-primer has a critical bearing on protection of RT from thermal inactivation. RT RNase H activity rapidly alters the structure of the template-primer to forms less tightly bound by RT and thus less able to protect the enzyme at elevated temperatures. We also found that when comparing wild-type or mutant AMV RT with the respective M-MLV RT, the avian enzymes retained more DNA synthetic activity at elevated temperatures than murine RTs. Enzyme, template-primer interaction again played the most significant role in producing these differences. AMV RT binds much tighter to template- primer and has a much greater tendency to remain bound during cDNA synthesis than M-MLV RT and therefore is better protected from heat inactivation.
Gerard
Gary F
GF
Invitrogen Corporation, 7335 Executive Way, Frederick, MD 21704, USA. gary.gerard@invitrogen.com
Potter
R Jason
RJ
Smith
Michael D
MD
Rosenthal
Kim
K
Dhariwal
Gulshan
G
Lee
Jun
J
Chatterjee
Deb K
DK
D009189:Avian Myeloblastosis Virus / Q000201:enzymology; D001667:Binding, Competitive; D017931:DNA Primers; D018076:DNA, Complementary / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000378:metabolism; D004795:Enzyme Stability; D006207:Half-Life; D008979:Moloney murine leukemia virus / Q000201:enzymology; D009154:Mutation; D011489:Protein Denaturation; D012313:RNA / Q000235:genetics / Q000378:metabolism; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013696:Temperature; D013698:Templates, Genetic; D014158:Transcription, Genetic
D017931:DNA Primers; D018076:DNA, Complementary; D011994:Recombinant Proteins; D012313:RNA; D012194:RNA-Directed DNA Polymerase; D004259:DNA-Directed DNA Polymerase