The role of template-primer in protection of reverse transcriptase from thermal inactivation

Nucleic Acids Res. 2002 Jul 15;30(14):3118-29. doi: 10.1093/nar/gkf417.


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.

Publication types

  • Comparative Study

MeSH terms

  • Avian Myeloblastosis Virus / enzymology
  • Binding, Competitive
  • DNA Primers
  • DNA, Complementary / genetics
  • DNA-Directed DNA Polymerase / metabolism
  • Enzyme Stability
  • Half-Life
  • Moloney murine leukemia virus / enzymology
  • Mutation
  • Protein Denaturation
  • RNA / genetics
  • RNA / metabolism
  • RNA-Directed DNA Polymerase / chemistry*
  • RNA-Directed DNA Polymerase / genetics
  • RNA-Directed DNA Polymerase / metabolism*
  • Recombinant Proteins / chemistry
  • Recombinant Proteins / metabolism
  • Temperature
  • Templates, Genetic
  • Transcription, Genetic


  • DNA Primers
  • DNA, Complementary
  • Recombinant Proteins
  • RNA
  • RNA-Directed DNA Polymerase
  • DNA-Directed DNA Polymerase