2'-Fluoro- and 2'-amino-2'-deoxynucleoside 5'-triphosphates as substrates for T7 RNA polymerase

Biochemistry. 1992 Oct 13;31(40):9636-41. doi: 10.1021/bi00155a016.

Abstract

2'-Fluoro- and 2'-amino-2'-deoxynucleoside 5'-triphosphates have been investigated as substrates for T7 RNA polymerase. Michaelis-Menten kinetic parameters are reported for the incorporation of 2'-fluoro-2'-deoxyuridine, 2'-fluoro-2'-deoxycytidine, and 2'-amino-2'-deoxyuridine into runoff transcripts. The 2'-amino derivative of uridine is a better substrate than the 2'-fluoro derivative. Gel electrophoretic analysis shows that full-length transcripts with a length of 2500 nucleotides can be obtained with the analogues, although a considerable amount of shorter fragments accompanies the full-length product. In keeping with the kinetic analysis, the 2'-aminouridine triphosphate gives a cleaner product than the 2'-fluoro analogue. Transcription of two tRNA genes shows that such shorter templates can be transcribed to full-length products essentially without premature termination with any of the analogues.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Autoradiography
  • DNA-Directed RNA Polymerases / metabolism*
  • Deoxyribonucleotides / metabolism*
  • Electrophoresis, Polyacrylamide Gel
  • Kinetics
  • RNA, Transfer / genetics
  • Substrate Specificity
  • Transcription, Genetic
  • Viral Proteins

Substances

  • Deoxyribonucleotides
  • Viral Proteins
  • RNA, Transfer
  • bacteriophage T7 RNA polymerase
  • DNA-Directed RNA Polymerases