2'-Fluoro- and 2'-amino-2'-deoxynucleoside 5'-triphosphates as substrates for T7 RNA polymerase.
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.
Aurup
H
H
Max-Planck-Institut für experimentelle Medizin, Göttingen, FRG.
Williams
D M
DM
Eckstein
F
F
D001345:Autoradiography; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D003854:Deoxyribonucleotides / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D007700:Kinetics; D012343:RNA, Transfer / Q000235:genetics; D013379:Substrate Specificity; D014158:Transcription, Genetic; D014764:Viral Proteins
D003854:Deoxyribonucleotides; D014764:Viral Proteins; D012343:RNA, Transfer; C062210:bacteriophage T7 RNA polymerase; D012321:DNA-Directed RNA Polymerases