Mutant T7 RNA polymerase is capable of catalyzing DNA primer extension reaction

• Published in: FEBS letters Vol. 423; no. 2; pp. 189 - 192
• Main Authors: Rusakova, E.E, Tunitskaya, V.L, Memelova, L.V, Kochetkova, S.V, Kostyuk, D.A, Kochetkov, S.N
• Format: Journal Article
• Language: English
• Published: England Elsevier B.V 20-02-1998
• Subjects: Bacteriophage T7 RNA polymerase; Bacteriophages - enzymology; Bacteriophages - genetics; Deoxyribonucleotides - metabolism; DNA Primers - chemistry; DNA Primers - genetics; DNA Primers - metabolism; DNA-Directed DNA Polymerase - genetics; DNA-Directed DNA Polymerase - metabolism; DNA-Directed RNA Polymerases - genetics; DNA-Directed RNA Polymerases - metabolism; Mutant; Oligodeoxyribonucleotides - biosynthesis; Primer extension; Viral Proteins; Bacteriophage T7 RNA polymerase; Mutant; Primer extension
• ISSN: 0014-5793; 1873-3468
• DOI: 10.1016/S0014-5793(98)00058-1

The mutant T7 RNA polymerase (T7 RNAP), containing two substitutions (Y639F, S641A) was earlier shown to utilize both rNTP and dNTP in a transcription-like reaction. In this report the ability of the enzyme to catalyze DNA primer extension reaction was demonstrated. The efficiency of the reaction essentially depended on the type of the primer sequence, and was significantly higher if the primer coincided with the T7 promoter non-coding sequence. In this case the primer extension reaction proceeded along with de novo RNA synthesis. The length of the product did not exceed 8 nucleotides, indicating that the primer extension reaction proceeds according to the mechanism of the T7 RNAP-catalyzed abortive transcription.