On the fidelity of transcription by escherichia coli RNA polymerase

Abstract

The enzymes that carry out template-directed synthesis of DNA (DNA polymerase and reverse transcriptase) were shown to participate in the selection of complementary nucleotides as proved by the effect of enzyme structure on the fidelity in copying polynucleotide templates [ 1,2]. Temperature-sensitive mutants affecting the structural gene of DNA polymerase are characterized by increased mutability [3-61. A difference has been observed between nucleotide misincorporation of in vitro DNA synthesis by normal and mutant enzyme [I]. Mutational alterations of DNA-dependent RNA polymerase have not revealed such a dependence [7]. No difference in the fidelity of in vitro transcription was observed when intrinsic Zn2+ of RNA polymerase was substituted by Co’+ [8]. The data available on the accuracy of base-selection during transcription are conflicting [7,9111. The values of misincorporation vary substantially in different reports. In the case of poly(AU) synthesis on a poly [d(AT) X d(AT)] template the frequency of GMP incorporation was shown to be 9.5 X lo4 [9] and 2.3 X IO-’ [IO]. The frequency of misincorporation of GMP into poly(A) synthesized on a poly(dT) template varies between 3.3 X lo-’ [l l] and 6.4 X lo4 [7]. With synthetic polyribonucleotides as a template, the variations are still greater. To study the role of RNA polymerase in ensuring the fidelity of transcription, in this work we chose RNA polymerase rifampicin resistant (rifr) mutants of Escher&h& coEi B/r, rpoB402, rpoB403 and rpoB409 [ 12,131, which are characterized by increased variance within one of the phenotypic parameters