No abstract

A mutant T7 RNA polymerase (T7 RNAP) having two amino-acid substitutions (Y639F and $641A) is altered in its specificity towards nucleotide substrates, but is not affected in the specificity of its interaction with promoter and terminator sequences. The mutant enzyme gains the ability to utilize dNTPs and catalyze RNA and DNA synthesis from circular supercoiled plasmid DNA. DNA synthesis can also be initiated from a single stranded template using a DNA primer. Another T7 RNAP mutant having only the single substitution $641A loses RNA polymerase activity but is able to synthesize DNA.Key words: T7 RNA polymerase; Mutagenesis; dNTP utilization; DNA polymerizing activity distribution of hydroxyl-containing amino-acid residues. Specifically, a serine residue is present at position 641 in T7 RNAP (and at corresponding positions in related RNAPs), while in DNA polymerases (DNAP) no such regularity is observed [5]. As $641 is the hydroxyl-bearing amino-acid residue closest to Y639 we have asked whether the hydroxyl groups of these two residues may be involved in the interactions of enzyme with NTP and, specifically, in discrimination between dNTP and rNTP as potential substrates. To test this, we have generated mutant enzymes with phenylalanine in place of tyrosine at position 639, alanine in place of serine in position 641 and a double mutant bearing both of these substitutions. The substrate specificity and other features of the latter two proteins were found to be quite surprising.

Deoxyribonucleotide-containing RNA-like polynucleotides (dcRNAs) were synthesized by mutant T7 RNA polymerase and their structures confirmed by sequencing. dcRNAs annealed with a 20mer oligodeoxyribonucleotide primer were tested as templates/primers in the reverse transcription reaction catalyzed by HIV-1 reverse transcriptase (RT). All dcRNAs were shown to be efficient templates for both wild-type RT and RT mutants, containing 'AZT-resistant' mutations. Differences in the patterns of the DNA products of RNA- and dcRNA-driven reverse transcription were demonstrated. The kinetic characteristics for dcRNAs utilization were compared with the corresponding parameters for RNA/DNA and DNA/DNA templates/primers. The respective K m values for dcRNAs appear to be intermediate between those for RNA and DNA templates. A correlation equation connecting apparent K m value for template/primer and the number of deoxyribonucleotide substitutions in RNA template is proposed.

Synthesis of deoxynucleotide-containing RNA-like single-stranded polynucleotides (dcRNAs) using the Y639F, S641A mutant of T7 RNA polymerase (T7 RNAP) was studied. A number of different T7 promoter-containing plasmids were tested as templates for dcRNA synthesis. The dcRNA synthesis efficiency strongly depended on the sequence of the first 8^10 nucleotides immediately downstream of the promoter and increased with the distance of the first incorporated dNMP from the transcription start. The incorporation of dGMP which is obligatory for most T7 promoters in positions +1^+2(3) was practically negligible. Using the constructed plasmid pTZR7G containing seven dG links in the non-coding chain immediately downstream of the promoter, the synthesis of all possible dcRNAs (except dG-containing) was achieved with high yields.z 1998 Federation of European Biochemical Societies.

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.z 1998 Federation of European Biochemical Societies.