[Photoaffinity modification of bacteriophage T7 DNA-dependent RNA polymerase by the reaction product containing the azido derivative of UTP].

The possibility of using the 5-[3-(E)-(4-azido-2,3,5,6-tetrafluorobenzamido)-propenyl-1]-UTP (N3-TFBP-UTP) as the affinity modificator of bacteriophage T7 DNA-dependent RNA polymerase was demonstrated. The UTP derivative used was rather efficient substrate substituting UTP in the transcription reaction performed by the enzyme. The UV treatment of "stopped" reaction complex formed using three of four substrate ribonucleotides, allow to obtain the covalent binding between the enzyme and the reaction product of 9 nucleotides length.

Mapping of T7 RNA polymerase active site with novel reagents--oligonucleotides with reactive dialdehyde groups.

Oligonucleotides of a novel type containing 2'-O-beta-ribofuranosyl-cytidine were synthesized and further oxidized to yield T7 consensus promoters with dialdehyde groups. Both types of oligonucleotides were tested as templates, inhibitors, and affinity reagents for T7 RNA polymerase and its mutants. All oligonucleotides tested retained high affinity towards the enzyme.

Synthesis of mixed ribo/deoxyribopolynucleotides by mutant T7 RNA polymerase.

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.

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

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.