Memelova Lv

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

Oligonucleotides of a novel type containing 2′‐O‐β‐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. Wild‐type T7 RNA polymerase and most of the mutants did not react irreversibly with oxidized oligonucleotides. Affinity labeling was observed only with the promoter‐containing dialdehyde group in position (+2) of the coding chain and one of the mutants tested, namely Y639K. These results allowed us to propose the close proximity of residue 639 and the initiation region of the promoter within initiation complex. We suggest the oligonucleotides so modified may be of general value for the study of protein‐nucleic acid interactions.

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. 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.

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. 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.

The studies of cooperative regions in T7 RNA polymerase

The heat denaturation of bacteriophage T7 RNA polymerase (T7RNAP) was studied by scanning microcalorimetry. The thermodynamic parameters of the denaturation were estimated within the pH range 6–9. The analysis of the denaturation curves showed the presence of two cooperative parts of the T7RNAP molecule melting according to the ‘all‐or‐none’ principle. The molecular masses of these parts were determined as 22 and 77 kDa. These values are close to the molecular masses of protein domains obtained from X‐ray diffraction and limited trypsinolysis data. The smaller N‐terminal domain was shown to increase the thermostability of the ‘catalytic’ C‐terminal domain within the intact T7RNAP molecule.

Localization of The Initiation Site in T7 RNA Polymerase by Affinity Labeling

Abstract The oligonucleotide duplexes containing modified CMP residues with the additional β-D-ribofuranosyl group were used to study the interactions between T7 RNA polymerase (T7 RNAP) and its promoter. Wild-type enzyme did not undergo the affinity labelling with these reagents. However the use of Y639K T7 RNAP mutant resulted in the specific covalent binding thus suggesting the location of the a.a. residue in pos. 639 within the transcription initiation site.