Kochetkov Sn

Elongation complexes of Thermus thermophilus RNA polymerase that possess distinct translocation conformations

We have characterized elongation complexes (ECs) of RNA polymerase from the extremely thermophilic bacterium, Thermus thermophilus. We found that complexes assembled on nucleic acid scaffolds are transcriptionally competent at high temperature (50–80°C) and, depending upon the organization of the scaffold, possess distinct translocation conformations. ECs assembled on scaffolds with a 9 bp RNA:DNA hybrid are highly stable, resistant to pyrophosphorolysis, and are in the posttranslocated state. ECs with an RNA:DNA hybrid longer or shorter than 9 bp appear to be in a pretranslocated state, as evidenced by their sensitivity to pyrophosphorolysis, GreA-induced cleavage, and exonuclease footprinting. Both pretranslocated (8 bp RNA:DNA hybrid) and posttranslocated (9 bp RNA:DNA hybrid) complexes were crystallized in distinct crystal forms, supporting the homogeneity of the conformational states in these complexes. Crystals of a posttranslocated complex were used to collect diffraction data at atomic resolution.

Role of a histidine residue in the active site of cyclic AMP-dependent histone kinase

The cyclic AMP-dependent histone kinase [ 1 ] is an important enzyme that carries out phosphorylation of lysine-rich histones, in particular, histone Hl . At the present time the molecular mechanism of phosphotransferase reaction is still unknown. In studying individual stages of the enzymic reaction it seems important to elucidate which functional groups of the active site participate in substrate binding and the catalytic act. This paper is concerned with the investigation of ATP binding in the active site of the histone kinase catalytic subunit and further transfer of the phosphate residue to a protein substrate.

Substrate properties of C' -methyl UTP derivatives in T7 RNA polymerase reactions. Evidence for N-type NTP conformation

The number of synthetic UTP analogues containing methyl groups in different positions of the ribose moiety were tested as substrates for T7 RNA polymerase (T7 RNAP). Two of these compounds (containing substituents in the 5′ position) were shown to be weak substrates of T7 RNAP. 3′Me‐UTP was neither substrate nor inhibitor of T7 RNAP while 2′Me‐UTP was shown to terminate RNA chain synthesis. Conformational analysis of the analogues and parent nucleotide using the force‐field method indicates that the allowed conformation of UTP during its incorporation into the growing RNA chain by T7 RNAP is limited to the χ angle range of 192–256° of N‐type conformation.

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.

Interaction of HIV-1 Reverse Transcriptase and T7 RNA Polymerase with Phosphonate Analogs of NTP and Inorganic Pyrophosphate

We have examined the interaction of human immunodeficiency virus reverse transcriptase (HIV RT) and T7 RNA polymerase (T7 RNAP) with modified nucleoside triphosphates and inorganic pyrophosphate (PPi) analogs containing nonhydrolyzable bisphosphonate groups. We have synthesized a number of derivatives of bisphosphonic acid having different aromatic and nonaromatic side substituents, as well as the NTP derivatives whose incorporation into the growing nucleotide chain during the polymerization reaction results in formation of bisphosphonates as leaving groups. The competitive character of inhibition of both enzymes has been revealed for all the compounds under study, and the inhibition constants have been estimated. One of PPianalogs containing a bulky aromatic substituent is characterized by similar inhibition constants for both T7 RNAP and RT. The universal character of this inhibitor can serve as evidence for a similar structure of the NPT-binding sites in the two polymerases. It has been shown that nonsubstituted methylenebisphosphate is a better leaving group than that containing additional methyl and hydroxyl groups. The NTP analogs are very weak inhibitors of T7 RNAP, whereas HIV RT is more sensitive to this type of compounds. On the basis of the X-ray crystallographic data on the T7 RNAP complex with a template and NTP, we have modeled the binding of some derivatives of bisphosphonic acid in the active center of the enzyme. The peculiarities observed in the model correlate well with the experimental data on inhibition.

Random mntagenesis of the gene for bacteriophage T7 RNA polymerase

Random mutagenesis of the gene for bacteriophage T7 RNA polymerase was used to identify functionally essential amino acid residues of the enzyme. A two-plasmid system was developed that permits the straightforward isolation of T7 RNA polymerase mutants that had lost almost all catalytic activity. It was shown that substitutions of Thr and Ala for Pro at the position 563, Ser for Tyr571, Pro for Thr636, Asp for Tyr639 and of Cys for Phe646 resulted in inactivation of the enzyme. It is noteworthy that all these mutations are limited to two short regions that are highly conservative in sequences of monomeric RNA polymerases.

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.

On the functional role of the Tyr-639 residue of bacteriophage T7 RNA polymerase

Substitution or Asp for a Tyr residue normally present at position 639 of the bacteriophage T7 RNA polymerase leads to a drastic drop in the enzymatic activity. This mutation does not affect the enzyme‐promoter interaction but decreases the ability of the RNA polymerase to discriminate between GTP and ATP molecules, resulting in a decrease in the rate of the incorporation of the nucleotide into the RNA chain.

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.

Tyr-571 is involved in the T7 RNA polymerase binding to its promoter

The in vitro studies of three T7 RNA polymerase point mutants suggest that substitutions of Ala and Thr for Pro‐563 and of Ser for Tyr‐571 have little effect on the enzyme catalytic competence, but result in its inability to utilize the promoter. Both P563A and P563T mutants retain the promoter‐binding ability, whereas the promoter affinity of the Y571S mutant drops drastically.