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Oligonucleotides of a novel type containing 2P-O-L Lribofuranosyl-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 proteinnucleic acid interactions.z 1999 Federation of European Biochemical Societies.

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.

Bacteriophage T7 RNA polymerase was covalently modified by 5'-[4-fluorosulfonyl)benzoyl]adenosine (4-FSOzBzAdo). The modified enzyme lacks the ability to catalyze RNA synthesis from the 410 promoter of bacteriophage T7; both promoter and GTP binding being markedly decreased. The mild hydrolysis of the ester bond of 4-FSOzBzAdo within the covalent enzyme-inhibitor complex restores the RNA synthesis at a lower rate.Sequence studies show that Lys172 is the target of modification by 4-FS02BzAdo. This residue, which is situated in the polypeptide region connccting two domains of RNA polymerase, was shown to be the primary site of the limited proteolysis occurring inWe propose that Lys172 is located outside the active site. Once this residue has reacted with 4-FSO2BzAdo, the nucleoside moiety of the analog is fixed in the NTP-binding site of the active centre and prevents binding of the substrates. Here, Lys172 per se is not important for the activity but serves as an 'anchor' for binding of the inhibitor.Bacteriophage T7 RNA polymerase (T7RP) is a suitable model for studying the physicochemical characteristics of transcription. In contrast to most of the known RNA polymerases, this enzyme is composed of one protein subunit with a molecular mass of 98 kDa. T7RP transcribes phage T7 late genes recognising a unique sequence from 17 bp to 23 bp long (for different promoter types) [l, 21. The primary structure of the gene coding for T7RP is known [3], but the threedimensional structure of the enzyme has not been yet established. Only limited evidence is available about some functionally essential amino acid residues which apparently include Cys347 and Lys631 [4 -61.We showed earlier that S'-[4-(fluorosulfonyl)benzoyl]-adenosine (4-FS02BzAdo) irreversibly inhibited T7RP and the time-dependent decrease in activity correlated with the incorporation of a 14C-labeled 4-FS02BzAdo into the protein which reached 1 mol/mol T7RP [7]. The aim of the present work was to localize the site of T7RP modification by 4-FSOzBzAdo and to study the effect of modification on the functioning of the enzyme.Correspondence to S. N .

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