RNA polymerase was treated in the presence of promoter-containing templates with 16 affinity reagents, derivatives on NMPs, NDPs and NTPs with reactive substituents at the terminal phosphate. This treatment was followed by addition of a pyrimidine [cx-~'P]NTP. Due to 'catalytic competence' of some of the residues of the affinity reagents bound covalently near the active center at the first stage, active-center-catalyzed synthesis of a phosphodiester bond occurred, and radioactive residues with the general formula -pNbN (where 5 = radioactive phosphate) appeared covalently attached to the enzyme. Such affinity labelling was super-selective because affinity reagent residues bound outside the active center were not elongated and thus remained non-radioactive.Labelling took place only when the combination of the reagent and [U-~'P]NTP corresponded to the sequence of nucleotides of the promoter. With reagents having short 'arms', only the fl subunit was labelled; the targets were His and/or Lys residues. With reagents having longer 'arms', the G subunit was also labelled.The value of information obtained by means of affinity modification of proteins (for review see [l]) depends on its selectivity, i.e. on the ratio of the extents of modification of amino acid residues inside and outside the ligand-binding center. It is difficult to achieve high selectivity with large enzymes and with analogues of substrates having moderate affinities to active centers. Escherichia coli RNA polymerase is a very large enzyme (relative molecular mass 500000) with moderate affinity to substrates '(Km z 0.01 mM). It is not surprising that numerous attempts to study its functional topography by means of affinity labelling with reactive analogues of substrates have given relatively poor results.Selectivity of affinity labelling may be increased by using differential labelling [2, 31, analogues of transition states [4, 51, and suicide substrates [6, 71. Certain basic disadvantages of the differential labelling technique make it practically inapplicable to RNA polymerase. As for the two other techniques, they depend upon availability of very specific reagents, and it is not yet clear what the structure of such reagents should be in the case of RNA polymerase.

Pt(2+)-containing derivatives of oligodeoxyribonucleotides were used to evaluate the ligand affinity to the template sites of Klenow fragment of DNA polymerase I from E. coli and DNA polymerase alpha from human placenta. The values of Kd and Gibb's energy (delta G degree) for the complexes of oligodeoxyribonucleotides and their derivatives with the template sites of these enzymes were determined from the effects protecting the enzyme from inactivation by Pt(2+)-containing oligonucleotides. Kd and delta G degree values of the complexes made by DNA polymerases and orthophosphate, triethylphosphate, d(pC)n, d(pT)n, d(pG)n, d(pA)n (where n = 1-25), heterooligonucleotides of various length and structure, and oligothymidylates with partially and completely ethylated internucleotide phosphates were evaluated. The obtained data enabled us to suggest 19-20 mononucleotide units of the template to interact with the protein. Only one template internucleotide phosphate forms a Me(2+)-dependent electrostatic contact (delta G = -1.1...-1.7 kcal/mol) and a hydrogen bond (delta G = -4.4...-4.9 kcal/mol) with the enzyme. It is likely that the mononucleoside units of the template form hydrophobic contacts with the enzymes. The efficiency of such interaction changes with the hydrophobicity of the bases: C less than T less than G approximately A. For both homo- and heterooligonucleotides the contributions of nucleoside units to the affinity of the templates to the enzymes is due to the complementary interactions with the primers. A hypothetical model for the template-primer interaction with DNA polymerases is suggested.

SummaryThe interaction of EcoRI with different oligodeoxyribonucleotides (ODNs) was analyzed using the method of the slow step-by-

The values of K d and Gibbs energy (ΔG°) have been measured for complexes of the template site of DNA polymerase I Klenow fragment with the homo‐oligonucleotides d(pC) n , d(pT) n , d(pG) n and d(pA) n and hetero‐oligonucleotides of various structures and lengths. These parameters were evaluated from the protective effect of the oligonucleotide on enzyme inactivation by the affinity reagents d(Tp)2C[Pt2+(NH3)2OH](pT)7 and d[(Tp)2C(Pt2+(NH3)2OH)p]3T of the template site. The present results and previously reported data [(1985) Biorg. Khim. 13, 357–369] indicate that the nucleoside components of the template form complexes as a result of their hydrophobic interactions with the enzyme. Only one template internucleotide phosphate forms an Me2+‐dependent electrostatic contact and a hydrogen bond with the enzyme. The 19–20‐nucleotide fragments of the template appear to interact with the protein molecule.