The nucleotide sequence of a cloned DNA coding for the 35-kDa polypeptide of the eye lens of the frog (Rana temporaria) has been determined. The sequence without connectors and poly(A) tract is 889 nucleotides in length and shows no homology with sequences coding for other classes of crystallins: cy-, &, y-or S-crystallins. The sequence contains one reading frame 675 nucleotides in length, an apparently intact 3'-non-translated region with the polyadenylation signal sequence and a poly(A) tract; the 5'-nontranslated region is lost along with part of the coding region; this accounts for about l/4 of the total mRNA length. The secondary structure prediction according to the Ptitsin-Finkelstein method shows the presence of predominantly P-strands with only a few a-helical regions. We conclude that the 35-kDa polypeptide from the frog eye lens belongs to a new class of eye lens crystallins for which we propose the name c-crystallin. Recombinant

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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