A method is proposed for localization of the sites of affinity labelling of the p subunit of Escherichiu coli RNA polymerase. The principle of this method is similar to that of the methods of rapid sequencing of nucleic acids.The polypeptide bearing a radioactive affinity label at one of the amino acid residues is subjected to shortterm treatment with cyanogen bromide. The conditions of this reaction are selected in such a way that less than one cleavage occurs on average per polypeptide chain. Two series of radioactive peptides are formed, one involving all the possible N-terminal peptides and the other the C-terminal peptides. The distribution of the lengths of these peptides is studied by means of gel electrophoresis and compared with the theoretical ones based on the known amino acid sequence of the p subunit. Obviously, the affinity label resides between the C-terminus of the shortest N-tcrminal radioactive peptide and the N-terminus of the shortest C-terminal radioactive peptide. In order to increase reliability and resolution of the method, partial trypsinolysis may be employed.The evidence obtained suggests that lysine residues over the regions 1036-1066, 1234-1242, and histidine-1237 are situated in the nearest neighbourhood to, or directly involved in the formation of the active center of initiating substrate binding of the p subunit of E. coli RNA polymerase.Our earlier communications [l -91 have described highly selective affinity labelling of Escherichiu coli RNA polymerase according to the following scheme:where E is RNA polymerase, XpNl an affinity reagent which is an analogue of an initiating substrate and pp6N2 is a pyrimidine [a-"'PINTI'. Treatment according to this scheme is performed on the complex of the enzyme with a promoter. The high selectivity of the affinity labelling is due to t p fact that the covalent binding of the radioactive residue -pN2 at the second stage takes place under catalytic action of the center of phosphodiester bond synthesis of the enzyme. Recently the same approach has been successfully applied to RNA polymerase I1 of wheat germ [lo] and to RNA polymerase of T7 coliphage [Ill.In the case of E. coli RNA polymerase, treatment with a majority of reagents (in combination with a pyrimidine pp6N) resulted in selective labelling of the p subunit. Some of the targets of labelling were identified as Lys and His residuesThe amino acid sequence of the /I subunit is known [12]. We attempted to localize the sites of affinity labelling by traditional methods like complete trypsinolysis followed by isolation and sequencing of radioactive peptides, but these attempts have failed because of the small yield of affinity labelling. Therefore we developed a new method for the localization. The principle of this method has been reported briefly earlier [4, 6-9, 13, 141; it is based upon the random cleavage approach widely used for rapid sequencing of nucleic acids [15, 161 and involves random limited cleavage at methionine residues followed by gel electrophoresis of radioactive peptides...
The cleavable homobifunctional reagent dichloro[N,N,K,W-tetrakis(2-aminoethyl)-1,6-hexamethylenediamminediplatinum(lI)] dichloride was used for studying rRNA-protein cross-links in free 35S-labelled 70 S ribosomes and within initiation complex ribosome AUGU, fMet-tRNAr". It was shown that the sets of proteins cross-linked to 16 S and 23 S rRNA in free 70 S ribosomes and in 70 S initiation complex do not differ significantly. The authors are the first to demonstrate most of the 23 S rRNA-protein cross-links and some 16 S rRNA-protein cross-links, in particular those with L7/L12 protein.
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