[Highly selective affinity labeling of a promoter in a complex with E. coli RNA-polymerase by alkylating derivatives of initiating substrates].
The complex [promoter A2 X E. coli RNA polymerase] was treated with phosphoamides, derivatives of 4-[N-methyl, N-(2-chloroethyl)]-aminobenzylamine and guanosine-5'-mono-, di-, and triphosphates with the alkylating group attached to the terminal phosphates. After this, [alpha-32P]CTP was added. Residues of the affinity reagents bound covalently at the first stage were elongated by radioactive -pC residues due to the catalytic action of the active centre of RNA polymerase. Affinity labelled were beta-and sigma-subunits of the enzyme, and the promoter. The affinity label was localized on -pGpC residues. A guanine residue was alkylated in the promoter as suggested by radioactivity elimination kinetics. As the data obtained and the previously known length of the reagent (maximum distance between the alpha-phosphorus atom of the reagent and the point of alkylation is less than 0.6 nm) indicate, there is a direct rather than protein-mediated contact between the template and the substrate within the complex [promoter X RNA polymerase].
Grachev
M A
MA
Lukhtanov
E A
EA
Mustaev
A A
AA
D000345:Affinity Labels; D000477:Alkylating Agents; D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D011401:Promoter Regions, Genetic; D013379:Substrate Specificity
D000345:Affinity Labels; D000477:Alkylating Agents; D012321:DNA-Directed RNA Polymerases
Vysokoselektivnoe affinnoe mechenie promotora v komplekse s RNK-polimerazoĭ E. coli alkiliruiushchimi proizvodnymi initsiruiushchikh substratov.