Studies of the functional topography of Escherichia coli RNA polymerase

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Abstract
Amidation of the 5''-phosphate group of the heptanucleotide pdApdApdApdTpdCpdGprC and of its derivatives of the general formula (pdN)npdGprC ( n = 0-5) with imadazole, N-methylimidazole, and 4-dimethylaminopyridine afforded a series of phosphorylating affinity regents. The parent oligonucleotides of this series complementary to promoter A2 of T7 phage over the region (-5 to +2) are known to be efficient primers of the synthesis of RNA by Escherichia coli RNA polymerase with promother A2 as template. Treatment of the complex RNA-polymerase .cntdot. promoter-A2 with affinity reagents followed by addition of [.alpha.-32P]UTP resulted in labelling of RNA polymerase by the residues -(pdN)npdGprC.ovrhdot.prU (.ovrhdot.p = radioactive phosphate). This affinity labelling was highly selective because elongation of the covalently bound residues (pdN)npdGprC by .ovrhdot.prU residues was catalyzed by the acive center of RNA polymerase. The most efficient reagents were N-methylimidazolides. A dramatic change of the pattern of labelling of the subunits .beta., .beta.'', and .sigma. took place with changing n. Maximum labelling of the .beta. subunit occurred at n = 1 and of the .sigma. subunit at n = 5. The targets in both the subunits were His residues. The .alpha. subunit was not specifically labelled.