Studies of the functional topography of Escherichia coli RNA polymerase

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 imidazole, N-methylimidazole, and 4-dimethylaminopyridine afforded a series of phosphorylating affinity reagents. 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 promoter A₂ as template. Treatment of the complex RNA-polymecase · promoter-A₂ with affinity reagents followed by addition of [α-³²P]UTP resulted in labelling of RNA polymerase by the residues -(pdN)_npdGprC   rU (   + radioactive phosphate). This affinity labelling was highly selective because elongation of the covalently bound residues (pdN)_npdGprC by    rU residues was catalyzed by the active center of RNA polymerase. The most efficient reagents were N-methylimidazolides. A dramatic change of the pattern of labelling of the subunits β, β', and σ took place with changing n. Maximum labelling of the β subunit occurred at n= 1 and of the σ subunit at n= 5. The targets in both the subunits were His residues. The α subunit was not specifically labelled.