[Affinity modification of E. coli RNA-polymerase in a complex with the promoter by phosphorylating derivatives of...

Oligonucleotides 2 to 7 nucleotide residues long, complementary to the codogenic strand of T7 promoter A2, have been synthesized; all of them contained a ribo-unit at the 3'-end. They were converted into 5'-(N-methyl)phosphoimidazolides, and the affinity reagents obtained were allowed to bind covalently to RNA polymerase in the presence of a promoter. Some of the nucleotide residues covalently attached occupied proper positions relative to the active centre of the phosphodiester bond synthesis and on addition of [alpha-32P]UTP were elongated, so that highly selective affinity labelling occurred. With oligonucleotides of various lengths, different distribution of the label between beta, beta' and sigma subunits of RNA polymerase took place. Most efficient was labelling of beta-subunit by the residue--pCpGpCpU, and of sigma-subunit by the residue--pApApApTp-CpGpCpU (p--radioactive phosphorus atom). In both cases, the amino acid residues labelled were histidines.