Early steps in the path of nascent ribonucleic acid across the surface of ribonucleic acid polymerase, determined by photoaffinity labeling

The photoaffinity probes β-(4-azidophenyl) adenosine 5′-diphosphate (N₃PhppA) and β-(4-azidophenyl) adenylyl-(3′-5′)-uridine 5′-diphosphate (N₃PhppApU) were used to determine the RNA polymerase subunit contacts made by the 5′ ends of three nascent RNA chains. Ternary enzyme-poly[d(A-T)]·oligonucleotide complexes were prepared in which the nascent oligonucleotide contained a photoaffinity label at the 5′ end and a ³²P radiolabel only at the 3′ end. The length of the RNA was fixed at two, three, or four nucleotides. Photolysis of the ternary complexes was followed by dissociation, polyacrylamide gel electrophoresis, autoradiography, and scintillation counting. With a dinucleotide probe, the enzyme subunits labeled were β′ (71%) and σ (21%). Photolysis of the ternary complex containing trinucleotide RNA also resulted in labeling of the β′ (64%) and σ (35%) subunits. With a tetranucleotide, the β′ subunit was very heavily labeled (88%), and a small amount of labeling of the β (7%) and σ (4%) subunits was observed. The α subunit was not labeled with any of the probes. These results imply that a conformational change, possibly involving dissociation of the σ subunit, occurs in the enzyme as the ribonucleotide is elongated from a tri- to a tetranucleotide.