Transcription initiation by Escherichia coli RNA polymerase at the gene II promoter of M13 phage: stability of ternary complex, direct photocrosslinking to nascent RNA, and retention of sigma subunit.

The initial stages of transcription have been characterized using a template containing the gene II promoter region of M13 phage. Initiation of transcription in the presence of all four nucleotides gives rise to the 140-residue run-off transcript, with a minor pause at the RNA hexamer stage. Cycling, leading to the accumulation of significant amounts of short oligonucleotides [1], was not observed. An RNA hexamer GUUUUU was the sole product when GpU and UTP were used and the ternary complex with the hexamer was stable and resistant to high salt (0.4 M) and S1 nuclease attack. After direct ultraviolet photocrosslinking of the RNA hexamer to RNA polymerase in the ternary complex, the radioactive label incorporation into various subunits was determined by autoradiography after sodium tetradecyl sulfate gel electrophoresis to be as follows: sigma, 86%; beta, 14%; beta' and alpha, negligible. Both electrophoresis and sucrose gradient centrifugation experiments indicate that the sigma subunit is not released from the ternary complex when either the RNA hexamer or the 140-residue RNA is synthesized on this template, even though the complexes are stable.