RNA polymerase. Direct evidence for two active sites involved in transcription.

The [alpha-32P]3'-azido-3'-deoxyxylofuranosyl analogues of 5'-ATP and 5'-GTP were used as photoaffinity probes to derivatize two distinctly different active sites of the DNA-dependent RNA polymerase obtained from Escherichia coli. The sites derivatized by these probes are both located on the beta subunit. Radioactive 32P containing cyanogen bromide peptides produced from the derivatized beta subunits were observed to give either one of two distinct patterns by high pressure liquid chromatography or TLC analysis. These sites are used alternately for the synthesis of successive phosphodiester bonds. These results support our rotational translocation model for transcription.