Mechanism of RNA polymerase II-specific initiation of transcription in vitro: ATP requirement and uncapped runoff transcripts

The ATP analog 5′-adenylyl imidodiphosphate (AMP-PNP) inhibits transcription of specific genes by the RNA polymerase II contained in whole cell extracts, not only with promoters that contain A as the first nucleotide of the transcript, but also with those that initiate transcripts with G or U. The analog AMP-PNP (a competitive inhibitor of ATP) probably acts at the level of initiation of transcription, but it can be used for elongation by RNA polymerase II in isolated nuclei or in the whole cell extract. AMPPNP and the other imidotriphosphates have little effect on purified HeLa cell RNA polymerase II initiation and elongation of transcription. Since RNA polymerase III in the crude system both initiates and elongates transcripts with AMP-PNP, we conclude that the availability of the β-γ bond of ATP is an indispensable requirement for faithful and specific in vitro initiation only by RNA polymerase II in the whole cell extract. Uncapped U- or G-initiated transcripts were obtained in the presence of UMPPNP or GMP-PNP, the respective imidodiphosphate analogs. The presence of the 5′-terminal imidotriphosphate at the same oligonucleotide as the cap for U-initiated precursors established that transcription initiation and capping occur at the same site. Capping is not required for transcription by RNA polymerase II in the in vitro system. Methylation of the 2′ ribose of the initiating nucleotide does not occur on the imidonucleotide containing 5′ ends of adenovirus EIV or murine leukemia virus long terminal repeat.