RAP30/74 (transcription factor IIF) is required for promoter escape by RNA polymerase II

RNA polymerase II-associating proteins (RAP30 and RAP74) are subunits of the transcription factor called variously RAP30/74, TFIIF, beta gamma, and FC. This factor is required for accurate transcription by RNA polymerase II, in addition to other basal transcription factors. Using recombinant human RAP30 and RAP74, the functions of these subunits have been tested separately during the initiation and elongation phases of transcription. RAP30 is required to form a Sarkosyl-resistant complex at 0.25% Sarkosyl, so RAP30 is required for initiation. RAP74, however, stimulates transcription when added after Sarkosyl, indicating that RAP74 is dispensable for initiation. The same result is obtained using a pulse-chase protocol in which accurately initiated RNA is labeled during a short pulse, followed by a chase with excess unlabeled nucleoside triphosphates. RAP30 is required in order to label the transcript during the pulse, but RAP74 is not. RAP74 must be added during the chase, however, in order to obtain a short runoff transcript. The following conclusions can be drawn from these experiments: 1) RAP30 is an initiation factor; 2) RAP74 is not required for ATP hydrolysis in initiation, which precedes phosphodiester bond formation; 3) RAP74 is not required for template strand separation; 4) RAP74 is not required to initiate phosphodiester bond formation; and 5) RAP74 is required for very early elongation.