Functional analysis of Drosophila factor 5 (TFIIF), a general transcription factor.

Factor 5 is a Drosophila RNA polymerase II initiation factor that also affects the elongation phase of transcription. We have used a cDNA encoding the large subunit of factor 5 (F5a) to produce recombinant F5a (rF5a). Antibodies directed against peptides deduced from the sequence of the F5a cDNA recognized rF5a and the large subunit of factor 5 purified from Kc cells. A chimeric human/fly factor composed of the small subunit of human TFIIF (RAP30) and rF5a stimulated elongation by Drosophila RNA polymerase II when assayed using a dC-tailed template. In addition, the chimeric human/fly factor functioned during initiation in either the Drosophila or human system. Therefore, the structure of the large subunit of TFIIF is sufficiently conserved from human to fly to allow functional interaction with both the small subunit of TFIIF and RNA polymerase II from either species. Analysis of deletion mutants of F5a indicated that almost all of the protein was required for initiation while only the NH2-terminal region was required for stimulating transcriptional elongation. A comparison of our results with those obtained with RAP74 suggest that the carboxyl terminal region of the protein may be involved in interactions with RNA polymerase II or other factors during initiation.