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 en coding 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 pur ified from Kc cells. A chimeric human/fly factor composed of the small subunit of human TFIIF (RAP30) and rF5a stimulated elongation by Dros ophila RNA polymerase II when assayed using a dC-tailed template. In a ddition, the chimeric human/fly factor functioned during initiation in either the Drosophila or human system. Therefore, the structure of th e large subunit of TFIIF is sufficiently conserved from human to fly t o allow functional interaction with both the small subunit of TFIIF an d RNA polymerase II from either species. Analysis of deletion mutants of F5a indicated that almost all of the protein was required for initi ation while only the NH2-terminal region was required for stimulating transcriptional elongation. A comparison of our results with those obt ained with RAP74 suggest that the carboxyl terminal region of the prot ein may be involved in interactions with RNA polymerase II or other fa ctors during initiation.