The broad aim of this research is to determine the role of human transcription factor IIF (TFIIF) in initiation and elongation. TFIIF appears to have a key role in isomerization of RNA polymerase II initiation and elongation complexes, supporting an activated state of RNA polymerase II for transcription. Mutations in the L155 to M177 region of the RAP74 subunit of TFIIF have startlingly similar effects on initiation and elongation. In initiation, TFIIF appears to induce promoter DNA to wrap around RNA polymerase II. TFIIF may isomerize elongation complexes by enhancing DNA bending through the RNA polymerase II active site.
Specific aims are directed toward testing conformational models for TFIIF function in initiation and elongation.
Other aims are directed toward analysis of DNA wrapping and helix untwisting in transcriptional mechanisms. Highly sensitive assays for TFIIF functions have been developed to monitor transcription complex. Abortive initiation, productive initiation, and runoff transcription assays will be done to analyze the roles of TFIIF in initiation. Transcription complexes will be probed with chemical modification reagents that are specific for single-stranded DNA to correlate maintenance of an open complex with transcriptional function. During elongation, RNA polymerase II may extend an RNA chain according to a branched kinetic pathway in which the enzyme partitions between an activated conformation for elongation and an inactive form. Elongation experiments will test this hypothesis. DNA wrapping around RNA polymerase II and the general transcription factors will be analyzed by fluorescence energy transfer and photocrosslinking studies. The role of TFIIF in this process will be analyzed. TFIIF is also proposed to have a role in DNA bending through the RNA polymerase II active site and in DNA untwisting. Helix untwisting is thought to result from constraint of the DNA between a DNA bend formed at the TATA box by TBP and TFIIB and a second DNA bend through the RNA polymerase II active site formed in part by TFIIF and TFIIE. DNA topology assays and gel mobility shift assays will be used to determine roles for TFIIF and TFIIE in conformational isomerization of the pre-initiation complex.
