by
Wade Powell
Adviser: Daniel
Reines, Ph.D.
A Dissertation submitted to the Faculty of the Graduate
School of Emory University in partial fulfillment of the requirements for
the degree of Doctor of Philosophy
1996
SUMMARY
RNA polymerase II can encounter transcriptional blocks during the elongation phase of RNA synthesis and become arrested. Transcription elongation factor SII (TFIIS) rescues arrested enzymes by activating an RNA cleavage activity intrinsic to RNA polymerase II.
In vivo and in vitro analysis of yeast RNA polymerase II enzymes containing mutations in the second largest subunit (RPB2) showed that the barnase homology region, which shares sequence similarity with barnase and several other bacterial ribonucleases, does not represent the active site of SII-stimulated RNA cleavage. Additional RPB2 mutant enzymes purified from 6-azauracil-sensitive yeast strains displayed increased arrest in vitro. The most severe effect result from mutation rpb2-10, which occurs in a domain associated with nucleotide binding. The elongation rate of this mutant enzyme is slower than wild type, suggesting that slow elongation contributes to the arrest-prone phenotype. This work was the first to relate 6-azauracil sensitivity to distinct, elongation-related defects in vitro.
Additional work employed RNA polymerase II and general initiation factors purified from rat liver, and a chromatographic scheme was devised for the purification of rat liver SII. Cleavage and read-through properties of elongation complexes treated with the detergent sarkosyl, a specific inhibitor of SII-dependent elongation, showed that one round of transcript cleavage is insufficient for full read-through at an arrest site, suggesting that cleavage allows the enzyme multiple attempts at read-through.
Cross-linking studies showed that SII contacts nascent RNA within the elongation complex. 4-thio-UMP, a photoactive nucleotide analog, was incorporated at the 3' end of radiolabeled transcripts and cross-linked to SII and RNA polymerase II subunits. SII cross-linking required the presence of RNA within the elongation complex and was not detectable between SII and free RNA. This work showed that the 3' end of RNA is near the SII binding site on RNA polymerase II and suggests that SII may activate RNA cleavage by positioning the transcript in the enzyme's active site.