A Study of Potential Factors Involved in Post-Recruitment Regulation at CYC1 in Saccharomyces cerevisiae

Abstract

Transcription is one of the most critical processes a cell undergoes to control cellular function.1 The three basic steps of transcription are initiation, elongation, and termination.2 It was previously believed that at all genes, the rate-limiting step between initiation and elongation was the recruitment of the enzyme RNA polymerase II (Pol II) to the promoter site of the gene. However, in recent years, much evidence has arisen that at many genes, the rate-limiting step occurs after Pol II recruitment. It has been proposed that genes are either recruitment regulated or post-recruitment regulated, respectively.2,3 At post-recruitment regulated genes, Pol II has been shown to be “paused” after catalyzing 20-40 nucleotides down the promoter site, unable to complete elongation. 3-5 This signifies that other factors function to pause Pol II at the promoter-proximal site and later, when induced, to release it. Post-recruitment regulation is evolutionarily conserved from bacteria to humans, 3-5 and in Drosophila melanogaster, a basic model has been proposed for this type of regulation.6 Complete understanding of the mechanisms of the regulation of transcription after initiation is still a lofty goal for molecular biologists and biochemists. The experiments presented continue the work by examining potential proteins’ involvement in post-recruitment regulation in the species Saccharomyces cerevisiae. With the aid of bioinformatics, the lab has suggested eleven deletion strain-viable proteins as potential candidates, and the list is growing. (Bur2 of the Bur complex; Cdc73, Leo1, Paf1, and Rtf1 of the Paf1 complex; Ctk1, Ctk2, and Ctk3 of the Ctk complex; Spt4 of the Spt4/Spt5 complex; and Pol II subunits Rpb4 and Rpb9). Experiments successfully demonstrated that four of these proteins, Rpb4, Rpb9, Leo1, and Spt4, affect the levels of transcript produced at the gene CYC1, which is known to be post-recruitment regulated. Deletion strains of these four proteins were cultured and frozen at timepoints previous to and after CYC1 transcription induction by H2O2. An S1 nuclease digestion assay followed by Northern blotting and visualization allowed the comparison of the deletion strains’ CYC1 transcript levels with wild-type levels at three time points. As hypothesized, deviation from the wild-type levels were observed in all four deletion strains tested, indicating the proteins’ of interest involvement in transcription regulation at a post-recruitment regulated gene. Possible functions of the four proteins have been discussed and specific future experiments described.