Open Access Dissertation
Date of Award
In budding yeast, as in higher eukaryotes, transcription of protein coding genes is executed by a highly specialized, conserved polymerase called RNA polymerase II (RNAPII). The transcription cycle of RNAPII has four major steps: initiation, elongation, termination, and reinitiation. The successful accomplishment of each of these steps requires a number of accessory factors. Many of these factors operate at multiple steps in the transcription cycle. The major focus of this study was to examine the function of Clp1, which is an RNA processing factor operating at the 3′ end of genes, in the transcription cycle. Clp1 is one of the four subunits of the CFIA 3′ end processing complex. It is the least investigated CFIA subunit. The role of the other three subunits of the CFIA complex in 3' end processing and termination of transcription is well documented. Here we investigate the role of Clp1 in the initiation as well as the termination of transcription. We used a temperature-sensitive mutant of Clp1 to assess its function. We demonstrated a direct role for this factor in the termination of transcription of CHA1. We used three different approaches; TRO assay, RNAPII-ChIP assay, and strand specific RT-PCR, to demonstrate the termination function of Clp1. In addition, we showed that Clp1 is also involved in the early steps of the transcription. Our results strongly suggest that Clp1 participates in promoter-associated transcription. We provide multiple lines of evidence in support of a role for Clp1 at the 5′ end of genes. First, the presence of Clp1 in the vicinity of the promoter region implies its involvement early in the transcription cycle. Second, the decrease in RNAPII density near the promoter without a parallel decrease in the level of the GTFs suggested a role for Clp1 in reinitiation of transcription. Third, an increase in 5' initiated antisense divergent transcripts in the Clp1 mutant supports a role for the factor in providing directionality to the promoter-bound polymerase. To assess the generality of the observed functions of Clp1, we investigated the role of Clp1 in the transcription cycle on a genomewide scale using GRO-Seq approach. Our results show that the number of transcriptionally active genes decreased by at least two-fold in the clp1mutant. The GRO-Seq results strongly suggest a genomewide function for Clp1 in the termination of transcription, and indicate that Clp1 is required for the pausing of RNAPII that is a pre-requisite for the termination of transcription. We also observed a dramatic increase in 3' initiated antisense transcription in the absence of a functional Clp1 protein. Using the chromosome conformation capture approach, CCC, we observed a role for Clp1 in gene loop formation. We found a strong correlation between the Clp1 function in gene looping, and its role in promoter-associated transcription which implies gene looping as the means through which this factor is exerting its functions at the 5′ end of genes.
Al-Husini, Nadra, "An Investigation Into The Role Of Cfia 3' End Processing Complex In The Termination And Initiation/reinitiation Of Transcription" (2013). Wayne State University Dissertations. 868.