RNA polymerase II (Pol II) is the enzyme responsible for the synthesis of all mRNAs in eukaryotic cells. As the central component of the eukaryotic transcription machinery, Pol II is the final target of transcription regulatory pathways. While the role for different Pol II associated proteins, co-activators and general transcription factors (GTFs) in regulation of transcription in response to different stimuli is well studied, a similar role for some subunits of the core Pol II is only now being recognized. The studies reported in this thesis address the role of the fourth largest subunit of Pol II, Rpb4, in transcription and stress response using Saccharomyces cerevisiae as the model system. Rpb4 is closely associated with another smaller subunit, Rpb7 and forms a dissociable complex (Edwards et al. 1991). The rpb4 null mutant is viable but is unable to survive at extreme temperatures (>34ºC and <12ºC) (Woychik and Young, 1989). This mutant has also been shown to be defective in activated transcription and unable to respond adequately to several stress conditions (Pillai et al. 2001; Sampath and Sadhale, 2005). In spite of wealth of available information, the exact role of Rpb4 in transcription process remains poorly understood. In the present work, we have used genetic, molecular and biochemical approaches to understand the role of Rpb4 as described in three different parts below:
I. Role of Rpb4 in various pathways related to Transcription Elongation
The genome-wide recruitment study of RNA pol II in presence and absence of Rpb4 has indicated role of Rpb4 in transcription elongation (Verma-Gaur et al. 2008). However, a recent proteomics based report has argued against it (Mosley et al. 2013). To address this conflict and understand Rpb4 functions, we monitored recruitment of RNA pol II on a few individual long genes in wild type and rpb4∆ cells. It was observed that RNA pol II recruitment on genes with longer coding regions is not significantly affected in rpb4∆ as compared to wild type thus ruling out role of Rpb4 in transcription elongation of these genes. However, our genetic interaction studies have shown a strong interaction (synthetic lethality) between RPB4 and the PAF1 and SPT4 genes, the products of which code for well-known transcription elongation factors. The studies based on Rpb4 overexpression in mutants for elongation factors, 6-Azauracil sensitivity of cells, effect of Dst1 overexpression in rpb4∆ cells and mitotic recombination rate in rpb4∆ cells have indicated functional interactions of Rpb4 with many of the transcription elongation factors.
II. Studies on Genetic and Functional Interactions of Rpb4 with SAGA Complex in Promoter- Specific Transcription Initiation
To carry out transcription, RNA pol II depends on several general transcription factors, mediators, activators, co-activators and chromatin remodeling complexes. In the present study, we explored the genetic and functional relationships between Rpb4 and the SAGA complex of transcription machinery, to gain some insight on the role of Rpb4 during transcription. Our chromatin immunoprecipitation data suggest that RNA pol II does not associate with promoters of heat shock genes during transcription activation of these heat stress induced genes in absence of Rpb4. SAGA coactivator complex is required for RNA pol II recruitment and transcription activation of these genes (Zanton and Pugh, 2004). However, recruitment of the SAGA complex at promoters of these heat shock genes was not affected in rpb4∆ cells after heat stress. Our genetic interaction analysis between RPB4 and components of SAGA complex (spt20∆) showed synthetic lethality indicating that fully functional Rpb4 and SAGA complex are required for cellular functions in the absence of heat stress and the simultaneous deletion of factors in the two complexes leads to cell death.
III. Role of Rpb4 in phosphorylation cycles of Rpb1-CTD
The C-Terminal Domain (CTD) of Rpb1 protein of RNA pol II undergoes several rounds of phosphorylation cycles at Ser-2 and Ser-5 residues on its heptad repeats during transcription. These phosphorylation marks are to be erased before the start of next round of transcription. Using protein pull down assay, we observed that hyperphosphorylated form of Rpb1 is reduced in rpb4∆ as compared to that seen in wild type cells among the free RNA pol II molecules. The level of Rpb2 protein was unaffected in both wild type and rpb4∆. These preliminary data hints at role of Rpb4 in the regulation of Rpb1 phosphorylation.
| Identifer | oai:union.ndltd.org:IISc/oai:etd.ncsi.iisc.ernet.in:2005/2624 |
| Date | January 2013 |
| Creators | Deshpande, Swati |
| Contributors | Sadhale, Parag, Vijayaraghavan, Usha |
| Source Sets | India Institute of Science |
| Language | en_US |
| Detected Language | English |
| Type | Thesis |
| Relation | G26007 |
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