Thesis (Ph. D.)--West Virginia University, 2005. / Title from document title page. Document formatted into pages; contains viii, 179 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 156-179).

O estudo sobre a cinética de incorporação de uridina em ácidos nucleicos permitiu estabelecer que o tamanho do \"pool\" de precursores permanece constante na presença de concentrações de uridina exógena acima de aproximadamente1 µM. Concluiu-se ainda que todo o sistema de tomada de uridina, medido pela sua incorporação em ácidos nucleicos, apresenta um Km de 5,1 µM e opera a uma velocidade máxima de 11 pmoles/min/l,3 x 107 células. Um estudo análogo, em presença de rifampicina, possibilitou calcular que a meia vida de RNAs mensa - geiros em B. subtilis é de 2 minutos e que 44% da radiatividade incorporada em RNA num determinado instante se encontra na fração de RNA estável (ribossômico e de transferência). A análise do mecanismo de transcrição dos genes para RNA ribossômico foi abordada por meio do estudo do alongamento de cadeias de rRNA já iniciadas, em presença de rifampicina. As relações iniciais de radiatividade incorporada em rRNA 16S e 23S, quando rifampicina e uridina tritiada são adicionadas concomitantemente, bem corno a cinética de decaimento de marcação presente em ambas as espécies de rRNA sugerem que os genes para RNA ribossômico 16S e 23S são cotranscritos nessa ordem, em B. subtilis. Levando-se em consideração essas e outras evidências, propõe-se o seguinte relacionamento estrutural para a unidade de transcrição: [Obs.: Ver no arquivo em PDF] Os resultados experimentais foram comparados com modelos teóricos descritos no Apêndice. A existência de um mecanismo de cotranscrição para os genes responsáveis pela síntese de rRNA em procariotos e eucariotos parece sugerir que esse mecanismo, de fundamental importância, instalou-se precocemente e foi mantido durante a evolução. / The kinetics of uridine incorporation into nucleic acids has shown that the precursor pool size remains constant in the presence of exogenous uridine concentrations above 1 µM, approximately. It has also been shown that the whole system of uridine uptake, as measured by the incorporation of uridine into nucleic acids, has an apparent Km of 5.1 µM and operates at a maximal rate of 11 pmoles/min/ 1.3 x 107 cells. The incorporation, in the presence of rifampicin, made it possible to calculate the half life of the messenger RNA\'s in B.subtilis as being 2 minutes. In any given instant, 44% of the radioactivity incorporated into RNA belongs to the stable RNA fraction (ribosomal and transfer). The analysis of the ribosomal RNA genes transcription mechanism was undertaken by a study of the rRNA chain elongation in the presence of rifampicin. The initial ratios of radioactivity incorporated in 16S and 23S rRNA\'s, when rifampicin and tritiated uridine are concomitantly added, and the decay kinetics of the radioactivity present in both rRNA species, suggest that the 16S and 23S rRNA genes are cotranscribed, in that order, in B.subtilis. Taking into consideration these and other evidences, the following structural relationships are proposed for the transcriptional unit: The experimental results were compared with theoretical models described in the Appendix. The existence of a cotranscription mechanism for the rRNA genes in prokaryotes and eukaryotes seems to suggest that such mechanism, being of fundamental importance, established itself very early and was maintained through evolution.

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 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 properly in several stress conditions (Pillai et al., 2001; Sampath and Sadhale, 2005). In spite of wealth of available information, the exact role of Rpb4 remains poorly understood. In the present work, we have used genetic, molecular and biochemical approaches to understand the role of Rpb4 as described in four different parts below: i) Studies on Genetic and Functional Interactions of Rpb4 with SAGA/TFIID Complex to Confer Promoter- Specific Transcriptional Control To carry out transcription, Pol II has to depend on several general transcription factors, mediators, activators, and co-activators and chromatin remodeling complexes. In the present study, we tried to understand the genetic and functional relationship of Rpb4 with some of the components of transcription machinery, which will provide some insight into the role of Rpb4 during transcription. Our microarray analysis of rpb4∆ strain suggests that down regulated genes show significant overlap with genes regulated by the SAGA complex, a complex functionally related to TFIID and involved in regulation of the stress dependent genes. The analysis of combination of double deletion mutants of either the SAGA complex subunits or the TFIID complex with rpb4∆ showed that both these double mutants are extremely slow growing and show synthetic growth phenotype. Further studies, including microarray analysis of these double mutants and ChIP (chromatin immunoprecipitation) of Rpb4 and SAGA complex, suggested that Rpb4 functions together with SAGA complex to regulate the expression of stress dependent genes. ii) Study of Genome Wide Recruitment of Rpb4 and Evidence for its Role in Transcription Elongation Biochemical studies have shown that Rpb4 associates sub-stoichiometrically with the core RNA polymerase during log phase but whether recruitment of Rpb4 is promoter context dependent or occurs only at specific stage of transcription remains largely unknown. Having discovered that Rpb4 can recruit on both TFIID and SAGA dominated promoters, it was important to study the genome wide role of Rpb4. Using ChIP on chip experiments, we have carried out a systematic assessment of genome wide binding of Rpb4 as compared to the core Pol II subunit, Rpb3. Our analysis showed that Rpb4 is recruited on coding regions of most transcriptionally active genes similar to the core Pol II subunit Rpb3 albeit to a lesser extent. This extent of Rpb4 recruitment increased on the coding regions of long genes pointing towards a role of Rpb4 in transcription elongation of long genes. Further studies showing transcription defect of long and GC rich genes, 6-azauracil sensitivity and defective PUR5 gene expression in rpb4∆ mutant supported the in vivo evidence of the role of Rpb4 in transcription elongation. iii) Genome Wide Expression Profiling and RNA Polymerase II Recruitment in rpb4∆ Mutant in Non-Stress and Stress Conditions Structural studies have suggested a role of Rpb4/Rpb7 sub-complex in recruitment of different factors involved in transcription (Armache et al., 2003; Bushnell and Kornberg, 2003). Though only few studies have supported this aspect of Rpb4/Rpb7 sub-complex, more research needs to be directed to explore this role of Rpb4/Rpb7 sub-complex. To study if Rpb4 has any role in recruitment of Pol II under different growth conditions, we have studied genome wide recruitment of Pol II in the presence and absence of Rpb4 during growth in normal rich medium as well as under stress conditions like heat shock and stationary phase where Rpb4 is shown to be indispensable for survival. Our analysis showed that absence of Rpb4 results in overall reduced recruitment of Pol II in moderate condition but this reduction was more pronounced during heat shock condition. During stationary phase where overall recruitment of Pol II also goes down in wild type cells, absence of Rpb4 did not lead to further decrease in overall recruitment. Interestingly, increased expression levels of many genes in the absence of Rpb4 did not show concomitant increase in the recruitment of Pol II, suggesting that Rpb4 might regulate these genes at a post-transcriptional step. iv) Role of Rpb4 in Pseudohyphal Growth The budding yeast S. cerevisiae can initiate distinct developmental programs depending on the presence of various nutrients. In response to nitrogen starvation, diploid yeast undergoes a dimorphic transition to filamentous pseudohyphal growth, which is regulated through cAMP-PKA and MAP kinase pathways. Previous work from our group has shown that rpb4∆ strain shows predisposed pseudohyphal morphology (Pillai et al., 2003), but how Rpb4 regulates this differentiation program is yet to be established. In the present study, we found that disruption of Rpb4 leads to enhanced pseudohyphal growth, which is independent of nutritional status. We observed that the rpb4∆/ rpb4∆ cells exhibit pseudohyphae even in the absence of a functional MAP kinase and cAMP-PKA pathways. Genome wide expression profile showed that several downstream genes of RAM signaling pathway were down regulated in rpb4∆ cells. Our detailed genetic analysis further supported the hypothesis that down regulation of RAM pathway might be leading to the pseudohyphal morphogenesis in rpb4∆ cells.