RNA polymerase II, the polymerase responsible for transcribing protein coding genes in eukaryotes, possesses an ability to discriminate between correct (complementary to the DNA template) and incorrect substrates (selectivity), and as well as remove incorrect substrates that have been erroneously incorporated into the nascent RNA transcript (proofreading). Although these features of pol II are not as robust as those observed for DNA polymerases, the accurate utilization of genetic information is of obvious importance to the cell. The role of the small RNA polymerase II subunit Rpb9 in transcriptional proofreading was assessed in vitro. Transcription elongation complexes in which the 3'-end of the RNA is not complementary to the DNA template have a dramatically reduced rate of elongation, which provides a fidelity checkpoint at which the error can be removed. The efficiency of such proofreading depends on competing rates of error propagation (extending the RNA chain without removing the error) and error excision, a process that is facilitated by TFIIS. In the absence of Rpb9, the rate of error propagation is increased by 2- to 3-fold in numerous sequence contexts, compromising the efficiency of proofreading. In addition, the rate and extent of TFIIS-mediated error excision is also significantly compromised in the absence of Rpb9. In at least some sequence contexts, Rpb9 appears to enhance TFIIS-mediated error excision by facilitating efficient formation of a conformation necessary for RNA cleavage. If a transcription error is propagated by addition of a nucleotide to the mismatched 3'-end, the rate of further elongation increases but remains much slower than that of a complex with a fully base-paired RNA, which provides a second potential fidelity checkpoint. The absence of Rpb9 also affects both error propagation and TFIIS-mediated error excision at this potential fidelity checkpoint in a manner that compromises transcriptional fidelity. The trigger loop, a mobile structural element of the largest subunit of RNA polymerase II is important for maintaining fidelity. The pol II specific toxin α-amanitin targets the trigger loop, and was used to distinguish trigger loop -independent and -dependent Rpb9 functions, in vitro. Rpb9 decreases the correct nt extension rate when trigger loop movement is restricted by α-amanitin. This occurs in the context of a RNA with a matched or mismatched 3’-end, which indicates that Rpb9’s contribution to correct nt extension occurs in a manner independent of the trigger loop. In addition, the effect on mismatch extension indicates that the trigger loop is not required for Rpb9 to facilitate recognition of proofreading ‘checkpoints’ after mismatches occur. Rpb9 also decreases the rate of misincorporation, but this effect is dependent on the trigger loop. Rpb9’s role in selectivity was tested by utilizing several assays to estimate nt discrimination. Rpb9 does not have a significant effect on nt discrimination for the sequence contexts tested, which suggests the role Rpb9 plays in fidelity is in large part due to its proofreading capabilities. Lastly, the charged residues of Rpb9’s C-terminal “loop” region, proposed in the prevailing model to be important for trigger loop interaction, are dispensable for Rpb9 function in vivo and in vitro.

Diss. (sammanfattning) Umeå : Univ., 2004. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Missouri--Columbia, 1996. / "May 1996." Typescript. Vita. Includes bibliographical references (l. 108-124). Also available on the Internet.

Regulation of gene expression by RNA polymerase (RNAP) is an essential ability of living organisms, required for their adaption to a changing environment and ultimately enabling their survival. Interaction of RNAP with ribonucleic acids (DNA or RNA) is crucial for transcription and its regulation. This Doctoral Thesis contains two projects addressing interactions of RNAP with nucleic acids: (i) Transcription of modified DNA templates and (ii) Ms1, a small RNA (sRNA) from M. smegmatis. (i) We investigated the influence of modifications in the major groove of DNA on bacterial transcription in vitro. We found out that transcription of modified DNA templates is influenced on the transcription initiation level and that the promoter sequence is important for the effect of the modifications. Furthermore, we successfully performed transcription switch ON and OFF in vitro by bioorthogonal reactions. This regulation of transcription by artificial DNA modifications has a future in biotechnologies and/or medical therapy. (ii) Regulators of transcription are also small non-coding RNAs. These molecules have an important role in gene expression regulation among prokaryotes and eukaryotes. Ms1 is an sRNA found in mycobacteria. It makes a complex with the RNAP core and it is abundant in stationary phase (in amounts...

The aim of this thesis was to study the steric influence of the base-modified nucleoside triphosphates (NTPs) on the enzymatic incorporation into RNA, as well as to study their inhibitory effect on different viral RNA polymerases in vitro. Their parent nucleosides and prodrug derivatives were also prepared and their antiviral activity evaluated. In the first part of the thesis, NTPs bearing groups varying in size from small methyl and ethynyl substituents via medium-size phenyl and benzofuryl groups, up to large dibenzofuran ring were prepared. Aromatic substituents were installed via Suzuki coupling on iodinated triphosphates or, in the case of modified guanosines, by the phosphorylation of modified nucleosides. Methyl and ethynyl NTPs were prepared via Pd-catalyzed coupling with AlMe3 and Sonogashira coupling, respectively, followed by the phosphorylation of modified nucleoside. To examine their incorporation into RNA by T7 RNA polymerase, templates coding for 35mer RNA containing one, three or seven modifications were designed. Modified pyrimidine triphosphates worked well for all the sequences, while the biggest dibenzofuryl group was not accepted in the difficult sequence with seven modifications. In the case of AR TPs dibenzofuryl modification did not incorporate at all, while other...

Indiana University-Purdue University Indianapolis (IUPUI) / During RNA Polymerase II (RNAPII) transcription, a dynamic network of protein-protein interactions (PPIs) coordinates the regulation of initiation, elongation, and termination. Taking a proteomics approach to study RNAPII transcription can offer a comprehensive view of the regulatory mechanisms mediated by PPIs within the transcription complex. However, traditional affinity purification mass spectrometry (APMS) methods have struggled to quantitatively capture many of the more dynamic, less abundant interactions within the elaborate RNAPII transcription interactome. To combat this challenge, we have developed and optimized a quantitative AP-MS based method termed Disruption-Compensation (DisCo) Network Analysis that we coupled with Tandem Mass Tag (TMT) labeling. In this application, TMT-DisCo was applied to investigate the PPIs that regulate RNAPII transcription. In the first study, TMT-DisCo network analysis was used to analyze how perturbation of subunits of four major transcription elongation regulators —Spt6, Spt5 (DSIF), Cdc73 (PAF-Complex), and Spt16 (FACT)— affect the RNAPII PPI network. TMT-DisCo was able to measure specific alterations of RNAPII PPIs that provide insight into the normal functions of Spt6/Spt5/Cdc73/Spt16 proteins within the RNAPII elongation complex. The observed changes in the RNAPII interactome also reveal the distinct mechanisms behind the phenotypes of each perturbation. Application of TMTDisCo provides in vivo, protein-level insights into synthetic genetic interaction data and in vitro structural data, aiding in the understanding of how dynamic PPIs regulate complex processes. The second study focused on the essential RNAPII CTD phosphatases, Ssu72 and Fcp1. TMT-DisCo captures how the ssu72-2 allele affects the ability of RNAPII to proceed through elongation, resulting in more arrested RNAPII that requires proteasomal degradation. Reduction of Ssu72 phosphatase activity shifts cells away from RNAPII reinitiation/ recycling and toward de novo expression and newly assembled RNAPII, aided by chaperones. RNAPII in fcp1-1 cells was observed to increase in interaction with the 26S proteasome, as well as TFIID and mRNA capping enzyme. These data support a model of the nuclear proteasome functioning as a chaperone during transcription initiation, as the fcp1-1 allele leads to inefficient formation of a pre-initiation complex with a hyperphosphorylated RNAPII CTD. / 2024-08-16

<p> This thesis describes the isolation and investigation of a DNA-dependent RNA polymerase from the extreme halophile Halobacterium cutirubrum.</p> <p> The enzyme system was analyzed under conditions of very high ionic strengths which are characteristic of the internal salt concentrations of extreme halophiles and at much lower ionic strengths found in conventional bacterial systems. The enzyme was found to have activity in a wide range of salt concentrations when attached to its DNA template in the form of a DNA-Membrane-Protein complex. The enzyme, however, lost the ability to function at high ionic strengths when freed from this complex.</p> <p> The properties of the isolated DNA-dependent RNA polymerase from the halophile were then compared to the properties of the same enzyme isolated from the non-halophilic bacterium, Eschericia coli. Both enzymes were found to have the same approximate molecular weights and to share the same substrate requirements. The enzymes differed, however, in their response to inhibitors specific for RNA synthesis. </p> / Thesis / Master of Science (MSc)

Transcription of the ribosomal genes accounts for the majority of transcription in the cell due to the constant high demand for ribosomes. The number of proteins synthesized correlates with an effective ribosomal biogenesis, which is regulated by cell growth and proliferation. In the work presented in this thesis, we have investigated the ribosomal RNA genes 45S and 5S rRNA, which are transcribed by RNA Pol I and RNA Pol III, respectively. The focus of this work is the chromatin remodelling complex B-WICH, which is composed of WSTF, the ATPase SNF2h and NM1. We have studied in particular its role in ribosomal gene transcription. We showed in Study I that B-WICH is required to set the stage at rRNA gene promoters by remodelling the chromatin into an open, transcriptionally active configuration. This results in the binding of histone acetyl transferases to the genes and subsequent histone acetylation, which is needed for ribosomal gene activation. Study II investigated the role of B-WICH in transcription mediated by RNA polymerase III. We showed that B-WICH is essential to create an accessible chromatin atmosphere at 5S rRNA genes, which is compatible with the results obtained in Study 1. In this case, however, B-WICH operates as a licensing factor for c-Myc and the Myc/Max/Mxd network. Study III confirmed the importance and the function of the B-WICH complex as an activator of ribosomal genes. We demonstrated that B-WICH is important for the remodelling of the rDNA chromatin into an active, competent state in response to extracellular stimuli, and that the association of the B-WICH complex to the rRNA gene promoter is regulated by proliferative and metabolic changes in cells. The work presented in this thesis has confirmed that the B-WICH complex is an important regulator and activator of Pol I and Pol III transcription. We conclude that B-WICH is essential for remodelling the rDNA chromatin into a transcriptionally active state, as required for efficient ribosomal gene transcription. / <p>At the time of the doctoral defense, the following papers were unpublished and had a status as follows: Paper 2: Manuscript. Paper 3: Manuscript.</p><p> </p>

The process of eukaryotic transcription and its regulation has been an interesting area of research for decades. With more insights into the process of transcriptional regulation of genes, studies have revealed a transcriptional regulation at the level of RNA polymerase II in response to nutritional stress. Further studies in our laboratory and others’, using Saccharomyces cerevisiae as a model system, had shown that two subunits of core RNA polymerase II, RPB4 and RPB7 play a crucial role in response to nutritional starvation. Similarly, these proteins are also known to play important roles in stress response in higher eukaryotes. Additionally, altering levels of Rpb4 and Rpb7 can differentially affect starvation response in S. cerevisiae (Singh et al., 2007). Multiple tissue blot analyses had shown that both these subunits are differentially expressed in different human tissues more significantly in heart, kidney and brain (Khazak et al., 1995; Khazak et al., 1998; Schoen et al., 1997). These findings have led us to investigate in Dictyostelium discoideum, a cellular slime mold, the possible role of these subunits during starvation-induced development. D. discoideum cells exist as unicellular amoebae in soil. In this organism, growth and differentiation phases are distinctly separated, which is an advantage for investigating the functions of these subunits during growth and development. Cells respond to nutritional starvation by undergoing a series of morphological changes coordinated with transcriptional changes giving rise to a terminally differentiated structure referred to as fruiting body which has live spores suspended on top of stalk of dead cells. Though starvation-induced development is accompanied by differential expression of genes, few studies related to the transcription machinery, RNA polymerase II have been reported so far. Purification and presence of all three RNA polymerases from D. discoideum had been reported earlier but the details of their structures and regulation have not been explored in detail (Pong and Loomis, 1973; Renart et al., 1985). One interesting observation reported by Lam and colleagues (Lam et al., 1992) was that CTD of the largest subunit of RNA polymerase II, Rpb1, is highly conserved with 24 heptapeptide repeats and expression of RPB1 transcript was regulated during development. Thus, we carried out experiments to characterize Rpb4 and Rpb7, two subunits of D. discoideum RNA polymerase II to understand any role of these subunits during development. Identification of Rpb4 and Rpb7, two subunits of D. discoideum RNA polymerase II To identify the homologs of S. cerevisiae Rpb4 and Rpb7 in D. discoideum, we employed bioinformatics and genetic approaches. Firstly, we searched D. discoideum database for all protein sequences of S. cerevisiae RNA polymerase II subunits. We could obtain sequences homologous to all twelve subunits in D. discoideum. Among the 12 subunits of D. discoideum RNA polymerase II, we chose to characterize two subunits, DdRpb4 and DdRpb7. We cloned the open reading frames of these two genes from D. discoideum Ax2 cells and cloned them in yeast expression vectors for complementation studies. In S. cerevisiae, Rpb4 is a non-essential protein but rpb4∆ cells show abnormal phenotypes. Few phenotypes of rpb4∆ cells, such as temperature sensitivity, defective in response to nutritional starvation and defective in activated transcription, were employed to identify the D. discoideum homolog of ScRpb4 (Woychik and Young, 1989; Pillai et al., 2001: Pillai et al., 2003). We observed that DdRPB4 can rescue temperature sensitivity corroborated with its ability to activate transcription from HSE containing promoters and sporulation defects of Scrpb4Δ mutant to the wild type. However, DdRPB4 can rescue neither the defect in activated transcription of GAL10 and INO1 promoters nor the elongated morphology exhibited by Scrpb4Δ mutant. On the other hand, we observed that DdRPB7 can complement the lethality associated with ScRPB7 deletion and can partially rescue the phenotypes associated with Scrpb4∆ strain similar to ScRPB7 (Sharma and Sadhale, 1999; Singh et al., 2004). Taken together, we have identified D. discoideum Rpb4 and Rpb7 as bona fide homologs of S. cerevisiae Rpb4 and Rpb7, respectively. Analysis of Rpb4 and Rpb7 in D. discoideum Since yeast RNA polymerase II subunits, Rpb4 and Rpb7, play an important role in the regulation of genes responsive to starvation stress, we carried out experiments to characterize Rpb4 and Rpb7 during growth and starvation-induced development in D. discoideum. Temporal and spatial expression profiles show avaried but similar pattern of RPB4 and RPB7 transcripts during D. discoideum development. We observed similarity between ScRpb4 and DdRpb4 in its ability to interact with DdRpb7 and to localise in both nuclear and cytoplasmic compartments. Attempts to knock out or reduce the levels of DdRpb4 and DdRpb7 by homologous recombination and antisense approaches, respectively, failed. However, since altering levels of Rpb4 and Rpb7 in S. cerevisiae can affect different stress response pathways, we had used overexpression to alter the level of Rpb4 and analysed its effect on D. discoideum development. We overexpressed DdRpb4 as GFP fusion protein in Ax2 cells and observed that D. discoideum cells overexpressing DdRpb4 showed normal growth and development similar to the wild type protein. Interestingly, we observed that Ax2 cells overexpressing DdRpb4 have drastically reduced levels of the endogenous protein. Thus, we have identified a post-transcriptional control on the level of Rpb4 in D. discoideum. Role of S. cerevisiae Rpb4/Rpb7 subcomplex in stress In S. cerevisiae, Rpb4 and Rpb7 interact with each other and carry out important functions (Choder, 2003; Sampath and Sadhale, 2004). Employing the functional conservation of Rpb4 and Rpb7 across various model systems, we further investigated the role of the subcomplex in S. cerevisiae. Since Rpb7 is an essential gene, we have generated rpb7Δstrain in the presence of plasmids expressing Rpb7 or its homologs. We have generated a S. cerevisiae strain lacking both RPB4 and RPB7 and introduced Rpb4 and Rpb7 homologs from either D. discoideum or C. albicans. We analysed these strains under stresses such as high temperature and nutrient starvation. The results of these experiments have provided how the differences in Rpb4 and Rpb7 proteins and their ability to form a subcomplex could be reflected in differential stress responses. Besides the high functional conservation of these proteins, their interaction with other regulatory proteins might also be critical for a proper response to nutritional stress.

Thesis (Ph. D.)--University of Washington, 1997. / Vita. Includes bibliographical references (leaves [68]-77).