München, Univ., Diss., 2003. / Computerdatei im Fernzugriff.

München, Univ., Diss., 2003. / Computerdatei im Fernzugriff.

University, Diss., 2003--München.

<p>Eukaryotic RNA polymerase II (RNAPII) not only synthesizes mRNA, but also coordinates transcription-related processes through the post-translational modification of its unique C-terminal repeat domain (CTD). The CTD is an RNAPII specific extension of the enzyme's largest subunit and consists of multiple repeating heptads with the consensus sequence Y₁S₂P₃T₄S₅P₆S₇. In <italic>Saccharomyces cerevisiae (Sc)</italic>, RNAPII committed to productive elongation is phosphorylated at the S₂ positions of the CTD, primarily by CTDK-I (composed of the CDK-like Ctk1, the cyclin-like Ctk2, and Ctk3) the principal elongation-phase CTD kinase in <italic>Sc</italic>. Although responsible for the bulk of S₂ phosphorylation <italic>in vivo</italic>, Ctk1 coexists with the essential kinase Bur1 which also contributes to S₂ phosphorylation during elongation. In higher eukaryotes there appears to be only one CTD S₂ kinase: P-TEFb, which had been suggested to reconstitute the activity of both of the <italic>Sc</italic> S₂ CTD kinases. Based on comparative genomics, we hypothesized that the previously-unstudied <italic>Drosophila</italic> CDK12 (dCDK12) and little-studied human CDK12 and CDK13 (hCDK12 and hCDK13) proteins are CTD elongation-phase kinases, the metazoan orthologs of yeast Ctk1. Using fluorescence microscopy we show that the distribution of dCDK12 on formaldehyde-fixed polytene chromosomes is virtually identical to that of hyperphosphorylated RNAPII, but is distinct from that of P-TEFb. Chromatin immunoprecipitation experiments confirm that dCDK12 is present on the transcribed regions of active <italic>Drosophila</italic> genes in a pattern reminiscent of a S₂ CTD kinase. Appropriately, we show that dCDK12, hCDK12, and hCDK13 purified from nuclear extracts manifest CTD kinase activity <italic>in vitro</italic> and associate with CyclinK, implicating it as the cyclin subunit of the kinase. Most importantly we demonstrate that RNAi knockdown of dCDK12 in <italic>Drosophila</italic> cell culture and hCDK12 in human cell lines alters the phosphorylation state of the CTD. In an effort to further characterize the transcriptional roles of human CDK12/CyclinK we overexpress, purify to near homogeneity, and characterize, full-length hCDK12/CyclinK. Additionally, we also identify hCDK12 associated proteins via mass spectrometry, revealing interactions with multiple RNA processing factors, and attempt to engineer an analog sensitive CDK12 human cell line. Overall, these results demonstrate that CDK12 is a major elongation-associated CTD kinase, the ortholog of yCtk1. Our findings clarify the relationships between two yeast CDKs, Ctk1 and Bur1, and their metazoan homologues and draw attention to major metazoan CTD kinase activities that have gone unrecognized and unstudied until now. Furthermore, the results suggest that hCDK12 affects RNA processing events in two distinct ways: Indirectly through generating factor-binding phospho-epitopes on the CTD of elongating RNAPII and directly through binding to specific factors.</p> / Dissertation

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.

Universiẗat, Diss., 2000--München.

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

En réponse à son environnement composé de signaux endogènes et exogènes, une cellule doit pouvoir adapter son transcriptome, et cela à travers une modulation fine de l'expression de ses gènes. Les mécanismes permettant une telle adaptation reposent sur de multiples paramètres, entre autre l'organisation du génome, que ce soit au niveau de sa séquence primaire ou de son organisation au sein de la chromatine qui est un support pour l'intégration de nombreuses informations (structurelles et épigénétiques). De plus, l'organisation tridimensionnelle du noyau cellulaire apporte des contraintes physiques et fonctionnelles qui contribuent également à ces régulations. Afin de comprendre comment toutes ces informations peuvent être intégrées lorsqu'un signal régule la transcription d'un ensemble de gènes colinéaires («cluster» de gènes), nos études se sont focalisées sur la description et dissection des mécanismes impliqués dans la régulation coordonnées de gènes œstrogéno-dépendant par le récepteur aux œstrogènes (ER) et ses facteurs pionniers (FOXA1, FOXA2 et GATAs) dans des cellules cancéreuses d'origine mammaire. Dans ce cadre, nous nous sommes plus particulièrement intéressés au cluster TFF, situé sur le bras long du chromosome 21, incluant le gène modèle TFF1, en utilisant des techniques d'analyse à grande échelle (ChIP-chip, ChIP-seq, 4C et analyses transcriptomiques). / A given cell has to be able to adapt its fate and homeostasis in response to endogenous and exogenous signals. This adaptation occurs through finely tuned regulations of genes' expressions leading to the variation of their transcriptomes. Multiple parameters have to be integrated in order to provide such mechanisms of regulation. First, the primary sequence of the genome and its organization into chromatin are major regulatory components that harbor genetic, structural and epigenetic information. Second, the three-dimensional organization of the genome into the nucleus brings both physical and functional constraints that also contribute towards these regulatory processes. Here, we engaged a work aiming to understand and dissect how these several levels of information are integrated during the transcriptional regulation of colinear genes (cluster of genes) by the same signal. We took as a model the coordinated regulation of the estrogen-sensitive TFF cluster driven by the estrogen receptor (ER) and its pioneering factors (FOXA1, FOXA2 and GATAs) in mammary cancer cells. This cluster is located within the long arm of the chromosome 21, and contains the gene model termed TFF1. We used large-scale methods (ChIP-chip, ChIP-seq, 4C and microarray transcriptomic analyses) to decipher these dynamic mechanisms.