Regulation of RNA Polymerase II CTD Phosphatase in S. Cerevisiae PDF Download
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Author: Susanne Jutta Hoheisel
Publisher:
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Category :
Languages : en
Pages : 328
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Author: Susanne Jutta Hoheisel
Publisher:
ISBN:
Category :
Languages : en
Pages : 328
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Author: Ulku Uzun
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Category :
Languages : en
Pages :
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Author: Lei Zhang
Publisher:
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Category : Messenger RNA.
Languages : en
Pages : 288
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Author: David Jansma
Publisher:
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Category :
Languages : en
Pages : 0
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RNA polymerase (RNAP) uses ribonucleoside triphosphates as a substrate to form RNA chains. The RNA is a faithful copy of one strand of a double-stranded DNA template along which RNAP moves while making the RNA. This process (called transcription) is highly regulated such that DNA-encoded genes are transcribed at a large variety of rates and at different times in response to cues within and outside the cell. The goal of my thesis work was to investigate the mechanisms that regulate the synthesis of RNAP in yeast cells. I focused on the form of RNAP (RNAPII) of the yeast, 'Saccharomyces cerevisiae', that is responsible primarily for transcribing genes that encode proteins. I have shown that a 10-fold reduction in the level of the largest subunit of RNAPII, and likely the level of RNAPII itself, causes slow growth, temperature-sensitivity, and the inability to grow on medium lacking inositol. Hence, the level of RNAPII must be carefully maintained for normal cell growth. I next examined elements that control the synthesis of RNAPII. I have demonstrated that the DNA sequences which are upstream of the genes encoding the two largest subunits of RNAPII, namely 'RPO21' and 'RPO22', contain binding sites for two abundant transcription factors called Abf1p and Reb1p, and thymidine-rich sequences downstream of these binding sites. Both the binding sites and the T-rich regions are important for the expression of these genes. An examination of the upstream sequences of other RNAPII subunit genes revealed binding sites for Abf1p and Reb1p as well as nearby thymidine-rich sequences. This may indicate that there is a mechanism for the coordinate synthesis of RNAPII subunit genes. I sought evidence for a feedback regulatory mechanism that may control the synthesis of RNAPII. Either the underproduction of Rpo21p, or the depletion of Fcp1p, an RNAPII phosphatase that has a critical role in transcription, leads to a 5-fold increase in the expression of a reporter gene that is controlled by 'RPO21' regulatory sequences. The increase is not observed with other subunits. I discuss the implications of these results and future directions.
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Category :
Languages : en
Pages :
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Author: Jason Nicholas Kuehner
Publisher:
ISBN:
Category :
Languages : en
Pages : 188
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![Regulation and Variation of Subunits of RNA Polymerase II in Saccharomyces Cerevisiae [microform] PDF](https://books.google.com/books/content/images/frontcover/v_7KygAACAAJ?fife=w80-h100)
Author: David Jansma
Publisher: National Library of Canada = Bibliothèque nationale du Canada
ISBN: 9780612411791
Category :
Languages : en
Pages : 400
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Author: Michael J. Berna (Sr.)
Publisher:
ISBN:
Category : Genetic transcription
Languages : en
Pages : 166
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The C-terminal domain (CTD) of the RNA polymerase II (RNAPII) subunit Rpb1 must exist in a hypophosphorylated state prior to forming a competent transcription initiation complex. However, during transcription, specific kinases and phosphatases act on the RNAPII CTD to regulate its phosphorylation state, which serves to recruit sequence-specific and general transcription factors at the appropriate stage of transcription. A key phosphatase involved in this process, Rtr1 (Regulator of Transcription 1), was shown to regulate a key step important for transcription elongation and termination. Although the role that Rtr1 plays in regulating RNAPII transcription has been described, the mechanism involved in the recruitment of Rtr1 to RNAPII during transcription has not been elucidated in yeast. Consequently, the present work utilized both affinity purification schemes in Saccharomyces cerevisiae and mass spectrometry to identify key Rtr1-interacting proteins and post-translational modifications that potentially play a role in recruiting Rtr1 to RNAPII. In addition to RNAPII subunits, which were the most consistently enriched Rtr1-interacting proteins, seven proteins were identified that are potentially involved in Rtr1 recruitment. These included PAF complex subunits (Cdc73, Ctr9, Leo1), the heat shock protein Hsc82, the GTPase Npa3, the ATPase Rpt6, and Spn1. Indirect evidence was also uncovered that implicates that the CTDK-I complex, a kinase involved in RNAPII CTD phosphorylation, is important in facilitating interactions between Rtr1, RNAPII, and select transcription factors. Additionally, a putative phosphorylation site was identified on Ser217 of Rtr1 that may also play a role in its recruitment to RNAPII during transcription.
Author: John Jun Kang
Publisher:
ISBN:
Category :
Languages : en
Pages : 444
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Author: Michael S. Kobor
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Category :
Languages : en
Pages :
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The form of RNA polymerase II (RNAPII) that binds preferentially to promoters is not extensively phosphorylated on the carboxy-terminal heptapeptide repeat domain (CTD) of its largest subunit. The CTD becomes phosphorylated during or shortly after initiation and elongating RNAPII generally has a phosphorylated CTD. Prior to or following transcriptional termination, dephosphorylation of the CTD presumably must occur to regenerate the hypophosphorylated form of RNAPII that is capable of reinitiating transcription. This thesis examines the function of the CTD phosphatase Fcp1p in the yeast 'Saccharomyces cerevisiae'. In chapter 2, it is shown that Fcp1 is an unusual eukaryotic protein phosphatase that is required for dephosphorylation of the CTD 'in vivo ' and for transcription by RNAPII 'in vivo'. These results suggest that Fcp1p is the founding member of a new class of protein phosphatases and acts as a general transcription factor 'in vivo'. In chapter 3, affinity chromatography is used to study the binding of Fcp1p to TFIIB and the RAP74 subunit of TFIIF. Fcp1p binds in a similar way to both of these factors. RAP74 and TFIIB have a short region of homology and amino acid changes in this region affect the binding to Fcp1p. The genes encoding RAP74 and Fcp1p interact 'in vivo'. Fcp1p can activate transcription when artificially tethered to a promoter and this effect is largely dependent on binding to RAP74. In chapter 4, it is shown that yeast strains with mutations in ' fcp1' grow much worse when the gene encoding the major CTD kinase Kin28p is also mutated. In contrast, inactivation of another CTD kinase encoded by the 'SRB10' gene suppresses the temperature-sensitivity and the sensitivity to certain cell cycle checkpoint inducing drugs of ' fcp1' mutant strains. These results therefore suggest that Fcp1p and Srb10p have opposing roles 'in vivo'. In chapter 5, analysis of the phosphorylation state of the CTD reveals that reduced Fcp1p activity results in a increased amount of the largest subunit of RNAPII but this subunit is not incorporated into functional enzyme and is largely degraded at a higher temperature.
