The role of Clp1 and Pcf11 in transcription and pre-mRNA 3’-end processing

Eukaryotic transcripts require a number of complex cotranscriptional modifications and processing events before translation to protein. Clp1 and Pcf11 are subunits of cleavage factor IA (CFIA), an essential component of the Saccharomyces cerevisiae pre-mRNA 3’-end processing machinery. The crystal s...

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Main Author: Hedden, J. J.
Published: University College London (University of London) 2012
Subjects:
572
Online Access:http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.568289
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spelling ndltd-bl.uk-oai-ethos.bl.uk-5682892016-11-18T03:18:16ZThe role of Clp1 and Pcf11 in transcription and pre-mRNA 3’-end processingHedden, J. J.2012Eukaryotic transcripts require a number of complex cotranscriptional modifications and processing events before translation to protein. Clp1 and Pcf11 are subunits of cleavage factor IA (CFIA), an essential component of the Saccharomyces cerevisiae pre-mRNA 3’-end processing machinery. The crystal structure of a Clp1-Pcf11 complex was determined previously and revealed the binding of ATP to a highly-conserved P-loop motif and a tight Pcf11-Clp1 interaction facilitated by a number of highly-conserved Pcf11 residues. Nonetheless, the biological function of both Clp1-ATP binding and the Pcf11-Clp1 interaction was not well understood. The work in this thesis combines an in vitro and in vivo investigation of the Clp-ATP and Clp-Pcf11 interactions in an effort to understand the function of these factors in transcription and pre-mRNA 3’-end processing. It is demonstrated that the interaction of ATP and Pcf11 with Clp1 are linked events: Loss of Clp1-ATP binding results in the abrogation of the Pcf11-Clp1 interaction and leads to Clp1 instability in vitro, and similarly, mutations that directly uncouple the Pcf11-Clp1 interaction also disrupt Clp1-ATP binding and cause Clp1 instability in vitro. An in vivo mutational analysis in S. cerevisiae revealed that both Clp1-ATP binding and the Pcf11-Clp1 interaction are essential for yeast survival. Further cell and immunoprecipitation studies demonstrated that one essential function of Clp1 is as a chaperone of Pcf11, and RT-qPCR analysis of mRNA from a sample set of yeast genes points to a role for these proteins in transcription and transcription termination rather than in poly(A) site selection.572University College London (University of London)http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.568289http://discovery.ucl.ac.uk/1369193/Electronic Thesis or Dissertation
collection NDLTD
sources NDLTD
topic 572
spellingShingle 572
Hedden, J. J.
The role of Clp1 and Pcf11 in transcription and pre-mRNA 3’-end processing
description Eukaryotic transcripts require a number of complex cotranscriptional modifications and processing events before translation to protein. Clp1 and Pcf11 are subunits of cleavage factor IA (CFIA), an essential component of the Saccharomyces cerevisiae pre-mRNA 3’-end processing machinery. The crystal structure of a Clp1-Pcf11 complex was determined previously and revealed the binding of ATP to a highly-conserved P-loop motif and a tight Pcf11-Clp1 interaction facilitated by a number of highly-conserved Pcf11 residues. Nonetheless, the biological function of both Clp1-ATP binding and the Pcf11-Clp1 interaction was not well understood. The work in this thesis combines an in vitro and in vivo investigation of the Clp-ATP and Clp-Pcf11 interactions in an effort to understand the function of these factors in transcription and pre-mRNA 3’-end processing. It is demonstrated that the interaction of ATP and Pcf11 with Clp1 are linked events: Loss of Clp1-ATP binding results in the abrogation of the Pcf11-Clp1 interaction and leads to Clp1 instability in vitro, and similarly, mutations that directly uncouple the Pcf11-Clp1 interaction also disrupt Clp1-ATP binding and cause Clp1 instability in vitro. An in vivo mutational analysis in S. cerevisiae revealed that both Clp1-ATP binding and the Pcf11-Clp1 interaction are essential for yeast survival. Further cell and immunoprecipitation studies demonstrated that one essential function of Clp1 is as a chaperone of Pcf11, and RT-qPCR analysis of mRNA from a sample set of yeast genes points to a role for these proteins in transcription and transcription termination rather than in poly(A) site selection.
author Hedden, J. J.
author_facet Hedden, J. J.
author_sort Hedden, J. J.
title The role of Clp1 and Pcf11 in transcription and pre-mRNA 3’-end processing
title_short The role of Clp1 and Pcf11 in transcription and pre-mRNA 3’-end processing
title_full The role of Clp1 and Pcf11 in transcription and pre-mRNA 3’-end processing
title_fullStr The role of Clp1 and Pcf11 in transcription and pre-mRNA 3’-end processing
title_full_unstemmed The role of Clp1 and Pcf11 in transcription and pre-mRNA 3’-end processing
title_sort role of clp1 and pcf11 in transcription and pre-mrna 3’-end processing
publisher University College London (University of London)
publishDate 2012
url http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.568289
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