Hedden, JJ;
(2012)
The role of Clp1 and Pcf11 in transcription and pre-mRNA 3’-end processing.
Doctoral thesis , UCL (University College London).
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Abstract
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.
| Type: | Thesis (Doctoral) |
|---|---|
| Title: | The role of Clp1 and Pcf11 in transcription and pre-mRNA 3’-end processing |
| Open access status: | An open access version is available from UCL Discovery |
| Language: | English |
| UCL classification: | UCL > Provost and Vice Provost Offices UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences UCL > Provost and Vice Provost Offices > School of Life and Medical Sciences > Faculty of Life Sciences > Div of Biosciences |
| URI: | https://discovery.ucl.ac.uk/id/eprint/1369193 |
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