Assembly of the mRNA 3'-end processing machinery
EMSL Project ID
24806
Abstract
We propose to use NMR to study the structural basis for the assembly of the protein complex responsible for the formation of mature 3'-ends of mRNAs by cleavage and polyadenylation, and the coupling of these reactions with transcription termination. Dissecting how transcription, 3'-end RNA processing and mRNA export are coupled at the molecular and structural level, as we propose to do, is an essential step to understanding gene expression.
The cleavage and polyadenylation apparatus is a complex macromolecular assembly, consisting of more than 50 proteins. In the first phase of the project, we propose to study individual protein-protein and protein-RNA complexes to understand the structural basis for the definition of the processing site and for the initiation of transcription termination. In the later stages, we will study how individual proteins become integrated into multiprotein assemblies by using advanced NMR methods (e.g. TROSY) to study protein components of this macromolecular machine.
The specific aims for the first years of the project are:
• Determine how RNA-binding proteins within the 3'-end processing complex are arranged on the pre-mRNA to achieve specific recognition of polyadenylation sites and initiate transcription termination. We propose to study the structural basis for RNA recognition by the 3'-end processing factor Rna15 and Hrp1 and by Npl3.
• Study the biochemical and structural basis interaction of the highly conserved C-terminal domain of Rna15 with Pcf11, a protein that is essential for both transcription termination and 3'-end processing.
• Study how different 3'-end processing and transcription termination factors are recruited to the C-terminal domain (CTD) of the polymerase as the enzyme reaches the 3'-end of genes. We will determine how transcription termination factors Rtt103 and Pcf11 interact with different phosphorylated forms of the polymerase CTD.
Project Details
Project type
Large-Scale EMSL Research
Start Date
2007-05-23
End Date
2010-09-30
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members
Related Publications
Leeper TC, X Qu, C Lu, C Moore, and G Varani. 2010. "Novel Protein-Protein Contacts Facilitate mRNA 3'-Processing Signal Recognition by Rna15 and Hrp1." Journal of Molecular Biology 401(3):334-49.
Simultaneous recognition of HIV-1 TAR RNA bulge and loop sequences by cyclic peptide mimics of Tat protein. Davidson A, TC Leeper, Z Athanassiou, K Patora-Komisarka, J Karn, JA Robinson, G Varani. doi:10.1073/pnas.0900629106. PNAS (2009) 106:29 11931-11936
The Box H/ACA snoRNP Assembly Factor Shq1p is a Chaperone Protein Homologous to Hsp90
Cochaperones that Binds to the Cbf5p Enzyme
Katherine S. Godin, Hélène Walbott, Nicolas Leulliot, Herman van Tilbeurgh and Gabriele Varani
doi:10.1016/j.jmb.2009.04.076 J. Mol. Biol. (2009) 390, 231–244