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Structural And Biochemical Studies On Rna Polymerase Elongation Complexes Bound To Universally Conserved Transcription Factor Nusg

Reference : PhD Albert WEIXLBAUMER

Publication de l'offre : 6 avril 2016

In order to convert genotype to phenotype every cell needs to retrieve the information stored in DNA. The first step, where DNA is copied into RNA, is called transcription. The central enzyme is a multi-subunit protein called RNA polymerase (RNAP). The core architecture of RNAP is universally conserved and thus the basic mechanisms underlying transcription are conserved from bacteria to humans. Due to its fundamental role, transcription
affects essentially every aspects of biology and a large number of regulatory processes target RNAP. Misregulation of transcription has severe effects and is implied in a growing list of human diseases. Thus, a detailed and mechanistic understanding of this vital process has important biological and
biomedical implications and requires a combination of biochemistry and structural biology. Transcriptional pausing plays an important regulatory role, but is also involved in termination and fidelity of transcription, making it particularly
interesting. In addition, it becomes increasingly clear that transcriptional pausing is critical in higher eukaryotes to regulate development and cell differentiation. Our lab is interested in transcription factors that modulate transcriptional pauses.

Only one transcription factor, called NusG in bacteria, is universally conserved. The eukaryotic homologue (Spt5 - it forms a heterodimer with Spt4 together known as DSIF in higher organisms) plays important roles in a process called promoter proximal pausing, which is involved in regulating development and cell differentiation. NusG has two essential functions: i) it reduces pausing, and increases elongation rates (in all kingdoms of life), and ii) it enhances transcription termination mediated by termination factor Rho in bacteria. Structural information in the context of RNAP is limited to archaeal complexes: a low resolution EM model of an RNAP Spt4/5 complex, and a crystal structure of Spt4/5 bound to a fragment of RNAP. These results are important but do not provide an atomic model of the entire complex and, more importantly, were not obtained with a canonical elongation complex (actively transcribing RNAP).

This is where the proposed work will contribute and several fundamental questions will be addressed:
i) How can NusG modulate processivity and translocation of RNAP?
ii) How is termination factor Rho recruited to the complex?
iii) How do NusG homologues (like E. coli RfaH) exert their functionally important sequence-specific interaction with DNA.
In addition, because of the high degree of conservation, information gained on NusG is highly relevant for our understanding of the basic transcription machinery in Archaea and Eukaryotes. For example, DSIF is part of the promoter-proximal paused RNAP II complex and stays associated with the
elongating form of RNAP II after release. Finally, NusG plays a role in rRNA transcription antitermination and could thus proof to be a valuable biomedical target.

First, functional complexes of bacterial RNAP from various species bound to DNA, RNA and NusG will be biochemically characterized in vitro. Considerable groundwork has been done in the host lab: RNAP and NusG from three species can be purified to homogeneity. Expression constructs
for proteins required for biochemical assays have been made and tested. Functional complexes can be formed as evident by RNA extension and gel-shift assays. We are currently setting up a biochemical assay to confirm NusG’s effect on pausing, elongation rates and termination following published methods.
Once the assays confirm functional complex formation, we will proceed with structural investigations. The prospective student will use a combination of biochemistry, X-ray crystallography and single particle cryo-EM to obtain detailed insights. The host institute is well equipped for this type of work and the thesis adviser has more than 10 years of experience with this approach.

- COMPETENCES SOUHAITEES : Students with a background in biology, biochemistry, chemistry, or biophysics are welcome to apply. The ideal candidate has prior experience in molecular biology (primer and protein expression construct design, PCR, restriction cloning, …) and biochemistry (protein expression and purification, biochemical assays, …) and shows a keen interest in the workings of molecular machines. Prior experience with
structural biology techniques and nucleic acid biochemistry is an added plus but not a requirement.

- EXPERTISES QUI SERONT ACQUISES AU COURS DE LA FORMATION : Students will learn standard molecular biology techniques and how to express and purify large, multi-subunit protein complexes using a variety of chromatographic techniques (affinity, ion-exchange, size-exclusion, hydrophobic interaction). Functional complexes (including DNA and RNA) will then be formed and a variety of biochemical and biophysical assays will
be used to characterize them (transcription assays, gel-shifts, pull-downs, radioactive assays, …). Structural biology techniques (X-ray crystallography, single particle cryo-EM, SAXS) will then be used to gain mechanistic insights.

Votre candidature

Date limite de candidature : 31 décembre 2016

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