Structural Biology of Coupled Transcription-Translation - Gabriel Demo
CEITEC MU CEITEC MU
Structural Biology of Coupled Transcription-Translation - Gabriel Demo

PhD Topics

1. Structural studies of various states of direct and bridged transcription-translation coupling in vitro and in vivo

Supervisor: Gabriel Demo,Ph.D.

Annotation:
Recent 30S subunit-RNA polymerase (RNAP) and expressome structures may represent interactions that occur at different steps of translation, e.g., during initiation and elongation. The aim is to structurally define the step-wise states of bacterial transcription-translation coupling and to determine whether accessory transcription factors or other proteins that link transcription and translation help to relocate RNAP on the ribosome - e.g., from its position in the 30S-RNAP structure to its position in the expressome structure.
Single-particle cryo-electron microscopy (cryo-EM) will be used to visualize transcription-translation coupling in vitro. Cryo-EM particle images from heterogeneous samples (in vitro reconstituted) will be classified into structurally homogeneous subsets by maximum-likelihood (ML) analyses. Packages such as Frealign and RELION with incorporated ML principles and extensive 3D-classification procedures will be used to resolve conformational heterogeneity.
Free 30S and 50S subunits mix with the nucleoid where they initiate co-transcriptional translation. Cellular co-localization of ribosomal subunits with RNAP protects the nascent mRNA and prevents undesirable backtracking of RNAP. The aim is to determine how is the coupling initiated within the nucleoid space and if the pioneer round of translation occurs within or near nucleoid space before the formation of polysomes that segregate from the nucleoid.
The RNAP-ribosome complexes will be visualized in situ at sub-nanometer resolution using cryo-electron tomography and sub-tomographic averaging of 3D volumes. E. coli cells will be flash-frozen directly onto EM grids allowing cellular structures to be studied in their near-native states. Ultra-thin sections will be prepared directly on the EM grid in the electron microscope by focused ion beam milling.
These studies can provide insight on how translating ribosomes preserve genome integrity by preventing RNAP from backtracking or pausing. This combined approach can convincingly show in vivo the transcriptional-translational apparatus in action with all players involved in the coupling mechanism.

Literature:

  1. R. Kohler, R. A. Mooney, D. J. Mills, R. Landick, P. Cramer, Architecture of a transcribing-translating expressome. Science 356, 194-197 (2017).
  2. G. Demo et al., Structure of RNA polymerase bound to ribosomal 30S subunit. eLife 6,(2017).
  3. Sanamrad et al., Single-particle tracking reveals that free ribosomal subunits are not excluded from the Escherichia coli nucleoid. Proc Natl Acad Sci U S A 111, 11413-11418 (2014).
  4. K. McGary, E. Nudler, RNA polymerase and the ribosome: the close relationship. Curr Opin Microbiol 16, 112-117 (2013).
  5. M. Jublot, M. Texier, Sample preparation by focused ion beam micromachining for transmission electron microscopy imaging in front-view. Micron 56, 63-67 (2014).

Keywords:
RNA polymerase, ribosome, coupling, structure, cryo-EM, cryo-ET

Requirements:
We are seeking a Ph.D. candidate who was trained in structural biology, mainly single-particle cryo-EM or cryo-electron tomography (cryo-ET), and is a motivated person with collaborative mindset. The candidate should be able to purify the components for structural studies of transcription-translation coupling.
PLEASE NOTE: before initiating the formal application process to doctoral studies, all interested candidates are required to contact Gabriel Demo (gabriel.demo@ceitec.muni.cz) for informal discussion.

 

2. Structural and functional identification of factors required to couple transcription and translation in bacteria

Supervisor: Gabriel Demo,Ph.D.

Annotation:
Whether specific cellular components—i.e., transcription, translation, or yet unidentified factors—are dedicated to transcription-translation coupling remains unknown. However, observations that E. coli transcription factors NusG and RfaH can interact with RNA polymerase (RNAP) and ribosome indicate that the transcriptional-translational coupling might be operon-dependent. Moreover, the involvement of ribosomal protein S1 in the transcription-translation coupling suggests that the transcriptional-translational apparatus can function on specific mRNAs. Thus, in each specific case the picture of transcriptional-translational coupling is incomplete.
The research involves the purification of components of transcriptional and translational machineries from E. coli and their reconstitution on specific DNA-RNA duplexes at different stages of transcription and translation To biochemically assess the roles of transcription or translation factors in coupled transcription-translation an in vitro experimental system will be used. To monitor the mRNA engagement in coupled transcription-translation, RelE printing (toe-printing) will be performed. RNA can be labelled by the incorporation of radiolabelled nucleotides during initial RNA N-mer synthesis. To monitor the protein synthesis and ribosome translocation, standard peptidyl transferase assays by including [35S]-labeled initiator fMet-tRNAfMet will be used.
Single-particle cryo-EM will be employed to determine high-resolution structures of RNAP-ribosome complexes assembled as described above. Atomic models of individual proteins and subunits determined by X-ray crystallography and single-particle cryo-EM will be fitted into electron density maps of the complexes obtained by single-particle cryo-EM.
To characterize cellular transcription-translation complexes and identify novel factors involved in the coupling, complexes will be immunoprecipitated from bacteria. Mass-spectrometry will be performed to identify proteins involved in coupling and RNA sequencing to identify mRNAs undergoing coupled transcription-translation.
Coupling RNAP to ribosome will help to understand how coupled transcription-translation is initiated, whether it is factor-dependent, and what mechanism is required to progress to translation elongation.

Literature:

  1. K. McGary, E. Nudler, RNA polymerase and the ribosome: the close relationship. Curr Opin Microbiol 16, 112-117 (2013).
  2. D. Castro-Roa, N. Zenkin, In vitro experimental system for analysis of transcription-translation coupling. Nucleic Acids Res 40, e45 (2012).
  3. B. M. Burmann et al., An alpha helix to beta barrel domain switch transforms the transcription factor RfaH into a translation factor. Cell 150, 291-303 (2012).
  4. B. M. Burmann et al., A NusE:NusG complex links transcription and translation. Science 328, 501-504 (2010)
  5. M. V. Sukhodolets, S. Garges, S. Adhya, Ribosomal protein S1 promotes transcriptional cycling. RNA 12, 1505-1513 (2006)

Keywords:
RNA polymerase, ribosome, coupling, toe-printing, in vitro assay, cryo-EM

Requirements:
We are seeking a Ph.D. candidate who holds MSc. in molecular biology or biochemistry and is a motivated person with collaborative mindset. The candidate should to be able to purify the components for transcription-translation coupling and run the biochemical assays. We will train the candidate in the field of cryo-EM and assist with cryo-EM data collection and structure determination.
PLEASE NOTE: before initiating the formal application process to doctoral studies, all interested candidates are required to contact Gabriel Demo (gabriel.demo@ceitec.muni.cz) for an informal discussion.