1. Structural studies of various states of direct and bridged transcription-translation coupling in vitro and in vivo
Supervisor: Gabriel Demo, Ph.D.
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 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 the 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.
R. Kohler et al., Architecture of a transcribing-translating expressome. Science 356, 194-197 (2017).
G. Demo et al., Structure of RNA polymerase bound to ribosomal 30S subunit. eLife 6, (2017).
M. W. Webster et al., Structural basis of transcription-translation coupling and collision in bacteria. Science 369, 1355-1359 (2020).
C. Wang et al., Structural basis of transcription-translation coupling. Science 369, 1359-1365 (2020).
F. J. O'Reilly et al., In-cell architecture of an actively transcribing-translating expressome. Science 369, 554-557 (2020).
Keywords: RNA polymerase, ribosome, coupling, structure, cryo-EM, cryo-ET
PLEASE NOTE: before initiating the formal application process to doctoral studies, all interested candidates are required to contact Gabriel Demo (firstname.lastname@example.org) for an informal discussion.