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

PhD Topics

1. Structal 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 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.

Recommended literature:

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

Requirements:
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.

 

2. Determination of structural mechanism of coupled viral transcription and host translation in virus infected mammalian cells

Supervisor: Gabriel Demo, Ph.D.

Annotation:

Whereas transcription and translation of cellular mRNAs are physically separated in mammalian cells, cytoplasmic viruses transcribe and translate their mRNAs in cytoplasmic viral factories in infected cells. Thus, transcription and translation of viral mRNAs might be directly coupled in vivo. For example, large double-strand DNA (dsDNA) viruses—i.e., poxviruses (e.g., vaccinia virus; VACV)—coordinate viral genome replication, transcription, translation and viral assembly within the viral transcription or replication factories in the cytoplasm of the host cells. The viral RNA polymerases (RNAPs) transcribe early mRNAs within the original viral core, but intermediate and late mRNAs are made within viral factories where they closely associate with host translation initiation and elongation factors and ribosomes. Taking into account the co-localization of host ribosomes with viral mRNAs in viral factories, the viruses are allowed to tightly coordinate viral genome replication, transcription, translation, and viral assembly to maximize the efficiency of the infection.

To reveal how dsDNA viruses couple transcription and translation of viral mRNAs in vivo, the presence of ribosomes at the periphery of viral factories in infected human (commercial HeLa and HEK-293) cells will be determined by immunofluorescence microscopy. To visualize ribosomes in viral factories in vivo at sub-nanometer resolution, correlative light (LM) microscopy will be combined with cryo-electron tomography (cryo-ET). In this case, correlative LM will facilitate the search for features of interest (i.e., viral RNAPs or host ribosomes).

To facilitate detection of viral RNAPs or host ribosomes by fluorescence microscopy, viruses or cells will be engineered to express ribosomes or viral RNAP fused to a miniSOG reporter. To study the coupled transcription-translation machinery in near native state, focused ion beam milling will be used to generate well-defined ultra-thin sections of infected mammalian cells. The ultra-thin sections of infected mammalian cells will be vitrified to assure that features of interest remain identical during both LM and EM imaging.

In the long term, the aim is to reconstitute the supramolecular complexes in vitro and visualize them at high resolution by single-particle cryo-EM. Firstly, to identify all players (novel factors) in coupled transcription-translation of viral mRNAs, a simple and rapid method of isolating the viral RNAP and associated proteins or protein complexes from infected cells would be developed to facilitate biochemical and structural investigations.

Detailed structural information will bring crucial insights into the mechanisms of viral pathogenesis and identify additional factors involved in coupling of viral transcription and host translation.

Recommended literature:

S. S. Broyles, Vaccinia virus transcription. J Gen Virol 84, 2293-2303 (2003).

B. Schramm, J. K. Locker, Cytoplasmic organization of POXvirus DNA replication. Traffic 6, 839-846 (2005).

J. A. den Boon, et al., Cytoplasmic viral replication complexes. Cell Host Microbe 8, 77-85 (2010).

M. Jublot, M. Texier, Sample preparation by focused ion beam micromachining for transmission electron microscopy imaging in front-view. Micron 56, 63-67 (2014).

P. de Boer, et al., Correlated light and electron microscopy: ultrastructure lights up! Nat Methods 12, 503-513 (2015).

Keywords: Poxviruses, viral RNA polymerase, ribosome, coupling, cryo-ET

Requirements:
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.