In this proposed Junior MASH proposal I will determine the mechanistic details and functional outcomes of coupled transcription-translation in bacteria and virus-infected mammalian cells using an interdisciplinary approach.

Results

Objective 1 - Identify factors required to couple transcription and translation in bacteria. We will biochemically identify components of RNAP•ribosome complexes—including transcription or translation factors—and investigate how they function in transcription-translation coupling. We will also determine if and how coupled transcription-translation depends on mRNA structure.
Objective 2 - Visualize various states of transcription-translation coupling in vitro and in vivo. We will use single-particle cryo-EM to determine high resolution structures of in vitro RNAP•ribosome complexes at multiple states of coupled transcription-translation: from translation initiation to elongation. The structures of in vitro complexes will be compared to in vivo complexes visualized by cryo-electron tomography (cryo-ET) imaging of the near-nucleoid space. These studies will help us understand how translating ribosomes preserve genome integrity by preventing RNAP from backtracking or pausing in bacteria, and whether an initial round of translation in the nucleoid guarantees the message for steady translation in cytoplasm.
Objective 3 - Investigate whether and how dsDNA viruses couple transcription and translation in mammalian cells. We will use cryo-ET of infected cells and apply sub-tomographic averaging to visualize viral RNAP and ribosomes at sub-nanometre resolution and determine whether and how dsDNA viruses including poxviruses and asfarvirus couple transcription and translation in viral factories. Detailed structural information will bring crucial insights into the mechanisms of viral pathogenesis and identify additional factors involved in coupling.

Sustainable Development Goals

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