Regulation of Translation – Gabriel Demo

In Bacteria and Archaea, newly transcribed RNAs are immediately bound by ribosomes, thus coupling transcription to translation. Indeed, recent cryo-electron microscopy (cryo-EM) studies report that RNA polymerase (RNAP) and ribosome may interact directly. This contrasts with eukaryotic transcription and translation which are physically separated by the nuclear envelope. Nevertheless, some double-strand DNA viruses replicate in cytoplasmic factories of infected eukaryotic cells, raising the possibility that viral transcription might be directly coupled to translation by host ribosomes in vivo.

To probe the mechanistic details and functional outcomes of coupled transcription-translation, we use an integrative approach that combines proteomics, molecular biology, biochemistry and structural biology. We aim to characterize transcription-translation coupling in E. coli and virus-infected cells in the context of ribosome interactions with RNAP, DNA, mRNA, and transcription or translation factors. The fundamental strength of our approach lies in the use of cryo-EM and cryo-electron tomography (cryo-ET) that will enable us to characterize various points of transcriptional-translational apparatus in bacteria and virus infected mammalian cells from the atomic resolution of individually reconstituted macromolecules in vitro (cryo-EM) to the supramolecular complexes in vivo (cryo-ET) within thin sections of bacterial or infected cells in near native conditions.

Little is yet known about crosstalk between nuclear transcription and cytoplasmic translation of mRNA in eukaryotes. Yet, in response to stress—e.g., glucose starvation or heat shock—some RNAP II subunits and eukaryotic translation factors act as mRNA coordinators that indirectly link transcription to translation. The biochemical and structural studies of transcription-translation coupling in bacteria may identify analogous stress responses in eukaryotes and point to mechanisms that challenge the long-standing dogma that eukaryotic transcription and translation are functionally separate processes.

The research of coupled transcription-translation will therefore not only significantly improve the understanding of gene regulation in bacterial and virus-infected mammalian cells, but also have useful clinical implications. For example, pharmacological approaches could be developed to treat dysregulated heat shock response, which is associated with numerous pathologies ranging from cancer to neurodegenerative conditions.

Identify factors required to couple transcription and translation in bacteria.

Biochemically identify components of RNAP•ribosome complexes—including transcription or translation factors—and investigate how they function in transcription-translation coupling. Additionally, determine if and how coupled transcription-translation depends on mRNA structure or cellular response to stress.

Visualize various states of transcription-translation coupling in vitro and in vivo.

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 the near-nucleoid space. These studies will help 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.

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-nanometer 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.