In vivo fluorescent tracking system of coupled transcription-translation
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| Year of publication | 2021 |
| Type | Conference abstract |
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| Description | The bacterial cellular space is not divided into sections by a membrane thus the ribosomes could initiate translation on nascent mRNA which is still transcribed by RNA polymerase (RNAP). The occurrence of coupled transcription-translation (CTT) in bacteria is broadly accepted due to in vitro structures describing CTT complexes. However, any in vivo evidence of CTTs in bacterial cells using live-cell imaging approaches is missing. Recent in vivo single-particle fluorescent studies focused on spatial organization of nucleoid in E. coli do investigate the localization of RNAP and ribosomes separately in a cell. Moreover, almost all fluorescently labelled ribosomal proteins in these studies, play a critical role in CTT (according to in vitro structures), making it impossible to track fully assembled CTTs. Here, we present a design of in vivo fluorescent system to monitor CTT in E. coli. For labelling, beta prime protein from RNAP, ribosomal proteins S20 and S16 from 30S small ribosomal subunit, and L9 and L17 from 50S large ribosomal subunit were selected as they do not interfere with CTT. Several sets of bacterial strains have been prepared with fused target proteins and fluorescent proteins (mCerulean, mVenus, mRFP1) by lamba-Red recombination in their endogenous loci. Confocal microscopy in combination with high-resolution fluorescence microscopy is used to look at the dynamics of this complex system in single cell. Due to CTT involvement in stress response, migration of ribosomal subunits and RNAP together with CTT occurrence is monitored in different stress conditions (heat- or cold-shock, oxidative stress, etc.) |
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