Single-Cell Biophysics: Measurement, Modulation, and Modeling

Single-Cell Biophysics: Measurement, Modulation, and Modeling

Poster Abstracts

11-POS Board 6 The Correlation between Bacterial Transcription and Translation at Single Molecule Level in Living Cells Ssu Ying Chen 1,2 , Chia-Fu Chou 1 , Yi-Ren Chang 2 . 1 Institute of Physics, Academia Sinica, Taipei, Taiwan, 2 Department of Physics, national Taiwan normal University, Taipei, Taiwan. Although most or all transcription is co-translational in bacteria, ~ 10-15% of translation is co- transcriptional. Since the spatial distribution of ribosomes displays strongly segregated from the nucleoid, 30s/50s ribosomal subunits need a circulation between ribosome-rich region and the nucleoid. It’s been suggested transertion enables expansion of the nucleoid and 30s/50s subunits moving into co-transcriptional translation region. To clarify if transertion plays a role in co- transcriptional translation, an in situ, time-resolved, single-molecule analysis would be beneficial in quantifying the syntheses of mRNA and polypeptide derived from a gene encoding a membrane protein in living cells. Hereby, such a real-time monitoring method was developed to investigate the spatiotemporal correlation between transcription and translation, and the role of transertion in co-transcriptional translation. We employed the labeling schemes of MS2 and SunTag to probe the syntheses of mRNA and polypeptides respectively, in which quantification is allowed by imaging the fluorescence emitted from the counter part of each tagging system, i.e., MCP-GFP and scFv-mCherry. Besides, exogenous, centromere-like DNA sequence parS was inserted to the flanking region of the gene encoding a specified membrane protein either in a single-copy plasmid or chromosome such that transcription events can be localized by imaging CFP-ParB fluorescence foci. In the present study, the events with co-localized fluorescence foci emitted from three tagging systems near the cell membrane that indicate the transertion were detected by TIRF microscopy and compared with other transcription and translation events. Our results demonstrate a feasible approach to probe the transertion events in living cells and further elucidate their spatiotemporal correlation in the process of co-transcriptional translation.

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