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

, Yi-Ren Chang

Institute of Physics, Academia Sinica, Taipei, Taiwan,

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.