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Nat Commun. 2018 Nov 30;9(1):5087. doi: 10.1038/s41467-018-07423-3.

Real-time assembly of ribonucleoprotein complexes on nascent RNA transcripts.

Author information

1
Department of Integrative Structural and Computational Biology, Department of Chemistry, and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA, 92037, USA.
2
Department of Structural Biology, Stanford University School of Medicine, CA, 94305, California, USA.
3
Department of Research and Development, Pacific Biosciences Inc, Menlo Park, CA, 94025, USA.
4
Department of Biochemistry, University of California, Riverside, CA, 92521, USA.
5
Department of Structural Biology, Stanford University School of Medicine, CA, 94305, California, USA. puglisi@stanford.edu.
6
Department of Integrative Structural and Computational Biology, Department of Chemistry, and The Skaggs Institute for Chemical Biology, The Scripps Research Institute, La Jolla, CA, 92037, USA. jrwill@scripps.edu.

Abstract

Cellular protein-RNA complexes assemble on nascent transcripts, but methods to observe transcription and protein binding in real time and at physiological concentrations are not available. Here, we report a single-molecule approach based on zero-mode waveguides that simultaneously tracks transcription progress and the binding of ribosomal protein S15 to nascent RNA transcripts during early ribosome biogenesis. We observe stable binding of S15 to single RNAs immediately after transcription for the majority of the transcripts at 35 °C but for less than half at 20 °C. The remaining transcripts exhibit either rapid and transient binding or are unable to bind S15, likely due to RNA misfolding. Our work establishes the foundation for studying transcription and its coupled co-transcriptional processes, including RNA folding, ligand binding, and enzymatic activity such as in coupling of transcription to splicing, ribosome assembly or translation.

PMID:
30504830
PMCID:
PMC6269517
DOI:
10.1038/s41467-018-07423-3
[Indexed for MEDLINE]
Free PMC Article

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