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.
Highlights
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
Transcription by E. coli RNA polymerase (RNAP) was initiated with nucleotide triphosphates (NTPs), omitting GTP, resulting in a complex with RNAP stalled at the first guanine nucleotide
To compare protein binding to a pre-transcribed and pre-folded RNA with binding to a nascent RNA emerging from the RNAP, we investigated the interaction of the r-protein S15 with the 3-helix junction composed of helices 20, 21, and 22 in the central domain of the E. coli 16 S ribosomal RNA (rRNA) (Fig. 5a, b)
Summary
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. Zero-mode waveguide (ZMW) fluorescence microscope technology allows single-molecule real-time dynamics of complex biological systems to be delineated at physiological ligand concentrations for thousands of single biomolecules simultaneously through four spectral channels[19,20]. This technology has been exploited for DNA sequencing[21], for studying translation by the ribosome[15,16] and for other applications[22]. We have developed assays that allow us to monitor simultaneously the progress and rate of transcription, the formation of full-length RNA transcripts, timing of transcriptional pausing at terminators, release of the DNA template, and specific binding of proteins at > 100 nM concentration and on hundreds to thousands of single rRNAs in parallel during a single experiment
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