Real-time assembly of ribonucleoprotein complexes on nascent RNA transcripts

Area of Science:

• Molecular Biology
• Biophysics
• Genetics

Background:

• Cellular protein-RNA complexes form on nascent transcripts.
• Observing real-time transcription and protein binding at physiological concentrations is challenging.
• Early ribosome biogenesis involves complex protein-RNA interactions.

Purpose of the Study:

• To develop a method for simultaneous real-time observation of transcription and protein binding.
• To investigate the co-transcriptional binding of ribosomal protein S15 to nascent RNA.
• To understand the influence of temperature and RNA folding on protein binding during transcription.

Main Methods:

• Utilized a single-molecule approach employing zero-mode waveguides.
• Simultaneously tracked transcription progress and ribosomal protein S15 binding to nascent RNA.
• Conducted experiments at physiological temperatures (35°C) and reduced temperatures (20°C).

Main Results:

• Observed stable binding of ribosomal protein S15 to the majority of single nascent RNAs at 35°C.
• Observed stable binding to less than half of nascent RNAs at 20°C.
• Identified transient binding or complete lack of binding in some transcripts, potentially due to RNA misfolding.

Conclusions:

• Established a foundational method for studying transcription coupled with co-transcriptional processes.
• Demonstrated temperature-dependent binding of ribosomal protein S15 to nascent RNA.
• Highlighted the role of RNA folding in co-transcriptional protein binding and complex formation.