Sequence-resolved detection of pausing by single RNA polymerase molecules.

Area of Science:

• Molecular Biology
• Biophysics

Background:

• Transcriptional pausing by RNA polymerase (RNAP) is a key regulatory mechanism in gene expression.
• Sequence-specific pause sites are known, but the nature of frequent, ubiquitous pauses during elongation remains unclear.

Purpose of the Study:

• To investigate the relationship between ubiquitous transcriptional pauses and DNA sequence.
• To characterize the properties of both known and ubiquitous RNAP pauses.

Main Methods:

• Utilized an ultrastable optical-trapping assay to monitor individual RNAP molecules.
• Employed engineered DNA templates with repeated sequences and known pause sites.

Main Results:

• Identified both known and ubiquitous pauses at precise, reproducible locations with base-pair accuracy.
• Found that ubiquitous pauses correlate with DNA sequences similar to regulatory pause sites.
• Pause lifetimes and efficiencies support a model where ubiquitous pauses represent a common intermediate state branching from the elongation pathway.

Conclusions:

• Ubiquitous transcriptional pauses are sequence-specific and linked to regulatory elements.
• A unified model explains RNAP pausing, involving a common elemental state for both known and ubiquitous pauses.