Closing and opening of the RNA polymerase trigger loop

Area of Science:

• Molecular Biology
• Biochemistry
• Structural Biology

Background:

• The RNA polymerase (RNAP) trigger loop (TL) is a mobile element within the RNAP active center.
• Crystal structures suggest the TL cycles between open and closed states to regulate nucleotide substrate binding.

Purpose of the Study:

• To detect and characterize the dynamic closing and opening of the RNAP TL in solution.
• To understand the functional role of TL dynamics in transcription fidelity and regulation.

Main Methods:

• Utilized single-molecule fluorescence resonance energy transfer (smFRET).
• Employed fluorescent probes attached to the TL and other RNAP or DNA components.

Main Results:

• Quantified TL closing and opening occurring on the millisecond timescale.
• Demonstrated TL dynamics serve as a checkpoint for NTP complementarity, ribo/deoxyribo identity, and triphosphate identity.
• Showed TL closing and opening is a target for inhibitors.
• Observed one TL cycle per nucleotide addition during transcription elongation.

Conclusions:

• The dynamic cycle of the RNAP TL is essential for accurate nucleotide selection during transcription.
• TL dynamics represent a critical regulatory mechanism and a potential target for therapeutic intervention.