Hinge action versus grip in translocation by RNA polymerase.

Area of Science:

• Molecular Biology
• Biophysics
• Structural Biology

Background:

• RNA polymerase (RNAP) is crucial for gene transcription, involving complex translocation steps.
• Understanding RNAP translocation mechanism is key to deciphering gene expression regulation.

Purpose of the Study:

• To elucidate the conformational mechanism of forward translocation by RNA polymerase.
• To investigate the role of specific protein domains and environmental factors in RNAP movement.

Main Methods:

• Molecular dynamics simulations of RNAP ternary elongation complex.
• Functional studies using exonuclease III assay to monitor RNAP translocation.
• Biochemical assays monitoring the effect of ions and RNA 3'-OH/H2 on RNAP sliding.
• Mutational analysis of the bridge helix in RNAP.

Main Results:

• Simulations revealed clamp closure and hinge-driven force generation, enabling RNAP sliding.
• The β flap tip helix and β' Zn finger engage RNA, guiding its exit.
• RNAP active site is coupled to the RNA exit channel and translocation.
• K+, Mg2+, and RNA 3'-OH/H2 significantly affect RNAP sliding, consistent with a sticky translocation mechanism.
• Mutational analysis identified a homeostatic hinge in the bridge helix crucial for conformational dynamics.

Conclusions:

• A conformational mechanism for RNAP forward translocation is proposed, involving coordinated movements of protein domains.
• Environmental factors like ions and RNA structure play critical roles in regulating RNAP translocation.
• The identified bridge helix hinge is essential for managing conformational changes during transcription.