Stringent control of the RNA-dependent RNA polymerase translocation revealed by multiple intermediate structures

Area of Science:

• Biochemistry
• Structural Biology
• Molecular Biology

Background:

• Polymerase activity involves nucleotide addition cycles with distinct conformational changes.
• Active site closure is understood, but translocation intermediates remain poorly characterized.
• RNA-dependent RNA polymerase (RdRP) is a key enzyme in RNA replication and related processes.

Purpose of the Study:

• To elucidate the structural mechanisms of translocation intermediates in RNA-dependent RNA polymerase (RdRP).
• To investigate the role of RdRP-unique motif G in regulating RNA template movement during translocation.
• To identify and characterize novel intermediate states mimicking the transition state of translocation.

Main Methods:

• X-ray crystallography to capture intermediate structures of RdRP.
• Site-directed mutagenesis of critical residues within motif G.
• Analysis of structural changes associated with forward and reverse translocation events.

Main Results:

• Three distinct types of translocation intermediate structures were captured.
• Motif G was identified as a critical element restricting RNA template movement, linked to the rate-limiting step of translocation.
• Mutations in motif G yielded intermediates potentially mimicking the transition state, revealing a novel template strand movement.

Conclusions:

• RdRP utilizes motif G to precisely control template positioning during translocation, ensuring efficient catalysis.
• The findings provide insights into the conserved mechanisms of nucleic acid polymerases.
• This work advances the understanding of polymerase translocation and its regulation.