Lineage-specific variations in the trigger loop modulate RNA proofreading by bacterial RNA polymerases

Area of Science:

• Molecular Biology
• Biochemistry
• Microbiology

Background:

• Bacterial RNA polymerase (RNAP) performs both nucleotide addition and RNA cleavage within the same active site.
• The molecular mechanism of RNAP RNA cleavage is less understood than nucleotide addition.
• RNAP from Deinococcus radiodurans exhibits higher intrinsic RNA cleavage activity than E. coli RNAP.

Purpose of the Study:

• To investigate the molecular basis for enhanced RNA cleavage activity in Deinococcus radiodurans RNAP.
• To understand the role of the trigger loop (TL) in modulating RNAP RNA cleavage.
• To explore the implications of altered RNA cleavage for bacterial stress adaptation.

Main Methods:

• Comparative analysis of RNAP from Deinococcus radiodurans and Escherichia coli.
• Site-directed mutagenesis to study the role of trigger loop (TL) amino acid substitutions.
• Biochemical assays to measure RNA cleavage activity.
• Investigating the effect of cleavage factors like GreA.

Main Results:

• Amino acid substitutions in the trigger loop (TL) are primarily responsible for the enhanced RNA cleavage activity in Deinococcus radiodurans RNAP.
• Deletion of the TL or the presence of GreA cleavage factors abolished the differences in RNA cleavage activity between the two RNAPs.
• TL substitutions appear to modulate RNA cleavage by altering TL folding and its interaction with the substrate RNA.

Conclusions:

• The trigger loop (TL) plays a critical role in regulating the intrinsic RNA cleavage activity of bacterial RNA polymerase (RNAP).
• Differences in TL structure and function contribute to variations in transcriptional proofreading efficiency among bacterial species.
• Enhanced RNA cleavage mediated by TL modifications may be a key factor in bacterial adaptation to stressful environments.