Role of the RNA polymerase trigger loop in catalysis and pausing

Area of Science:

• Molecular Biology
• Biochemistry
• Enzymology

Background:

• The trigger loop (TL) is a key RNA polymerase (RNAP) component involved in nucleotide addition.
• The folded alpha-helical hairpin (trigger helices, TH) is thought to be crucial for RNAP function.
• The precise roles of the TL/TH in transcript cleavage, catalysis, substrate selectivity, and pausing are not fully understood.

Purpose of the Study:

• To investigate the role of the trigger helices (TH) in transcript cleavage and nucleotide addition by RNA polymerase (RNAP).
• To determine the contribution of TH formation to catalysis, substrate selectivity, and pausing.
• To elucidate the mechanism by which the TL/TH influences RNAP activity.

Main Methods:

• In vitro assays using Escherichia coli RNAP with specific TL/TH alterations.
• Analysis of transcript cleavage, nucleotide addition, and pausing in response to TL/TH modifications.

Main Results:

• Transcript cleavage of backtracked RNA, both intrinsic and regulator-assisted, does not require TH formation.
• TH formation's main role in rapid nucleotidyl transfer is steric alignment of reactants, not acid-base catalysis.
• TL/TH alterations similarly affect pausing and nucleotide addition, suggesting TH formation is rate-limiting for escaping nonbacktracked pauses.

Conclusions:

• The trigger helices (TH) are not essential for transcript cleavage in RNA polymerase (RNAP).
• Steric alignment, rather than catalysis, is the primary function of TH formation in nucleotide addition.
• The TL/TH complex is not the sole determinant of substrate selectivity in RNAP.