E. coli RNA Polymerase Determinants of Open Complex Lifetime and Structure

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Transcription initiation in Escherichia coli involves RNA polymerase (RNAP) binding to promoter DNA, causing conformational changes.
• Initial binding leads to DNA bending and opening, forming a transient open complex (OC).
• The lifetime of these OCs is a critical regulatory target.

Purpose of the Study:

• To investigate the late conformational changes affecting OC stability.
• To determine the role of RNAP mobile elements (σ1.1, β' jaw, β' SI3) in OC dissociation kinetics.
• To understand how these elements regulate OC lifetime for different promoters (λPR, T7A1).

Main Methods:

• Analysis of OC dissociation kinetics using deletion variants of RNAP mobile elements (σ1.1, β' jaw, β' SI3).
• Comparison of OC lifetimes for wild-type and mutant RNAP with λPR and T7A1 promoters.
• Characterization of DNA binding within the RNAP cleft and jaw regions.

Main Results:

• Stable OCs involve downstream duplex DNA bound to the β' jaw and SI3, and the discriminator region bound in a positively charged cleft track.
• The σ1.1 element destabilizes OCs by competing for binding sites with DNA elements.
• Deleting σ1.1, β' jaw, or SI3 equalizes OC lifetimes for both λPR and T7A1 promoters, while DNA closing rates remain constant.

Conclusions:

• Late conformational changes stabilizing OCs are primary regulatory targets, similar to early DNA bending events.
• The intrinsic DNA opening/closing step is not a major target for regulation.
• RNAP mobile elements play crucial roles in modulating OC stability and promoter specificity.