The forward translocation position of Escherichia coli RNA polymerase is stabilized by KCl and Mg²⁺ and is strongly dependent on a bridge helix N‐terminal hinge beta’ 778‐GARKGL‐783 (944.2)

Abstract

Exonuclease (exo) III was used to probe the Escherichia coli RNA polymerase (RNAP) ternary elongation complex (TEC) downstream and upstream borders. In the absence of NTPs, RNAP appears to stall primarily in a post‐translocated state and to return slowly to a pre‐translocated state. Exo III mapping, therefore, appears inconsistent with an unrestrained thermal ratchet model for translocation. The forward translocation state is made more stable by elevating the KCl and Mg2+ concentrations into the physiological range (150 versus 40 mM KCl; 5 versus 0.5 mM Mg2+), indicating that, under natural conditions, translocation tends to stick in the post‐translocated position. RNAP mutants in the N‐terminal bridge helix hinge beta’ 778‐GARKGL‐783 strongly affect TEC translocation, NTP binding and stability. Mutations that stiffen the hinge (i.e. GARKGL‐>AARKAL) are strongly defective for holding the forward translocation register and for stable NTP binding but do not destabilize the TEC. Reminiscent of a ball and socket joint, the bulky R780 is surrounded by the beta fork, when the bridge helix is in a relatively straight conformation. An overly flexible hinge (i.e. K781A), however, may result in misaligning R780 within the fork resulting in low transcriptional activity. Molecular dynamics simulation indicates that the GARKGL hinge can completely unwind, but only if the beta’ trigger loop is in an open conformation. Grant Funding Source: NSF and NIH