Domain movements of the enhancer-dependent sigma factor drive DNA delivery into the RNA polymerase active site: insights from single molecule studies

Abstract

Recognition of bacterial promoters is regulated by two distinct classes of sequence-specific sigma factors, σ70 or σ54, that differ both in their primary sequence and in the requirement of the latter for activation via enhancer-bound upstream activators. The σ54 version controls gene expression in response to stress, often mediating pathogenicity. Its activator proteins are members of the AAA+ superfamily and use adenosine triphosphate (ATP) hydrolysis to remodel initially auto-inhibited holoenzyme promoter complexes. We have mapped this remodeling using single-molecule fluorescence spectroscopy. Initial remodeling is nucleotide-independent and driven by binding both ssDNA during promoter melting and activator. However, DNA loading into the RNA polymerase active site depends on co-operative ATP hydrolysis by the activator. Although the coupled promoter recognition and melting steps may be conserved between σ70 and σ54, the domain movements of the latter have evolved to require an activator ATPase.

The bacterial enhancer-dependent RNA polymerase

“…The structure, thus, suggests that promoter DNA entry into RNAP is blocked by RI–RIII of σ 54 and so must be re-organized by the bEBPs ATPase for DNA melting and/or the binding of melted out DNA to take place. This is consistent with FRET data showing that promoter DNA competes for the positioning of the RI relative to upstream and downstream promoter DNA ends [47]. …”

Dynamic action of the Sec machinery during initiation, protein translocation and termination revealed by single molecule fluorescence

“…PLs were immobilized on a glass supported lipid bilayer and imaged with a previously described TIRF set-up (Sharma et al, 2014) extended with msALEX illumination (Kapanidis et al, 2005). The alternation cycle consisted of 100 ms cyan (488 nm) and orange (594 nm) excitation periods, adding information about stoichiometry of dyes and thus allowed to filter out singly labelled molecules.…”

“…The laser alternation period was set to 40 μs (duty cycle of 40%) with intensity for the 488-nm laser ∼100 µW and the 594-nm laser intensity ∼90 μW. Data were collected using Three correction parameters: γ-factor, donor leakage into the acceptor channel and acceptor direct excitation by the donor excitation laser were employed and determined using polyproline standards of different length as FRET samples (Best et al, 2015;Sharma et al, 2014).…”

Biophysical Characterization of Chromatin Remodeling Protein CHD4

“…Single molecule experiments performed on custom-build TIRFM FRET instrument [ 34 ] using imaging rate fi ve frames per second.…”

“…Compute FRET effi ciency (proximity ratio) using consecutive single-step acceptor-donor photobleaching sequence [ 34 ] or spot intensities corrected for channel sensitivity (Fig. 4c ).…”