Abstract
The final sigma(54) subunit of the bacterial RNA polymerase requires the action of specialized enhancer-binding activators to initiate transcription. Here we show that final sigma(54) is able to melt promoter DNA when it is bound to a DNA structure representing the initial nucleation of DNA opening found in closed complexes. Melting occurs in response to activator in a nucleotide-hydrolyzing reaction and appears to spread downstream from the nucleation point toward the transcription start site. We show that final sigma(54) contains some weak determinants for DNA melting that are masked by the Region I sequences and some strong ones that require Region I. It seems that final sigma(54) binds to DNA in a self-inhibited state, and one function of the activator is therefore to promote a conformational change in final sigma(54) to reveal its DNA-melting activity. Results with the holoenzyme bound to early melted DNA suggest an ordered series of events in which changes in core to final sigma(54) interactions and final sigma(54)-DNA interactions occur in response to activator to allow final sigma(54) isomerization and the holoenzyme to progress from the closed complex to the open complex.
Highlights
Accessing the information in DNA often relies upon the action of DNA-binding proteins that are able to generate noncanonical B-DNA structures
We show that the presence of core RNA polymerase inhibits those changes in 54-DNA interactions that occur in response to activator, consistent with the view that tight binding to the early melted
We showed that purified 54 bound to the S. meliloti nifH promoter was able to isomerize if the DNA template had an unpaired sequence downstream of the GC element of the promoter [24]
Summary
DNA and Proteins—The promoter fragments used in this work were Escherichia coli glnHp2 from Ϫ60 to ϩ28 in which T at Ϫ13 was replaced by G and the Ϫ11/Ϫ10 sequence was replaced with a CA/TG mismatch to create a short unpaired DNA element next to a consensus GC (Ϫ13/Ϫ12) promoter element or a mismatched sequence between Ϫ11 and Ϫ6 (see Fig. 1). The activator was E. coli PspF lacking a functional C-terminal DNA-binding domain (PspF⌬HTH) [26]. DNA Binding Assays—End-labeled DNA (16 –100 nM) and 1 M 54 or ⌬I54 in a 10-l reaction in buffer containing 25 mM Tris acetate (pH 8.0), 8 mM magnesium acetate, 10 mM KCl, 1 mM dithiothreitol, and 3.5% (w/v) polyethylene glycol 8000 were incubated for 5 min at 30 °C. DNA Footprints—Binding reactions were conducted as described above; footprinting reagents were added; reactions were terminated; and bound and unbound DNAs were separated on native gels as described above. For DNase I footprints, 1.75 ϫ 10Ϫ3 units of enzyme (Amersham Pharmacia Biotech) was added to a 10-l binding reaction for 1 min, followed by addition of 10 mM EDTA to stop cutting. For KMnO4 footprinting, 4 mM fresh KMnO4 was added for 30 s, followed by 50 mM -mercaptoethanol to quench DNA oxidation
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