Key Roles of the Downstream
Mobile Jaw of <i>Escherichia coli</i> RNA Polymerase in
Transcription Initiation
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Abstract
Differences in kinetics of transcription initiation by
RNA polymerase
(RNAP) at different promoters tailor the pattern of gene expression
to cellular needs. After initial binding, large conformational changes
occur in promoter DNA and RNAP to form initiation-capable complexes.
To understand the mechanism and regulation of transcription initiation,
the nature and sequence of these conformational changes must be determined. <i>Escherichia coli</i> RNAP uses binding free energy to unwind
and separate 13 base pairs of λP<sub>R</sub> promoter DNA to
form the unstable open intermediate I<sub>2</sub>, which rapidly converts
to much more stable open complexes (I<sub>3</sub>, RP<sub>o</sub>).
Conversion of I<sub>2</sub> to RP<sub>o</sub> involves folding/assembly
of several mobile RNAP domains on downstream duplex DNA. Here, we
investigate effects of a 42-residue deletion in the mobile β′
jaw (ΔJAW) and truncation of promoter DNA beyond +12 (DT+12)
on the steps of initiation. We find that in stable ΔJAW open
complexes the downstream boundary of hydroxyl radical protection shortens
by 5–10 base pairs, as compared to wild-type (WT) complexes.
Dissociation kinetics of open complexes formed with ΔJAW RNAP
and/or DT+12 DNA resemble those deduced for the structurally uncharacterized
intermediate I<sub>3</sub>. Overall rate constants (<i>k</i><sub>a</sub>) for promoter binding and DNA opening by ΔJAW
RNAP are much smaller than for WT RNAP. Values of <i>k</i><sub>a</sub> for WT RNAP with DT+12 and full-length λP<sub>R</sub> are similar, though contributions of binding and isomerization
steps differ. Hence, the jaw plays major roles both early and late
in RP<sub>o</sub> formation, while downstream DNA functions primarily
as the assembly platform after DNA opening