Key Roles of the Downstream Mobile Jaw of Escherichia coli RNA Polymerase in Transcription Initiation

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. Escherichia coli RNAP uses binding free energy to unwind and separate 13 base pairs of λPR promoter DNA to form the unstable open intermediate I2, which rapidly converts to much more stable open complexes (I3, RPo). Conversion of I2 to RPo 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 I3. Overall rate constants (ka) for promoter binding and DNA opening by ΔJAW RNAP are much smaller than for WT RNAP. Values of ka for WT RNAP with DT+12 and full-length λPR are similar, though contributions of binding and isomerization steps differ. Hence, the jaw plays major roles both early and late in RPo formation, while downstream DNA functions primarily as the assembly platform after DNA opening.

Probing the protein-folding mechanism using denaturant and temperature effects on rate constants

“…The small effects of urea and Hofmeister salts on the rate and equilibrium constants for the step involving opening of 13 bp of duplex promoter DNA provide evidence that RNAP opens this region in the active site cleft (14,48,49). The large effects of urea, glycine betaine, and different Hofmeister salts on the subsequent step(s) that stabilize the initial open complex provide evidence for large-scale coupled folding and assembly of mobile elements of RNAP to form a clamp on the downstream duplex DNA (14,(48)(49)(50). Solutes are also excellent probes of intermediates and TS for RNA folding, because burial of base ASA is disfavored by all solutes whereas burial of backbone ASA is favored by some solutes (12,13 (Table S5).…”

Mechanism of transcription initiation and promoter escape by E . coli RNA polymerase

“…2, we propose that the T7A1(λP R ) hybrid promoter forms an OC structurally similar to that of λP R (λP R ). By contrast, promoters containing the T7A1 discriminator are structurally similar to the T7A1 WT OC, in which conformational changes within RNAP and DNA do not stabilize the OC to the same extent as λP R (λP R ) (1,29). We also propose that both the rrnB P1 discriminator and an additional upstream feature that distinguishes rrnB P1(rrnB P1) from T7A1(T7A1) and λP R (λP R ) may be responsible for the instability and short lifetime of its OC.…”

“…Stocks of heparin (50 mg/mL), DTT (0.1 M), and BSA (50 mg/mL) were filtered and stored at −20°C before use. Buffers and solutions used in filter-binding assays and permanganate footprinting are the same as used previously (SI Appendix) (1,29). The transcription buffer (TB) was 40 mM Tris base (pH 8.0), 5 mM MgCl 2 , 1 mM DTT, 0.1 mg/mL BSA, and 60 mM KCl.…”

“…For filterbinding assays and permanganate footprinting experiments, HTOP or HBOT was kinase-labeled using T4 polynucleotide kinase and γ-[ Nitrocellulose Filter-Binding Dissociation Assays. Dissociation kinetic assays were performed as previously described (29,33). RNAP-promoter OCs were formed by incubating 2-6 nM RNAP for 1 h in binding buffer (BB) (see SI Appendix, General Procedures for composition) at 37°C with γ-32 P-radiolabeled promoter DNA (∼36,000 cpm).…”

“…Instead, we find that the primary difference between promoter OCs with very different lifetimes is in the range of the lengths of the abortive RNAs produced by the fraction of nonproductive ITC at the promoter. It is possible that late-acting E. coli regulatory factors analogous to bacteriophage T7gp2 (55,56) exist that interact selectively with the different downstream structures of OCs with different lifetimes (1,29) to regulate initiation, but none have been discovered. We therefore propose that the longer abortive RNAs produced by long-lived, stable promoter OCs play regulatory roles.…”