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Results: 4

1.
Figure 2

Figure 2. Effect of RNAP mutations on dMyx activity. From: Transcription inactivation through local refolding of the RNA polymerase structure.

The half-maximal inhibitory concentration (IC50) values were measured in vitro with purified RNAP variants (see Methods). The data is for all variants tested in this study. The IC50 could not be determined for the highly resistant β’ K345A variant. The T. thermophilus residue numbers are shown in brackets. Errors are standard deviations of the best fit estimates for IC50 and were calculated by nonlinear regression of the RNA synthesis measurements versus dMyx concentration assuming a hyperbolic dependence.

Georgiy A. Belogurov, et al. Nature. ;457(7227):332-335.
2.
Figure 4

Figure 4. Mutations in switch-2 affect the open complex formation. From: Transcription inactivation through local refolding of the RNA polymerase structure.

Accessibility of the non-template DNA (blue) strand residues to permanganate modification probed as in Fig. 3a. Wild-type and mutant RNAPs differ in their patterns of reactivity in the absence of dMyx (top traces) but are nearly identical in the presence of 10 μM dMyx (bottom traces). Notably, β’Δ309–325 that removes the entire rudder loop (which is inserted in the same helix as switch-2, but is unlikely to interfere with the nucleic acids) has no effect on DNA melting, suggesting that a melting defect of a different rudder deletion19 might be due to changes in the adjacent switch-2 instead.

Georgiy A. Belogurov, et al. Nature. ;457(7227):332-335.
3.
Figure 1

Figure 1. Structure of the RNAP–Myx complex. From: Transcription inactivation through local refolding of the RNA polymerase structure.

The same colour scheme is used in all figures throughout this manuscript. The σ-subunit, bridge helix, trigger loop and the remainder of the RNAP molecule are in blue, magenta, cyan and grey, respectively. The switch-2 segments in the Myx-free and Myx-bound structures are in orange and green, respectively. dMyx is in black. The Mg2+ ion is shown as magenta sphere. a, The overall view of the complex is shown. CC, coiled–coil; CH, clamp helices. b, Close-up view of the dMyx binding site. c, Sequence alignment of the switch-2 segment from bacterial (bsu, Bacillus subtilis; eco, E. coli; mtu, Mycobacterium tuberculosis; tt, T. thermophilus), archaeal (pfu, Pyrococcus furiosis) and yeast Saccharomyces cerevisiae pol II (scII) enzymes. Substitutions constructed in this work are shown above the sequence in green. d, Schematic drawing of the protein–dMyx interactions. The polar and van der Waals interactions are shown as solid arrows and dashed lines, respectively. The mutated residues are indicated by the red boxes. e, f, Conformations of the switch-2 segment in the Myx-free (e) and Myx-bound (f) holo-RNAP structures. In the panels d and e the E. coli residue numbers are shown in blue.

Georgiy A. Belogurov, et al. Nature. ;457(7227):332-335.
4.
Figure 3

Figure 3. A mechanism of the dMyx action. From: Transcription inactivation through local refolding of the RNA polymerase structure.

a, Myx alters the contacts between RNAP and λPR promoter DNA. a, A linear DNA fragment encompassing positions −81 to +70 of the λPR promoter was generated by polymerase chain reaction (PCR); the non-template DNA strand was end-labelled with [32P]-γATP (see Methods). The sequence from −44 to +23 is shown. The −35 and −10 hexamers are indicated by black boxes, the start site (+1) is shown by a black dot. The top panel shows probing of the non-template strand by piperidine-induced cleavage of the permanganate-modified T residues. Reactivities of −10, −4, −3 and +3 residues (quantification described in Methods) are shown to the left of the gel and summarized above the promoter sequence where black and white arrows indicate high and low reactivity, respectively. The bottom panel shows protection of the non-template DNA strand from DNaseI digestion. The footprint boundaries in the promoter region shown are indicated on the gel and by black (RNAP alone) and white (RNAP with the inhibitor) bars below the promoter sequence; the dideoxy-sequencing ladder is shown for reference. In the gels shown, independent reaction repeats were analysed for consistency. b, Schematic drawing of the putative mechanism of the dMyx action. dwDNA, downstream DNA.

Georgiy A. Belogurov, et al. Nature. ;457(7227):332-335.

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