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. 2017 Nov 23;6:e32038. doi: 10.7554/eLife.32038

Figure 1. TSS selection exhibits first hallmark of scrunching--movement of RNAP leading edge but not RNAP trailing edge--both in vitro and in vivo.

(A) RNAP leading-edge and trailing-edge positions at promoters having GGG discriminator (TSS 7 bp downstream of −10 element; top) and CCT discriminator (TSS 9 bp downstream of −10 element; bottom). Changes in TSS selection result from changes in discriminator-sequence-dependent DNA scrunching. Gray, RNAP; yellow, σ; blue, −10-element nucleotides; dark purple, GGG-discriminator nucleotides; light purple, CCT-discriminator nucleotides; i and i + 1, NTP binding sites; arrow, TSS; boxes, DNA nucleotides (nontemplate-strand nucleotides above template-strand nucleotides; nucleotides downstream of −10 element numbered); red, trailing-edge Bpa and nucleotide crosslinked to Bpa; pink, leading-edge Bpa and nucleotide crosslinked to Bpa. Scrunching is indicated by bulged-out nucleotides. Distance between leading-edge and trailing-edge crosslinks is indicated below RNAP. (B) RNAP trailing-edge crosslinking (top), TSS (middle), and RNAP leading-edge crosslinking (bottom) for promoters having GGG discriminator and CCT discriminator, in vitro (lanes 5–6) and in vivo (lanes 7–8). Horizontal dashed lines relate bands on gel (left) to nucleotide sequences (right).

Figure 1.

Figure 1—figure supplement 1. Unnatural-amino-acid mutagenesis and protein-DNA photocrosslinking in vivo.

Figure 1—figure supplement 1.

Left column, procedure for mapping RNAP trailing edge relative to DNA in vivo. Right column, procedure for mapping RNAP leading edge relative to DNA in vivo. (A) Three-plasmid merodiploid system for co-production, in E. coli cells, of Bpa-labeled, decahistidine-tagged, mutationally inactivated, RNAP-derivative (gray, with red or pink circle indicating Bpa, triangle indicating decahistidine tag, and black X indicating mutational inactivation), in presence of unlabeled, untagged, wild-type RNAP (light gray). First plasmid carries gene for RNAP subunit with a nonsense (TAG) codon at the site for incorporation of Bpa (residue βʹT48 for trailing edge; residue βʹR1148 for leading edge); second plasmid carries genes for engineered Bpa-specific nonsense-supressor tRNA and Bpa-specific amino-acyl-tRNA synthetase; third plasmid carries promoter of interest; and chromosome carries wild-type RNAP subunit genes. (B) Procedure for protein-DNA photocrosslinking, entailing UV-irradiation of cells, lysis of cells, immobilized-metal-ion affinity chromatography (IMAC), denaturation, primer extension with 32P-5’-end-labeled primer (asterisk denotes label), electrophoresis, and storage-phosphor imaging. Brackets represent steps performed in cells.
Figure 1—figure supplement 2. Mutationally inactivated RNAP derivative traps RPo in presence of NTPs, enabling protein-DNA photocrosslinking of RPo in presence of NTPs.

Figure 1—figure supplement 2.

(A) RNAP leading-edge and trailing-edge positions for wild-type RNAP (WT; left) and mutationally inactivated RNAP (βʹD460A; right) at promoters having GGG discriminator (TSS 7 bp downstream of −10 element; top) and CCT discriminator (TSS 9 bp downstream of −10 element; bottom). Black X, mutational inactivation due to βʹD460A substitution. Other colors as in Figure 1A. (B) RNAP trailing-edge crosslinking (top), TSS (middle), and RNAP leading-edge crosslinking (bottom) for wild-type RNAP (WT; left) and mutationally inactivated RNAP (βʹD460A; right) at promoters having GGG discriminator and CCT discriminator, in absence and presence of NTPs. Horizontal dashed lines relate bands on gel (left) to nucleotide sequences (right).