Figure 1.

Mapping of the σ-DNA crosslink in paused TEC. (A) The lacUV5 pause-inducing sequence and the −10 promoter element consensus sequence (Keilty and Rosenberg, 1987). Matches to consensus are indicated in bold typeface. The +5G to A substitution is indicated (+5mut). An asterisk marks a position (+6T), which when substituted to A abolishes pausing (Brodolin et al, 2004). Transcription start sites (+1) are shown by arrows. (B) An autoradiogram of a denaturing gel showing ³²P-labeled RNA products produced during run-off transcription from the lacUV5 promoter fragments containing either WT or mutant (+5mut) pause-inducing sequence (PS). (C) Formaldehyde crosslinking of the +17 paused complexes formed at the WT or mutant (+5mut) ³²P-labeled lacUV5 promoter DNA. Crosslinked σ⁷⁰-DNA and β′-DNA complexes are labeled as σ and β′. (D) Mapping of the crosslinking site in the σ⁷⁰ subunit crosslinked to ³²P-labeled lacUV5 DNA in TEC16 by Met-specific chemical cleavage (σ-DNA). The panel on the right (σ_m) shows Met-specific cleavage of the σ⁷⁰ subunit ³²P-labeled at the C terminus. Continuous lines with arrows between panels σ_m and σ-DNA connect bands corresponding to the identical cleavage products. Positions of σ⁷⁰ Met residues, which when cleaved give rise to observed cleavage products, are indicated on the right side of the figure. Suffixes C or N refer to C- or N-terminal cleavage product, respectively. (E) The diagram shows the map of the σ⁷⁰ subunit; the positions of Met cleavage sites are indicated by arrows. ³²P-labeled peptides produced by cleavage at indicated σ⁷⁰ Met residues are shown beneath as lines. σ⁷⁰ region containing the crosslink site to DNA is indicated by a black bar.