Figure 4
Ss-LrpB binding to CRISPR A and CRISPR B leader regions. A. Zoomed average ChIP-chip binding profile of the CRISPR A/B genomic region. Below the profile, the genomic organization is schematically shown aligned to genomic positions. ORFs, mainly encoding CRISPR-associated and –related genes, are indicated as horizontal arrows, whereas the leader regions are depicted as grey boxes. Primary pre-crRNA TSSs are indicated by arrows. B. EMSA that was used for ‘in gel’ Cu-OP footprinting, using a 270-bp fragment representing the CRISPR B leader sequence. In this experiment, the bottom strand was 32P-labeled. Used protein concentrations are indicated. Populations of input DNA (I), free DNA (F) and bound DNA (B) are boxed in the same way as they were excised. C. Autoradiograph of the denaturing gel electrophoresis of footprinting samples. Direction of electrophoresis is indicated with an arrow, lanes contain either the I, B or F populations or the A + G and C + T Maxam-Gilbert sequencing ladders, as indicated. The protected region is indicated with a horizontal line and the corresponding sequence is given (5’ - > 3’). D. Alignment of the promoter-proximal CRISPR A and B leader sequences, with indication of the Ss-LrpB binding site, factor B recognition element (BRE), TATA box, first CRISPR repeat and primary TSS (arrow). Non-conserved residues are highlighted in grey; position numbering is with respect to the CRISPR B leader sequence.