Fig. 1
From: Target preference of Type III-A CRISPR-Cas complexes at the transcription bubble
Tethering of TthCsm to a transcription elongation complex (TEC) by a complementary nascent RNA transcript. a Schematic of the TthCsm complex and the TEC. TthCsm subunits are labeled. RNAP; RNA polymerase. The sequence in the RNA transcript that is complementary to the crRNA in TthCsm is highlighted in orange. A 5´-desthiobiotin moiety on the template strand of DNA in the TEC is labeled. b Schematic of the pull-down assay. The TEC was immobilized on streptavidin beads using a desthiobiotin group on the DNA. TthCsm was incubated with the TEC, after which the beads were washed to remove unbound complexes, and biotin was added to elute the TECs from the beads. c Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) of the purified T. thermophilus RNAP (α, β´, β, σ, ϖ) and TthCsm (Cas10/Csm1, Csm2-Csm5) complexes. A molecular weight marker was run for comparison in the leftmost lane and sizes are indicated in kilodaltons. d Pull-down assays were done with TECs containing a target (left), non-target (middle), or target RNA transcript added in trans (right). The elutions were analyzed by SDS-PAGE. The presence of RNAP and TthCsm subunits is indicated. The positions of the molecular weight markers in (c) are shown. Uncropped gel images for (c, d) are available online in the Source Data file
