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. 2020 Mar 24;295(19):6401–6412. doi: 10.1074/jbc.RA120.012641

Figure 1.

Figure 1.

Preparation and characterization of chemical roadblocking DNA templates. A and B, chemical structures of internal desthiobiotin–TEG (A) and amino linker (B) modifications. C, layout of internally modified DNA templates. The positions of the forward primer and the internally modified reverse primer are shown. The transcription start site (TSS) and U15 walk site are indicated. D, overview of internally modified (mod.) DNA template preparation. The position of the internally modified reverse primer is shown in purple. E, denaturing PAGE quality analysis of internally modified DNA template preparations (Modification +) alongside an unmodified (Modification −) positive control. The size marker is the Low Range ssRNA Ladder (New England Biolabs). F and G, single-round in vitro transcription of internal desthiobiotin–TEG–modified (F) and amino linker–modified (G) DNA templates. In both cases, RNAP stalls one nucleotide upstream of the modification site. E is representative of several DNA template quality control gels performed throughout this study. The experiments in F and G were performed once to precisely map critical bands for all subsequent experiments.