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. 2021 Jun 29;118(27):e2106388118. doi: 10.1073/pnas.2106388118

Fig. 1.

Fig. 1.

Use of MASTER to monitor primer-independent and primer-dependent transcription initiation. (A) Binding of initiating entities to DNA template-strand nucleotides in primer-independent and primer-dependent transcription initiation. (Top) RPo. (Bottom) Enlarged view of initiating entities bound to template-strand nucleotides in the RNAP active center. Dark gray, RNAP; yellow, σ; blue, −10-element nucleotides; purple, TSS nucleotides; light gray, RNA nucleotides; pink, primer-binding nucleotides at positions TSS−1, TSS−2 or TSS−3; white boxes, DNA nucleotides; NT, nontemplate-strand nucleotides; T, template-strand nucleotides. P−3, P−2, P−1, and P, RNAP active-center initiating entity binding sites; A, RNAP active-center extending NTP binding site. Unwound transcription bubble in RPo indicated by raised and lowered nucleotides. (B) Analysis of primer-independent and primer-dependent initiation using MASTER. (Top) DNA fragment containing MASTER template library. Light green, randomized nucleotides in the promoter region; dark green, transcribed-region barcode. (Bottom) 5′ RNA-seq analysis of RNA products generated from the promoter library by primer-independent, NTP-dependent initiation (Rpp treatment) and primer-dependent initiaion (PNK treatment).