Fractional‐nucleotide translocation in sequence‐dependent pausing by RNA polymerase: Single‐molecule picometer‐resolution nanopore tweezers (SPRNT)

Abstract

Single‐molecule picometer‐resolution nanopore tweezers (SPRNT) enables monitoring of translocation of a nucleic‐acid motor protein on a nucleic‐acid track with sequence registration, sub‐nucleotide spatial resolution, sub‐millisecond temporal resolution, and the ability to apply assisting and opposing forces.We have demonstrated the ability of SPRNT to monitor translocation of RNA polymerase (RNAP) relative to the DNA template strand, the DNA non‐template strand, and the RNA product strand in transcription, and we have applied SPRNT to analyze RNAP translocation in transcription elongation and sequence‐dependent transcriptional pausing.The results show that sequence‐dependent transcriptional pausing involves a “half‐translocated” state in which RNAP translocates relative to the DNA template strand by 0.5 nucleotides. The “half‐translocated” state is unable to bind the next NTP substrate, presumably because RNAP translocation by 0.5 nt does not align the next template‐strand nucleotide with the RNAP active‐center NTP addition site. The half‐translocated state interconverts rapidly (~0.1–1 s) with the “pre‐translocated” state and converts slowly (~1 s) to the NTP‐binding‐competent “post‐translocated state.” In the presence of saturating NTPs, conversion to the post‐translocated state is followed by pause escape. In the presence of limiting NTPs, conversion to the post‐translocated state is followed by return to interconverting half‐translocated and pre‐translocated states and continued pausing.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.