Forward Translocation Is the Natural Pathway of RNA Release at an Intrinsic Terminator  Thomas J Santangelo, Jeffrey W Roberts  Molecular Cell  Volume 14, Issue 1, Pages 117-126 (April 2004) DOI: 10.1016/S1097-2765(04)00154-6

Figure 1 Forward Translocation Model of Intrinsic Termination (A) Hairpin formation drives RNAP and the transcription bubble forward in the absence of further synthesis, unwinding DNA ahead of the catalytic site and rewinding DNA at the upstream edge of the transcription bubble. After translocation of ∼4–6 nucleotides, only a minimal, weak RNA/DNA hybrid remains, the RNA 3′ terminus has been removed from the active site, and the ssRNA/RNAP interactions are lost; this combination ultimately results in transcript release. RNAP is shown in the pretranslocated position with the RNA 3′ terminus occupying the active site (boxed). The RNA is shown in red, the DNA is shown in black, and RNAP is a gray oval. Hairpin sequences are overscored (DNA) or underscored (RNA). The crosslinked sequences are highlighted in cyan. The green oval represents E111Q EcoRI. The drawing is not meant to be to scale. (B) Cartoon of forward translocation, with or without propagation of the transcription bubble. The RNA transcript is shown as a red, solid line; the template strand as a solid, black line; the non-template strand as a broken, black line. RNAP is a gray freeform figure; the catalytic center is represented as a green sphere. Vertical lines emphasize translocation downstream (to the right in this orientation). Molecular Cell 2004 14, 117-126DOI: (10.1016/S1097-2765(04)00154-6)

Figure 2 Inhibiting Downstream Movement of the Transcription Bubble Decreases Termination Efficiency Transcription reactions comparing release on noncrosslinked (WT) and crosslinked templates (A and B). The percentage of complexes producing full run-off (RO) transcripts is shown. P, pellet, RNA associated with RNAP. S, supernatant, RNA released to solution. One minute time point is shown. The sequence of the templates is shown below. Bases involved in the crosslink are marked, and asterisks mark the major release positions, +104 and +105. The diagram outlines competing pathways; arrow size crudely represents reaction rate. F, fast release pathway involving movement of the transcription bubble; S, slow release pathway. Pathways blocked by the crosslinks (red, template A; green, template B) are shown. Molecular Cell 2004 14, 117-126DOI: (10.1016/S1097-2765(04)00154-6)

Figure 3 Interstrand DNA Crosslinks Decrease the Rate and the Overall Efficiency of Oligonucleotide-Mediated Transcript Release (A) Crosslinked bases in templates 1–6 are shown by dashes connecting the two strands. (B) Products of OMR assays from the WT and crosslinked template 1. (C) Efficiency of OMR on the WT template and template 1. Percentage of released transcripts after 5′ is plotted against oligo concentration. (D) Relative efficiency of OMR on crosslinked templates. The ratio of the oligo concentration required for 50% transcript release on the WT template versus each crosslinked template is given. Note that template 1 does not allow elongation to +106 and no value is given for this position. (E) Products of OMR assays comparing the rate of OMR on WT and template 1 at three different oligo concentrations. (F) Percentage of transcripts released to solution is plotted versus time for WT and template 1. (G) Relative rate of OMR WT and crosslinked templates. The curves were treated as single exponentials and the value given is the average ratio (WT to crosslinked template) of the decay constant, λ. Note that template 1 does not allow elongation to +106 and no value is given for this position. Molecular Cell 2004 14, 117-126DOI: (10.1016/S1097-2765(04)00154-6)

Figure 4 A Downstream Protein Roadblock Inhibits Oligonucleotide-Mediated Transcript Release and Decreases Intrinsic Termination Efficiency (A) The elongation complex with E111Q EcoRI (shown in green) immediately downstream. Oligos used to stimulate release are shown. (B) Efficiency of OMR for the 14 bp spacer after 5′ at increasing oligo concentration ± E111Q EcoRI. (C) Relative efficiency of OMR on templates with increasing spacer distance ± E111Q EcoRI. (D) RNA transcripts from templates with full terminators ± E111Q EcoRI downstream. P, pellet, RNA associated with RNAP. S, supernatant, RNA released to solution. (E) Relative termination efficiency is plotted versus distance separating RNAP and E111Q EcoRI. Molecular Cell 2004 14, 117-126DOI: (10.1016/S1097-2765(04)00154-6)

Figure 5 Hairpin Formation Can Drive RNAP through a Roadblock Products of transcription reactions using each template. Note that RNA transcripts with a hairpin migrate faster than equivalent length transcripts without a hairpin. Percentage readthrough (RT) of the roadblock (B) under each condition is shown below the figure. Molecular Cell 2004 14, 117-126DOI: (10.1016/S1097-2765(04)00154-6)

Figure 6 Effect of Interstrand DNA Crosslinks and E111Q EcoRI on Backtracking (A) OMR and cleavage assays with WT and template 1 are shown to compare the efficiency of transcript release at position +105 (T) and the lack of 3′-labeled cleavage product (*) on the crosslinked template. (B) OMR and cleavage assays on unmodified templates in the presence or absence of E111Q EcoRI immediately downstream of the elongation complex. Chase reactions contained all four NTPs and confirm that E111Q EcoRI serves as a protein roadblock. Transcription from λpR′ serves as a general size marker. Note the significant decrease of cleavage products in the presence of E111Q EcoRI, as well as the lack of OMR release of +105 in the presence of E111Q EcoRI. (C) Graph showing the percentage of +105 transcript released under each condition in (B). Molecular Cell 2004 14, 117-126DOI: (10.1016/S1097-2765(04)00154-6)