The elemental mechanism of transcriptional pausing
- University of Wisconsin-Madison, United States
Figures
Figure 1 with 3 supplements
The consensus elemental pause signal.
(A) The simplest elemental pause kinetic scheme and the biological roles of pausing. (B) Model of RNAP active site during the normal nucleotide addition cycle (green) consisting of RNA-DNA …
Figure 1—figure supplement 1
The elemental pause is a distinct offline state, not an on-pathway elongation intermediate.
(A) The complete consensus elemental pause scaffold (ntDNA #9563, tDNA #8334, RNA #8342; Supplementary file 1), identical to that used in Larson et al. (2014), except the template and nontemplate …
Figure 1—figure supplement 2
(A) Scaffold used to probe for biphasic escape kinetics at two sequential elemental pause sites (Tandem Consensus Elemental Pause Scaffold; tDNA #12623, ntDNA #12624, RNA #8342; Supplementary file 1).
(B) Pause assays on single or tandem consensus pause scaffold (Single Pause; left time course) or Tandem Pause Scaffold (right time course). Transcription was initiated by addition of NTPs to 100 µM …
Figure 1—figure supplement 3
(A) Model for RNA cleavage in pre-translocated or backtracked states following entry into the elemental pause.
Fast extension of ECsU14-G16 and fast interchange of half-translocated, pre-translocated, frayed, and 1 bp backtracked ePECs enables generation of 2-nt cleavage products even when the …
Figure 2 with 1 supplement
The elemental pause signal is multipartite.
(A,B) The consensus ePEC. The ePEC structure (pdb 6bjs; Kang et al., 2018a) is shown above the consensus pause sequence (Larson et al., 2014) color-coded as usFJ (blue), Hyb (red), dsFJ (green), and …
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Figure 2—source data 1
Fraction C17 pause RNA for wild-type and mutant pause signals.
- https://doi.org/10.7554/eLife.40981.009
Figure 2—figure supplement 1
(A) Quantitation of pause assays with scaffold variants described in Figure 2.
All pause assays were performed with the same RNAP preparation. (B) Plot of fast species lifetime (1/k-p, app) vs. slow species life (1/k-sp, app) for each scaffold variant. The high positive …
Figure 3 with 2 supplements
Translocation of the RNA:DNA hybrid is not rate-limiting for elemental pause escape.
(A) Scheme for translocation following CTP addition to control EC or ePEC scaffolds. The locations of 6-MI for both usFJ and dsFJ probes are indicated, but a probe was present in only one location …
Figure 3—figure supplement 1
Control EC scaffold used for 6-MI translocation assays in Figures 3 and 4.
Full sequences of oligonucleotides are given in Supplementary file 1. 6-MI is present at the locations indicated in Figures 3 and 4.
Figure 3—figure supplement 2
Reconstitution of ECs and PECs for 6-MI translocation assays.
(A) Experimental schematic for the assay of translocation and nucleotide addition. (B) Nucleotide addition for samples used in translocation assays of usFJ 6MI complexes (EC, left; ePEC, right). …
Figure 4
Translocation of the incoming template DNA limits elemental pause escape.
(A and B) Scaffolds used to reconstitute control EC and ePEC for fluorescence experiments. M, position of 6-MI. (C and D) Equilibrium fluorescence changes of control EC and ePEC, respectively, upon …
Figure 5 with 1 supplement
NTP binding but not TL folding is inhibited in the ePEC.
Scaffolds used for these experiments are shown in Figure 5—figure supplement 1. (A) Location of F937-736, P937-687, U937-1137, and U937-1139 Cys-pair reporters to test various conformations of the …
Figure 5—figure supplement 1
Scaffolds used for disulfide bond assays of trigger loop position shown in Figure 5.
Full sequences of oligonucleotides are given in Supplementary file 1. (A–C) For experiments on RNA 3′ OH-containing EC and PECs (Figure 5B–D), the full-length RNAs were used. For experiments on …
Figure 6 with 1 supplement
Restriction of clamp movement has less effect on ePEC than on hairpin-stabilized PEC.
(A) Location of disulfides used to restrict clamp movement or generate the Cys-triplet reporter (CTR; described in Hein et al., 2014; Kang et al., 2018b). (B) Example β-β′ disulfide mobilities …
Figure 6—figure supplement 1
Scaffolds used for CPR and CTR clamp-position assays shown in Figure 6.
Full sequences of oligonucleotides are given in Supplementary file 1. (A) Control EC scaffold used in Figure 6E,F (ntDNA#8847, tDNA #8848, RNA #8855). (B) ePEC scaffold used in Figure 6F …
Figure 7
β R542 in fork-loop two may contribute to a template base loading barrier in ePEC.
(A) Locations of β′K334 and βR542 in the ePEC. Relevant components of the ePEC are colored and labeled in a cutaway view of the active-site region of the ePEC. (B) Relative pause strength (PSP, Figur…
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Figure 7—source data 1
Fraction C17 pause RNA for wild-type and mutant pause signals.
- https://doi.org/10.7554/eLife.40981.019
Figure 8
Multistate model of elemental pausing.
(A) The small shift in RNAP modules observed in the ePEC (pdb 6bjs; magenta) relative to an EC (pdb 6alf; green) is depicted in the top central panel (Kang et al., 2018a; Kang et al., 2017). The …
Additional files
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Supplementary file 1
Oligonucleotides and plasmids used in this study.
- https://doi.org/10.7554/eLife.40981.022
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Transparent reporting form
- https://doi.org/10.7554/eLife.40981.023
