The mechanism of variability in transcription start site selection
- Rutgers University, United States
- Institut de Biologie de l’Ecole Normale Supérieure (IBENS), CNRS, INSERM, PSL Research University, France
- Ligue Contre le Cancer, France
- Institut Jacques Monod, CNRS, UMR7592, University Paris Diderot, France
Figures
Figure 1 with 2 supplements
TSS selection exhibits first hallmark of scrunching--movement of RNAP leading edge but not RNAP trailing edge--both in vitro and in vivo.
(A) RNAP leading-edge and trailing-edge positions at promoters having GGG discriminator (TSS 7 bp downstream of −10 element; top) and CCT discriminator (TSS 9 bp downstream of −10 element; bottom). …
Figure 1—figure supplement 1
Unnatural-amino-acid mutagenesis and protein-DNA photocrosslinking in vivo.
Left column, procedure for mapping RNAP trailing edge relative to DNA in vivo. Right column, procedure for mapping RNAP leading edge relative to DNA in vivo. (A) Three-plasmid merodiploid system for …
Figure 1—figure supplement 2
Mutationally inactivated RNAP derivative traps RPo in presence of NTPs, enabling protein-DNA photocrosslinking of RPo in presence of NTPs.
(A) RNAP leading-edge and trailing-edge positions for wild-type RNAP (WT; left) and mutationally inactivated RNAP (βʹD460A; right) at promoters having GGG discriminator (TSS 7 bp downstream of −10 …
Figure 2
TSS selection exhibits second hallmark of scrunching--change in size of transcription bubble.
(A) Magnetic-tweezers single-molecule DNA nanomanipulation (Revyakin et al., 2004; Revyakin et al., 2005; Revyakin et al., 2006). End-to-end extension (l) of a mechanically stretched, positively …
Figure 3 with 1 supplement
TSS selection downstream and upstream of the modal TSS involves, respectively, forward and reverse movements of RNAP leading edge.
(A) Ribotrinucleotide primers program TSS selection at positions 6, 7, 8, and 9 bp downstream of −10 element (UGG, GGA, GAA, and AAU). Cyan, green, orange, and red denote primers UGG, GGA, GAA, and …
Figure 3—figure supplement 1
Protein-DNA photocrosslinking in primer-programmed TSS selection: primer GGA yields same pattern of RNAP leading-edge and trailing-edge crosslinking as in absence of primer.
(A) RNAP leading-edge and trailing-edge positions and RNAP-dependent DNA unwinding with primers programming TSS selection at positions 6, 7, 8, and 9 bp downstream of −10 element (left panel), and …
Figure 4 with 2 supplements
TSS selection downstream and upstream of the modal TSS involves, respectively: increases and decreases in RNAP-dependent DNA unwinding.
(A) Use of single-molecule DNA nanomanipulation to define RNAP-dependent DNA unwinding. Single-molecule time traces with primers UGG, GGA, GAA, and AAU (positively supercoiled DNA in upper panel; …
Figure 4—figure supplement 1
Single-molecule DNA-nanomanipulation: shorter DNA fragment enables detection of RNAP-dependent DNA unwinding with single-base-pair resolution.
(A–B) Single-molecule time traces and transition-amplitude histograms with 2.0 kb DNA fragment [fragment length in previous work (Revyakin et al., 2004; Revyakin et al., 2005) and in Figure 2] (A), …
Figure 4—figure supplement 2
Single-molecule DNA-nanomanipulation: analysis of primer-programmed TSS selection with primer GGA.
Single-molecule time traces and transition-amplitude histograms with GGA, which programs TSS selection 7 bp downstream of the −10 element, and and with no primer, which also yields TSS selection 7 …
Figure 5
Energetic costs of scrunching and anti-scrunching: primer-concentration dependences of unwound-state lifetimes with primers UGG, GGA, GAA, and AAU.
(A) Single-molecule time traces at low (left) and high (right) primer concentrations. Black bars, lifetimes of unwound states (tunwound). Colors as in Figure 2B. (B) tunwound distributions and mean …
Figure 6 with 1 supplement
Energetic costs of scrunching and anti-scrunching: calculation of energetic costs and relationship between energetic costs and range and relative utilization of TSS positions.
(A) Coupled equilibria for promoter scrunching or anti-scrunching (Kscrunch) and primer binding (KNpNpN). (B) Equations for promoter binding (KB), promoter unwinding (k2/k-2), promoter scrunching or …
Figure 6—figure supplement 1
Single-molecule DNA-nanomanipulation: calculation of Kscrunch and ΔGscrunch in primer-programmed TSS selection with primers UGG, GGA, GAA, and AAU.
(A) Coupled equilibria for promoter scrunching or anti-scrunching (Kscrunch) and primer binding (KNpNpN) with primers UGG, GGA, GAA, and AAU. Rectangle with rounded corners highlights case of primer …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| strain, strain background(E. coli) | BL21(DE3) | New England BioLabs | Cat# C2527I | |
| strain, strain background(E. coli) | NiCo21(DE3) | New England BioLabs | Cat# C2529H | |
| strain, strain background (T. thermophilus) | HB8 | ATCC | Cat# ATCC 27634 | |
| recombinant DNA reagent | pUC18 | Thermo-Fisher | ||
| recombinant DNA reagent | pUC18-T20C2-lacCONS-CCT (plasmid) | this paper | progenitors: PCR of T. thermophilus HB8, pUC18 | |
| recombinant DNA reagent | pUC18-T20C2-lacCONS-CCT (plasmid) | this paper | progenitors: PCR of T. thermophilus HB8, pUC18 | |
| recombinant DNA reagent | pUC18-T20C2-lacCONS-CCT (plasmid) | this paper | progenitors: PCR of T. thermophilus HB8, pUC18 | |
| recombinant DNA reagent | pVS10 (plasmid) | PMID: 12511572 | ||
| recombinant DNA reagent | pIA900 (plasmid) | PMID: 25665556 | ||
| recombinant DNA reagent | pIA900-beta'R1148Bpa (plasmid) | PMID: 26257284 | ||
| recombinant DNA reagent | pIA900-beta'T48Bp | PMID: 26257284 | ||
| recombinant DNA reagent | pIA900-beta'R1148Bpa;beta'D460A | this paper | progenitor: pIA900 | |
| recombinant DNA reagent | pIA900-beta'T48Bpa;beta'D460A | this paper | progenitor: pIA900 | |
| recombinant DNA reagent | pEVOL-pBpF (plasmid) | PMID: 12154230; Addgene | cat# 31190 | |
| sequence-based reagent | UGG (oligoribonucleotide) | Trilink Biotechnologies | ||
| sequence-based reagent | GGA (oligoribonucleotide) | Trilink Biotechnologies | ||
| sequence-based reagent | GAA (oligoribonucleotide) | Trilink Biotechnologies | ||
| sequence-based reagent | AAU (oligoribonucleotide) | Trilink Biotechnologies | ||
| sequence-based reagent | JW 30 (oligodeoxyribonucleotide) | Integrated DNA Technologies | ||
| sequence-based reagent | JW 61 (oligodeoxyribonucleotide) | Integrated DNA Technologies | ||
| sequence-based reagent | JW 62 (oligodeoxyribonucleotide) | Integrated DNA Technologies | ||
| sequence-based reagent | JW 85 (oligodeoxyribonucleotide) | Integrated DNA Technologies | ||
| sequence-based reagent | LY10 (oligodeoxyribonucleotide) | Integrated DNA Technologies | ||
| sequence-based reagent | LY11 (oligodeoxyribonucleotide) | Integrated DNA Technologies | ||
| sequence-based reagent | S128a (oligodeoxyribonucleotide) | Integrated DNA Technologies | ||
| sequence-based reagent | S1219 (oligodeoxyribonucleotide) | Integrated DNA Technologies | ||
| sequence-based reagent | S1220 (oligodeoxyribonucleotide) | Integrated DNA Technologies | ||
| sequence-based reagent | RPOC820 (oligodeoxyribonucleotide) | Integrated DNA Technologies | ||
| sequence-based reagent | RPOC3140 (oligodeoxyribonucleotide) | Integrated DNA Technologies | ||
| sequence-based reagent | SbfRPOC50 (oligodeoxyribonucleotide) | Integrated DNA Technologies | ||
| sequence-based reagent | Taq_rpoC_F (oligodeoxyribonucleotide) | Integrated DNA Technologies | ||
| sequence-based reagent | Taq_rpoC_R (oligodeoxyribonucleotide) | Integrated DNA Technologies | ||
| sequence-based reagent | XbaRPOC4050 (oligodeoxyribonucleotide) | Integrated DNA Technologies | ||
| peptide, recombinant protein (E.coli) | RNAP polymerase core enzyme | PMID: 12511572 | ||
| peptide, recombinant protein (E.coli) | sigma70 | PMID: 9157885 | ||
| peptide, recombinant protein (E.coli) | RNAP core enzyme derivative beta'R1148Bpa | PMID: 26257284 | ||
| peptide, recombinantprotein (E.coli) | RNAP core enzyme derivative beta'T48Bpa | PMID: 26257284 | ||
| peptide, recombinantprotein (E.coli) | RNAP core enzyme derivative beta'R1148Bpa;D460A | this work | ||
| peptide, recombinant protein (E.coli) | RNAP core enzyme derivative beta'R1148Bpa;D460A | this work | ||
| peptide, recombinant protein | Dam methyltransferase | New England BioLabs | Cat# M0222S | |
| peptide, recombinantprotein | restriction endonuclease SbfI-HF | New England BioLabs | Cat# R3642S | |
| peptide, recombinantprotein | restriction endonuclease XbaI | New England BioLabs | Cat# R0145S | |
| peptide, recombinant protein | T4 polynucleotide kinase | New England BioLabs | Cat# M0201S | |
| antibody | anti-digoxigenin antibody | Sigma-Aldrich | Cat# 11333089001 | |
| commercial assay or kit | Dynabeads MyOne Streptavidin C1 | Thermo-Fisher | Cat# 650.01 | |
| commercial assay or kit | MagneHis protein purification system | Promega | Cat# V8500 | |
| commercial assay or kit | TRI reagent | Molecular Research Center | Cat# TR118 | |
| chemical compound,drug | carbenicillin | Gold Biotechnology | cat# c-103-25 | |
| chemical compound,drug | chloramphenicol | Gold Biotechnology | cat# c-105-25 | |
| chemical compound,drug | spectinomycin | Gold Biotechnology | cat# 22189-32-8 | |
| chemical compound,drug | streptomycin | Omnipur | cat# 3810-74-0 | |
| software, algorithm | SigmaPlot | Systat Software | ||
| software, algorithm | xvin | PMID: 16156080 |
Additional files
-
Supplementary file 1
Oligonucleotides
- https://doi.org/10.7554/eLife.32038.015
-
Transparent reporting form
- https://doi.org/10.7554/eLife.32038.016
