Bridged filaments of histone-like nucleoid structuring protein pause RNA polymerase and aid termination in bacteria
- University of Wisconsin–Madison, United States
- University of Nebraska Medical Center, United States
Figures
Figure 1 with 3 supplements
H-NS formed two different filaments depending on concentration.
(A) The H-NS-silenced E. coli bgl operon, encoding genes for ß-glucoside catabolism, contains an antisense promoter within bglF (PAS) (Peters et al., 2012). The 1.56-kb linear λPR-bgl DNA template …
Figure 1—figure supplement 1
Model of H-NS filaments.
H-NS is a minor groove DNA binding protein that first binds to A/T rich DNA as a dimer through a C-terminal DNA binding domain (right inset; PDB 2LEV [Cordeiro et al., 2011]), and can form filaments …
Figure 1—figure supplement 2
Interwound filaments formed preferentially in samples of ECs at 8 mM Mg2+ and 66 H-NS /kb.
(A) Lower magnification images of AFM as described and shown in Figure 1C, with the additional representative image of linear filaments formed in 8 mM Mg2+ and 200 H-NS/kb (high concentration H-NS). …
Figure 1—figure supplement 3
Temperature affected H-NS bridging interactions.
Native PAGE of H-NS complexes assembled at either 20°C (A) or 37°C (B) on 10 nM λPR-bgl template in 8 mM Mg2+. H-NS-DNA complexes were electrophoresed at 4°C (left panels), 20°C (middle panels), or …
Figure 2 with 3 supplements
H-NS dramatically decreased transcript elongation in vitro.
(A) In vitro transcription in the presence of 66 H-NS/kb or 200 H-NS/kb filaments at 20°C, 8 mM Mg2+, and 30 µM each NTP. ECs (10 nM) were formed at the end of the C-less cassette on the λPR-bgl …
Figure 2—figure supplement 1
Mapping of 3′ ends of pauses on λPR-bgl template.
(A–K) Pauses on the λPR-bgl template were could be mapped to nucleotide resolution up to ∼200 nucleotides downstream from the transcription start site using ladders generated by 3′-deoxy NTP …
Figure 2—figure supplement 2
Linear H-NS filaments had minimal effects on elongation.
(A) 10 nM halted ECs formed on the λPR-bgl template were incubated with H-NS in either 2 or 8 mM Mg2+ to reach equilibrium. 30 μM NTPs were added, time points were taken at 10, 20, 40, 60, 120, and …
Figure 2—figure supplement 3
H-NS effects on transcript elongation also occurred on a different template.
(A) The 1.27-kb linear pMK121 DNA template (λPR-bglF template) similarly contains the λPR promoter followed by a 26-nucleotide C-less cassette and includes a different portion of the bgl operon, the …
Figure 3 with 1 supplement
H-NS inhibited transcript elongation at physiological NTP concentrations (1 mM each NTP).
Densitometry profiles of transcripts produced at 20°C, 12 mM Mg2+ and 1 mM each NTP from the λPR-bgl template in bridged H-NS filaments (66 H-NS/kb) or in linear H-NS filaments (200 H-NS/kb). …
Figure 3—figure supplement 1
Electrophoretic gel image showing H-NS inhibited transcription elongation at physiological NTP concentrations (1 mM each NTP).
ECs (10 nM) formed on the λPR-bgl template were incubated with bridged H-NS (66 H-NS/kb) or linear filaments (200 H-NS/kb) in 12 mM Mg2+. 1 mM NTPs were added, and samples were removed at 0.66, 1, …
Figure 4 with 1 supplement
H-NS effects on transcript elongation were reduced at ≥30°C.
Densitometry profiles of transcripts produced at 25°C, 30°C, or 37°C, 8 mM Mg2+ and 30 μM each NTP from the λPR-bgl template in the presence of 66 H-NS/kb (bridged filaments). Samples were removed …
Figure 4—figure supplement 1
Electrophoretic gel image showing reduced H-NS effects on transcription elongation at ≥30°C.
10 nM-halted ECs formed on the λPR-bgl template were incubated with 66 H-NS/kb in 8 mM Mg2+ for 20 min at 25°C, 30°C, or 37°C. 30 μM NTPs were added, time points were taken at 2, 3, 4, 8, 16, and 32 …
Figure 5
Bridged H-NS filaments reformed upstream of ECs during transcription.
(A) Representative AFM images of ECs elongating through bridged filaments (66 H-NS/kb; 8 mM Mg2+; 20°C) sampled at either 8 or 16 min after addition of NTPs (30 µM each). ECs (10 nM) and H-NS …
Figure 6 with 3 supplements
Bridged H-NS filaments induced RNAP backtracking, which was rescued by GreB.
(A) Steps in pausing affected by the NusG-like N-terminal domain (NGN) of RfaH (RfaH-NTD) or Gre factors (e.g., GreB). Binding of the NGN RfaH-NTD (cyan) to the clamp domain (pink) of RNAP inhibits …
Figure 6—figure supplement 1
At 1 mM NTPs, NusG partially suppressed H-NS effects on pausing and GreA more significantly suppressed H-NS effects.
(A) A26 ECs (10 nM) formed on λPR-bgl were incubated with H-NS to form bridged filaments at 66 H-NS/kb and 12 mM Mg2+. GreA (500 nM) or NusG (75 nM) was added followed by NTPs (1 mM each), time …
Figure 6—figure supplement 2
Electrophoretic gel image showing that H-NS-induced RNAP backtracking was rescued by GreB.
PAGE corresponding to Figure 6, where reactions were assembled with 66 H-NS/kb and 50 nM GreB or 300 nM RfaH-NTD.
Figure 6—figure supplement 3
The H-NS stimulated C346 pause readily backtracked, whereas H-NS-resistant pausing at C370 occurred without obligate backtracking.
(A, B) Nucleic-acid scaffolds that enable EC reconstitution just upstream of sequences equivalent to the C346 H-NS-stimulated pause (A) and the C370 pause not stimulated by H-NS (B). Lowercase RNA, …
Figure 7 with 1 supplement
Stimulation of pausing by bridged H-NS filaments aided Rho-dependent termination.
(A, B) Densitometry profiles of transcripts produced at 28°C, 8 mM Mg2+, and 30 µM each NTP from the λPR-bgl template in bridged filaments (66 H-NS/kb) with or without 5 nM Rho (A) and with or …
Figure 7—figure supplement 1
Stimulation of pausing by bridged H-NS filaments aided Rho-dependent termination.
(A) Halted A26 ECs (10 nM) formed on the λPR-bgl template were incubated with bridged H-NS, 75 nM NusG, or 5 nM Rho at 8 mM Mg2+ at 28°C. Samples were then collected at 2, 4, 8, 16, and 32 min after …
Figure 8 with 1 supplement
Linear H-NS filaments did not aid in Rho-dependent termination.
(A) Densitometry profiles of transcripts produced at 28°C, 8 mM Mg2+, and 30 µM each NTP from the λPR-bgl template in linear filaments (200 H-NS/kb) with or without 5 nM Rho. Samples were removed at …
Figure 8—figure supplement 1
Linear H-NS Filaments did not aid in Rho termination.
(A) Halted A26 ECs (10 nM) formed on λPR-bgl template were incubated with either 66 H-NS/kb or 200 H-NS/kb with or without 5 nM Rho at 8 mM Mg2+ and 28°C. Samples were then collected at 2, 4, 8, 16, …
Figure 9
Models for H-NS effects on pausing, Rho termination, and DNA bridging.
(A) As RNAP elongates through bridged filaments, pause durations increase for one or both of two reasons: (i) the off-rate of bridged H-NS is slower than the elongation rate of RNAP, leading to a …
Tables
Table 1
Pause sites and their responses to H-NS and transcription factors
| Pause position | Pausing | Termination | Sequence | ||||||
|---|---|---|---|---|---|---|---|---|---|
| H-NS | RfaH | GreB | GreA | Rho | Rho + H-NS | Rho + NusG | Pause↓ | ||
| C134 | ↑ | ? | ? | ? | ? | ? | ? | CGCUGAUAACUCAAGC | UUUCUUCCUG |
| G162 | ↑ | ↓ | ↓ | ? | – | – | – | AAUUAAGGCUGAACUG | AAAUUUUAUU |
| U169 | ↑ | – | ↓ | ↓ | – | – | ↑ | GCUGAACUGAAAUUUU | AUUAAUUGCA |
| C213 | ↑ | – | ↓ | ↓ | – | – | – | GCGUGACACCUGCAAC | AUCCUCCAUA |
| C220 | ↑ | ↓ | – | ↓ | – | – | – | ACCUGCAACAUCCUCC | AUAUUUCCGC |
| U226 | ↑ | ↓ | – | ↓ | – | – | – | AACAUCCUCCAUAUUU | CCGCUCAUUU |
| C346 | ↑↑↑ | – | ↓ | – | – | ↑ | – | UAGCUGGAACUCUUUC | GGGUAAAGCC |
| C370 | – | ↓ | – | ↓ | ↑ | – | – | CCGCUGGAUAUCCCAC | AGCAACGGGU |
| C393, G394 | ↑ | ↓ | ↓ | ↓ | – | ↑ | ↑ | GGUUGGGCAGCAACAC | GUUUUGCUGA |
| U588, U589 | ↑↑↑ | – | ↓ | ↓ | ↑ | – | – | UCAAGGCAUACUCUUU | UUCUAUUCCA |
| A593 | – | – | – | – | – | – | – | GCAUACUCUUUUUCUA | UUCCACUUGA |
| G624 | ↑ | ↓ | ↓ | ↓ | – | – | – | UUCUUUCGCCAGCGCG | UUUUUGAAAG |
| G643 | ↑ | – | ↓ | ↓ | – | – | – | UUGAAAGCCAAUUCCG | CGCCCCAUGA |
| A746, U747 | – | – | ↓ | – | ↑ | – | ↑ | GCAAGGACCUUUUUUA | UAAACAAAAA |
| G926 | ↑ | – | – | – | – | ? | ? | AAUAUGACCAUGCUCG | CAGUUAUUAA |
| U996 | ↑ | – | ↓ | ↓ | – | ? | ? | CCAAUAAUUAAGUUAU | UGGGAUUUGU |
| U1011 | ↑ | ↓ | ↓ | ↓ | – | ? | ? | UUGGGAUUUGUCUGGU | GAAUUAUUUG |
| U1022, U1024 | ↑ | ↓ | ↓ | ↓ | – | ? | ? | GUCUGGUGAAUUAUUU | GUCGCUAUCU |
| U1079 (ops) | – | – | ↓ | ↓ | ↑ | ? | ? | CUAGUGGCGGUAGCGU | GCUUUUUUCA |
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Pause positions are given as 3′ RNA nucleotide identity and distance from the transcription start site as mapped by high-resolution PAGE (Figure 2—figure supplement 1). ↑, increased pause or termination. ↓, decreased pause or termination. In the sequences shown, pause 3′ ends are bold (under arrow) and the position corresponding to the incoming NTP is underlined.
