Amino-terminal sequences of sigmaN (sigma54) inhibit RNA polymerase isomerization

Abstract

In bacteria, association of the specialized sigmaN protein with the core RNA polymerase subunits forms a holoenzyme able to bind promoter DNA, but unable to melt DNA and initiate transcription unless acted on by an activator protein. The conserved amino-terminal 50 amino acids of sigmaN (Region I) are required for the response to activators. We have used pre-melted DNA templates, in which the template strand is unpaired and accessible for transcription initiation, to mimic a naturally melted promoter and explore the function of Region I. Our results indicate that one activity of Region I sequences is to inhibit productive interaction of holoenzyme with pre-melted DNA. On pre-melted DNA targets, either activation of sigmaN-holoenzyme or removal of Region I allowed efficient formation of complexes in which melted DNA was sequestered by RNA polymerase. Like natural pre-initiation complexes formed on conventional DNA templates through the action of activator, such complexes were heparin-resistant and transcriptionally active. The inhibitory sigmaN Region I domain functioned in trans to confer heparin sensitivity to complexes between Region I-deleted holoenzyme and pre-melted promoter DNA. Evidence that Region I senses the conformation of the promoter was obtained from protein footprint experiments. We suggest that one function for Region I is to mask a single-strand DNA-binding activity of the holoenzyme. On the basis of extended DNA footprints of Region I-deleted holoenzyme, we also propose that Region I prevents RNA polymerase isomerization, a conformational change necessary for access to and the subsequent stable association of holoenzyme with melted DNA.

Figure 1

Gel mobility-shift assays on R. meliloti nifH promoter DNA. (A) Effect of activator and/or nucleotides on heparin-resistant complex formation between σ^N- and ΔIσ^N-holoenzymes (Eσ^N and EΔIσ^N) and heteroduplex 1 DNA (see Table 1). Holoenzymes were at 100 nm, activator PspFΔHTH (lanes 3,4,6,7,10,13,14) at 4 μm, nucleotides at 1 mm and end-labeled DNA at 16 nm. Prior to gel loading, heparin (100 μg/ml) was added for 5 min. Reactions included salmon sperm DNA (455 ng/μl), which helped to reduce background smears. The percentage of DNA shifted is indicated in the histogram. (B) Stability of holoenzyme complexes on heteroduplex DNA. Following holoenzyme binding to DNA and incubation with PspFΔHTH and ddGTP where indicated, samples were taken prior to (time 0) and after addition of heparin for 1, 5, 10, and 20 min, and immediately loaded onto running native gels. Holoenzymes (Eσ^N, EΔIσ^N) alone (□,█), plus ddGTP (○,●), and plus ddGTP and PspFΔHTH (▵,▴). (C) Formation of heparin-resistant complexes with homoduplex DNA. Holoenzymes were at 250 nm, end-labeled 88-mer homoduplex DNA at 16 nm, PspFΔHTH at 4 μm and GTP at 4 mm (lanes 1,3,6). Prior to gel loading, heparin (lanes 2,3,5,6) was added for 5 min. The level of initial complexes (lanes 1,4) was unaffected by GTP plus PspFΔHTH (data not shown). (D) Region I sequences confer heparin sensitivity to holoenzyme. Holoenzymes (50 nm) containing end-labeled σ proteins (Eσ^N-³²P, EΔIσ^N-³²P) were incubated with or without 50 nm 88-mer homoduplex (lanes 3,4,11,12) and heteroduplex 1 (lanes 5–8,13,14), challenged with heparin for 5 min where indicated and loaded onto native gels. PspFΔHTH (4 μm) and GTP (4 mm) were added for activation (lanes 7,8). (H) Holoenzyme; (H+DNA) holoenzyme–DNA complex; (σ) free sigma.

Figure 1

Gel mobility-shift assays on R. meliloti nifH promoter DNA. (A) Effect of activator and/or nucleotides on heparin-resistant complex formation between σ^N- and ΔIσ^N-holoenzymes (Eσ^N and EΔIσ^N) and heteroduplex 1 DNA (see Table 1). Holoenzymes were at 100 nm, activator PspFΔHTH (lanes 3,4,6,7,10,13,14) at 4 μm, nucleotides at 1 mm and end-labeled DNA at 16 nm. Prior to gel loading, heparin (100 μg/ml) was added for 5 min. Reactions included salmon sperm DNA (455 ng/μl), which helped to reduce background smears. The percentage of DNA shifted is indicated in the histogram. (B) Stability of holoenzyme complexes on heteroduplex DNA. Following holoenzyme binding to DNA and incubation with PspFΔHTH and ddGTP where indicated, samples were taken prior to (time 0) and after addition of heparin for 1, 5, 10, and 20 min, and immediately loaded onto running native gels. Holoenzymes (Eσ^N, EΔIσ^N) alone (□,█), plus ddGTP (○,●), and plus ddGTP and PspFΔHTH (▵,▴). (C) Formation of heparin-resistant complexes with homoduplex DNA. Holoenzymes were at 250 nm, end-labeled 88-mer homoduplex DNA at 16 nm, PspFΔHTH at 4 μm and GTP at 4 mm (lanes 1,3,6). Prior to gel loading, heparin (lanes 2,3,5,6) was added for 5 min. The level of initial complexes (lanes 1,4) was unaffected by GTP plus PspFΔHTH (data not shown). (D) Region I sequences confer heparin sensitivity to holoenzyme. Holoenzymes (50 nm) containing end-labeled σ proteins (Eσ^N-³²P, EΔIσ^N-³²P) were incubated with or without 50 nm 88-mer homoduplex (lanes 3,4,11,12) and heteroduplex 1 (lanes 5–8,13,14), challenged with heparin for 5 min where indicated and loaded onto native gels. PspFΔHTH (4 μm) and GTP (4 mm) were added for activation (lanes 7,8). (H) Holoenzyme; (H+DNA) holoenzyme–DNA complex; (σ) free sigma.

Figure 1

Gel mobility-shift assays on R. meliloti nifH promoter DNA. (A) Effect of activator and/or nucleotides on heparin-resistant complex formation between σ^N- and ΔIσ^N-holoenzymes (Eσ^N and EΔIσ^N) and heteroduplex 1 DNA (see Table 1). Holoenzymes were at 100 nm, activator PspFΔHTH (lanes 3,4,6,7,10,13,14) at 4 μm, nucleotides at 1 mm and end-labeled DNA at 16 nm. Prior to gel loading, heparin (100 μg/ml) was added for 5 min. Reactions included salmon sperm DNA (455 ng/μl), which helped to reduce background smears. The percentage of DNA shifted is indicated in the histogram. (B) Stability of holoenzyme complexes on heteroduplex DNA. Following holoenzyme binding to DNA and incubation with PspFΔHTH and ddGTP where indicated, samples were taken prior to (time 0) and after addition of heparin for 1, 5, 10, and 20 min, and immediately loaded onto running native gels. Holoenzymes (Eσ^N, EΔIσ^N) alone (□,█), plus ddGTP (○,●), and plus ddGTP and PspFΔHTH (▵,▴). (C) Formation of heparin-resistant complexes with homoduplex DNA. Holoenzymes were at 250 nm, end-labeled 88-mer homoduplex DNA at 16 nm, PspFΔHTH at 4 μm and GTP at 4 mm (lanes 1,3,6). Prior to gel loading, heparin (lanes 2,3,5,6) was added for 5 min. The level of initial complexes (lanes 1,4) was unaffected by GTP plus PspFΔHTH (data not shown). (D) Region I sequences confer heparin sensitivity to holoenzyme. Holoenzymes (50 nm) containing end-labeled σ proteins (Eσ^N-³²P, EΔIσ^N-³²P) were incubated with or without 50 nm 88-mer homoduplex (lanes 3,4,11,12) and heteroduplex 1 (lanes 5–8,13,14), challenged with heparin for 5 min where indicated and loaded onto native gels. PspFΔHTH (4 μm) and GTP (4 mm) were added for activation (lanes 7,8). (H) Holoenzyme; (H+DNA) holoenzyme–DNA complex; (σ) free sigma.

Figure 1

Gel mobility-shift assays on R. meliloti nifH promoter DNA. (A) Effect of activator and/or nucleotides on heparin-resistant complex formation between σ^N- and ΔIσ^N-holoenzymes (Eσ^N and EΔIσ^N) and heteroduplex 1 DNA (see Table 1). Holoenzymes were at 100 nm, activator PspFΔHTH (lanes 3,4,6,7,10,13,14) at 4 μm, nucleotides at 1 mm and end-labeled DNA at 16 nm. Prior to gel loading, heparin (100 μg/ml) was added for 5 min. Reactions included salmon sperm DNA (455 ng/μl), which helped to reduce background smears. The percentage of DNA shifted is indicated in the histogram. (B) Stability of holoenzyme complexes on heteroduplex DNA. Following holoenzyme binding to DNA and incubation with PspFΔHTH and ddGTP where indicated, samples were taken prior to (time 0) and after addition of heparin for 1, 5, 10, and 20 min, and immediately loaded onto running native gels. Holoenzymes (Eσ^N, EΔIσ^N) alone (□,█), plus ddGTP (○,●), and plus ddGTP and PspFΔHTH (▵,▴). (C) Formation of heparin-resistant complexes with homoduplex DNA. Holoenzymes were at 250 nm, end-labeled 88-mer homoduplex DNA at 16 nm, PspFΔHTH at 4 μm and GTP at 4 mm (lanes 1,3,6). Prior to gel loading, heparin (lanes 2,3,5,6) was added for 5 min. The level of initial complexes (lanes 1,4) was unaffected by GTP plus PspFΔHTH (data not shown). (D) Region I sequences confer heparin sensitivity to holoenzyme. Holoenzymes (50 nm) containing end-labeled σ proteins (Eσ^N-³²P, EΔIσ^N-³²P) were incubated with or without 50 nm 88-mer homoduplex (lanes 3,4,11,12) and heteroduplex 1 (lanes 5–8,13,14), challenged with heparin for 5 min where indicated and loaded onto native gels. PspFΔHTH (4 μm) and GTP (4 mm) were added for activation (lanes 7,8). (H) Holoenzyme; (H+DNA) holoenzyme–DNA complex; (σ) free sigma.

Figure 2

Binding of σ proteins (σ) and their holoenzymes (Eσ) to 88-mer homoduplex and heteroduplex 1 DNA. Binding reactions were essentially as in Fig. 1A, except that 1.6 nm DNA was used and heparin and nonspecific DNA were omitted. (□) σ^N, (○) ΔIσ^N, (█) σ^N-holoenzyme, (●) ΔIσ^N-holoenzyme.

Figure 3

Region I sequences are sensitive to promoter DNA conformation. End-labeled σ^N (lanes 1–4) or holoenzyme prepared with end-labeled σ^N (lanes 5–7) were incubated for 10 min without DNA (lanes 2,5), with homoduplex DNA (lanes 3,6) or with heteroduplex DNA (lanes 4,7) and then cleaved with endoproteinase Glu-C (Casaz and Buck 1997). Numbers on the left indicate the amino acid at which cleavage has occurred.

Figure 4

Premelted sequences are sequestered in stable complexes but accessible to KMnO₄. (A) Gel mobility-shift assays of heteroduplex 1 DNA complexes. Binding reactions were conducted as in Fig. 1A, treated with S1 nuclease or KMnO₄ (see Material and Methods) and loaded onto running native gels. (B) Bound DNA (lanes 3–6); (P) undigested probe (lane 1); (F) digested probe (lane 2); (U) unbound DNA (lanes 3–6). (B,C) The DNA bands were isolated from native gels described in A, and both strands analyzed on sequencing gels [(B) top strand; (C) bottom strand]. The mismatch region between −10 and −1 is indicated. Markers (M) were generated by chemical cleavage of the DNA with piperidine following partial methylation with dimethylsulfate. Probe alone or cleaved with piperidine (without treatment with KMnO₄) are lanes marked P− and P+, respectively. Footprints of ΔIσ^N-holoenzyme plus PspFΔHTH and ddGTP were the same as ΔIσ^N-holoenzyme alone (not shown).

Figure 4

Premelted sequences are sequestered in stable complexes but accessible to KMnO₄. (A) Gel mobility-shift assays of heteroduplex 1 DNA complexes. Binding reactions were conducted as in Fig. 1A, treated with S1 nuclease or KMnO₄ (see Material and Methods) and loaded onto running native gels. (B) Bound DNA (lanes 3–6); (P) undigested probe (lane 1); (F) digested probe (lane 2); (U) unbound DNA (lanes 3–6). (B,C) The DNA bands were isolated from native gels described in A, and both strands analyzed on sequencing gels [(B) top strand; (C) bottom strand]. The mismatch region between −10 and −1 is indicated. Markers (M) were generated by chemical cleavage of the DNA with piperidine following partial methylation with dimethylsulfate. Probe alone or cleaved with piperidine (without treatment with KMnO₄) are lanes marked P− and P+, respectively. Footprints of ΔIσ^N-holoenzyme plus PspFΔHTH and ddGTP were the same as ΔIσ^N-holoenzyme alone (not shown).

Figure 4

Premelted sequences are sequestered in stable complexes but accessible to KMnO₄. (A) Gel mobility-shift assays of heteroduplex 1 DNA complexes. Binding reactions were conducted as in Fig. 1A, treated with S1 nuclease or KMnO₄ (see Material and Methods) and loaded onto running native gels. (B) Bound DNA (lanes 3–6); (P) undigested probe (lane 1); (F) digested probe (lane 2); (U) unbound DNA (lanes 3–6). (B,C) The DNA bands were isolated from native gels described in A, and both strands analyzed on sequencing gels [(B) top strand; (C) bottom strand]. The mismatch region between −10 and −1 is indicated. Markers (M) were generated by chemical cleavage of the DNA with piperidine following partial methylation with dimethylsulfate. Probe alone or cleaved with piperidine (without treatment with KMnO₄) are lanes marked P− and P+, respectively. Footprints of ΔIσ^N-holoenzyme plus PspFΔHTH and ddGTP were the same as ΔIσ^N-holoenzyme alone (not shown).

Figure 5

Holoenzyme complexes lacking Region I are transcriptionally active on heteroduplex DNA without activator. Holoenzymes (100 nm) with GTP or ATP (1 mm) in the presence or absence of PspFΔHTH (4 μm) were used to transcribe the 88-mer homoduplex and heteroduplex 1 (16 nm) templates. Core RNAP and holoenzyme containing the DNA nonbinding 1–424 σ derivative (Cannon et al. 1995b) were used as controls. The location of the DNA size markers are shown (arrows). (A) [α-³²P]UTP-labeled transcripts with GTP as the hydrolyzable nucleotide used by activator. (B) [γ-³²P]GTP-labeled transcripts with ATP as the hydrolyzable nucleotide used by activator.

Figure 5

Holoenzyme complexes lacking Region I are transcriptionally active on heteroduplex DNA without activator. Holoenzymes (100 nm) with GTP or ATP (1 mm) in the presence or absence of PspFΔHTH (4 μm) were used to transcribe the 88-mer homoduplex and heteroduplex 1 (16 nm) templates. Core RNAP and holoenzyme containing the DNA nonbinding 1–424 σ derivative (Cannon et al. 1995b) were used as controls. The location of the DNA size markers are shown (arrows). (A) [α-³²P]UTP-labeled transcripts with GTP as the hydrolyzable nucleotide used by activator. (B) [γ-³²P]GTP-labeled transcripts with ATP as the hydrolyzable nucleotide used by activator.

Figure 6

Region I-containing fragments confer heparin sensitivity in trans. (A) Different amino-terminal peptides of σ^N: 1–51, 1–63, 1–71, 1–71Δ (15 μm) and 1–56 (2.5 μm) were added to ΔIσ^N (200 nm) prior to core RNAP addition (100 nm) and heteroduplex 1 DNA (16 nm) was added last in a binding reaction. After incubation, heparin was added for 5 min and samples loaded onto native gels. The percentage of heparin-resistant DNA complexes is indicated. (B) Effect of Region I on heparin stability. Gel mobility shifts were conducted with ΔIσ^N-holoenzyme (100 nm), GTP (1 mm) and heteroduplex 1 DNA (16 nm; █) and Region I peptide (1–56, 2 μm) was added before ΔIσ^N-holoenyme assembly (▴) or after holoenzyme–heteroduplex complex formation (●). σ^N-Holoenzyme plus heteroduplex DNA, in which most complexes are heparin sensitive (see Fig. 1B), was used as a control (□). Samples were taken prior to (time 0) and after the addition of heparin for 1, 5, 10, 20 min.

Figure 6

Region I-containing fragments confer heparin sensitivity in trans. (A) Different amino-terminal peptides of σ^N: 1–51, 1–63, 1–71, 1–71Δ (15 μm) and 1–56 (2.5 μm) were added to ΔIσ^N (200 nm) prior to core RNAP addition (100 nm) and heteroduplex 1 DNA (16 nm) was added last in a binding reaction. After incubation, heparin was added for 5 min and samples loaded onto native gels. The percentage of heparin-resistant DNA complexes is indicated. (B) Effect of Region I on heparin stability. Gel mobility shifts were conducted with ΔIσ^N-holoenzyme (100 nm), GTP (1 mm) and heteroduplex 1 DNA (16 nm; █) and Region I peptide (1–56, 2 μm) was added before ΔIσ^N-holoenyme assembly (▴) or after holoenzyme–heteroduplex complex formation (●). σ^N-Holoenzyme plus heteroduplex DNA, in which most complexes are heparin sensitive (see Fig. 1B), was used as a control (□). Samples were taken prior to (time 0) and after the addition of heparin for 1, 5, 10, 20 min.

Figure 7

S1 nuclease footprints on linear DNA. Linear end-labeled templates prepared by primer extension were used. (A) σ^N and holoenzyme (amino acids 1–477); (B) ΔIσ^N and holoenzyme (amino acids 57–477). (Solid line) The extent of protection seen with holoenzyme (H) or σ (S); (broken line) weaker protection; (star) −10 cutting lost on holoenzyme binding. Markers were chemical cleavage G reactions. σ Proteins were at 1 μm, holoenzymes at 100 nm, activator PspFΔHTH at 4 μm, and GTP at 1 mm. Compared to σ^N-holoenzyme, the ΔIσ^N-holoenzyme displays an extended footprint in the direction of transcription (A vs. B). Activation of the σ^N-holoenzyme by PspFΔHTH (A) results in an extended footprint with increased protection from −5 to +7. The extended footprint to +20 observed with ΔIσ^N-holoenzyme is independent of activator and nucleotide. Reproducible footprints and cutting of promoter template DNA was observed with several different batches of S1 nuclease. A control with core RNAP did not produce a S1 footprint.

Figure 7

S1 nuclease footprints on linear DNA. Linear end-labeled templates prepared by primer extension were used. (A) σ^N and holoenzyme (amino acids 1–477); (B) ΔIσ^N and holoenzyme (amino acids 57–477). (Solid line) The extent of protection seen with holoenzyme (H) or σ (S); (broken line) weaker protection; (star) −10 cutting lost on holoenzyme binding. Markers were chemical cleavage G reactions. σ Proteins were at 1 μm, holoenzymes at 100 nm, activator PspFΔHTH at 4 μm, and GTP at 1 mm. Compared to σ^N-holoenzyme, the ΔIσ^N-holoenzyme displays an extended footprint in the direction of transcription (A vs. B). Activation of the σ^N-holoenzyme by PspFΔHTH (A) results in an extended footprint with increased protection from −5 to +7. The extended footprint to +20 observed with ΔIσ^N-holoenzyme is independent of activator and nucleotide. Reproducible footprints and cutting of promoter template DNA was observed with several different batches of S1 nuclease. A control with core RNAP did not produce a S1 footprint.

Figure 8

Gel mobility-shift, footprinting, and transcription assays (see Figs. 1, 4, and 7) show activator and nucleotide hydrolysis are required to efficiently form stable heparin-resistant σ^N-holoenzyme complexes on homoduplex (open complex, in which DNA is melted) and heteroduplex DNA. In contrast, ΔIσ^N-holoenzyme bypasses the activator requirement for stable complex formation on heteroduplex DNA but fails on homoduplex, regardless of the presence of activator. We propose that the inhibition of DNA melting associated with Region I (Wang et al. 1995, 1997a) is due to prevention of polymerase isomerization, necessary to reveal a single-strand DNA-binding activity in the holoenzyme required for its stable association with melted DNA (Fig. 4). The thermodynamic barrier to open complex formation (Wedel and Kustu 1995) at σ^N-dependent promoters is suggested to be associated with energetically unfavorable DNA strand separation.