Limits...
Escherichia coli RNA polymerase-associated SWI/SNF protein RapA: evidence for RNA-directed binding and remodeling activity.

McKinley BA, Sukhodolets MV - Nucleic Acids Res. (2007)

Bottom Line: Specifically, the formation of stable RapA-RNA intermediates in transcription and other, independent lines of evidence presented herein indicate that RapA binds and remodels RNA during transcription.Our results are consistent with RapA promoting RNA release from DNA-RNA polymerase-RNA ternary complexes; this process may be accompanied by the destabilization of non-canonical DNA-RNA complexes (putative DNA-RNA triplexes).Taken together, our data indicate a novel RNA remodeling activity for RapA, a representative of the SWI/SNF protein superfamily.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Biochemistry, Department of Chemistry, Lamar University, Beaumont, TX 77710, USA.

ABSTRACT
Helicase-like SWI/SNF proteins are present in organisms belonging to distant kingdoms from bacteria to humans, indicating that they perform a very basic and ubiquitous form of nucleic acid management; current studies associate the activity of SWI/SNF proteins with remodeling of DNA and DNA-protein complexes. The bacterial SWI/SNF homolog RapA-an integral part of the Escherichia coli RNA polymerase complex-has been implicated in remodeling post-termination DNA-RNA polymerase-RNA ternary complexes (PTC), however its explicit nucleic acid substrates and mechanism remain elusive. Our work presents evidence indicating that RNA is a key substrate of RapA. Specifically, the formation of stable RapA-RNA intermediates in transcription and other, independent lines of evidence presented herein indicate that RapA binds and remodels RNA during transcription. Our results are consistent with RapA promoting RNA release from DNA-RNA polymerase-RNA ternary complexes; this process may be accompanied by the destabilization of non-canonical DNA-RNA complexes (putative DNA-RNA triplexes). Taken together, our data indicate a novel RNA remodeling activity for RapA, a representative of the SWI/SNF protein superfamily.

Show MeSH

Related in: MedlinePlus

RapA promotes ATP-dependent separation of RNA from DNA in putative non-canonical DNA–RNA complexes. (A) PAGE-based demonstration of a non-canonical interaction between the double-stranded DNA probes (shown at the top) and (rA)20 RNA. The buffers used for the sample preparation, gel casting and running are indicated at the left. The 10 µl binding reactions in lanes 2 and 3, 5 and 6, 8 and 9, 11 and 12, 14 and 15 contained, respectively, 40 and 160 pmol DNA. Nucleic acid probes were purified as described in the Materials and Methods section. The overall design of nucleic acid probes used in this set of experiments mimics that described in Ref. (28). Note that the DNA probe shown in lanes 1–3 (5′-dA20dC4dT20) was utilized in the experiments with immobilized non-canonical DNA–RNA complexes described below. (B) RapA promotes destabilization of putative DNA–RNA triplexes in an ATP-dependent manner. A schematic of the experiment is illustrated in the top panel. RNA polymerase, S1, Hfq, NusA and RapA were isolated as described in the Materials and Methods section. Reaction components were present in the following concentrations: RNA polymerase and transcription factors, 200 nM; Tris–HCl (pH 7.5), 50 mM; NaCl, 200 mM; MgCl2, 5 mM; ATP, 0.2 mM. Kinetics of ATP-dependent (red bars) or ATP-independent (blue bars) disruption of DNA–RNA templates by RNA polymerase (lane 2), RapA (lane 5) or a 1:1 RNA polymerase–RapA complex (lane 6) in 10-min (open bars), 30-min (hatched bars) and 90-min (solid bars) reactions. Controls included: no proteins (lane 1); the polymerase-associated RNA-binding factor NusA (lane 3); NusA plus RNA polymerase (lane 4); the ribosomal protein S1 (lane 7); the Sm-like ATPase Hfq (lane 8) and S1 plus Hfq (lane 9). (C) Separation of RNA from DNA in putative triplexes by the RNA polymerase–RapA complex under conditions of excess RapA. Core RNA polymerase, 100 nM; RapA, 800 nM; Tris–HCl (pH 7.5), 50 mM; NaCl, 200 mM; MgCl2, 5 mM; ATP, 2 mM. Approximately 4000 c.p.m. of 32P-labeled DNA was used per reaction; data represent the results of four independent sets of experiments. Coomassie-stained samples of core RNA polymerase (5.6 µg) and RapA (1.4 µg) used in this experiment are shown below.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2175355&req=5

Figure 7: RapA promotes ATP-dependent separation of RNA from DNA in putative non-canonical DNA–RNA complexes. (A) PAGE-based demonstration of a non-canonical interaction between the double-stranded DNA probes (shown at the top) and (rA)20 RNA. The buffers used for the sample preparation, gel casting and running are indicated at the left. The 10 µl binding reactions in lanes 2 and 3, 5 and 6, 8 and 9, 11 and 12, 14 and 15 contained, respectively, 40 and 160 pmol DNA. Nucleic acid probes were purified as described in the Materials and Methods section. The overall design of nucleic acid probes used in this set of experiments mimics that described in Ref. (28). Note that the DNA probe shown in lanes 1–3 (5′-dA20dC4dT20) was utilized in the experiments with immobilized non-canonical DNA–RNA complexes described below. (B) RapA promotes destabilization of putative DNA–RNA triplexes in an ATP-dependent manner. A schematic of the experiment is illustrated in the top panel. RNA polymerase, S1, Hfq, NusA and RapA were isolated as described in the Materials and Methods section. Reaction components were present in the following concentrations: RNA polymerase and transcription factors, 200 nM; Tris–HCl (pH 7.5), 50 mM; NaCl, 200 mM; MgCl2, 5 mM; ATP, 0.2 mM. Kinetics of ATP-dependent (red bars) or ATP-independent (blue bars) disruption of DNA–RNA templates by RNA polymerase (lane 2), RapA (lane 5) or a 1:1 RNA polymerase–RapA complex (lane 6) in 10-min (open bars), 30-min (hatched bars) and 90-min (solid bars) reactions. Controls included: no proteins (lane 1); the polymerase-associated RNA-binding factor NusA (lane 3); NusA plus RNA polymerase (lane 4); the ribosomal protein S1 (lane 7); the Sm-like ATPase Hfq (lane 8) and S1 plus Hfq (lane 9). (C) Separation of RNA from DNA in putative triplexes by the RNA polymerase–RapA complex under conditions of excess RapA. Core RNA polymerase, 100 nM; RapA, 800 nM; Tris–HCl (pH 7.5), 50 mM; NaCl, 200 mM; MgCl2, 5 mM; ATP, 2 mM. Approximately 4000 c.p.m. of 32P-labeled DNA was used per reaction; data represent the results of four independent sets of experiments. Coomassie-stained samples of core RNA polymerase (5.6 µg) and RapA (1.4 µg) used in this experiment are shown below.

Mentions: Synthetic RNA oligonucleotides were obtained from Dharmacon. Nucleic acid probes were labeled at the 5′-end using T4 polynucleotide kinase (USB) and [Gamma 32P] ATP (MP Biomedicals), according to the USB protocol. Following the end-labeling procedure, the RNA probes were gel-purified on a denaturing 20% polyacrylamide gel (National Diagnostics). The gel-purification procedure for RNA probes was as follows. After PAGE, X-ray film was exposed to a ‘wet’ gel that had been covered with plastic wrap. Full-length RNA bands were visualized, marked and cut from the gel. The polyacrylamide gel slice containing the end-labeled RNA was then manually homogenized in an RNase-free, 1.5 ml Eppendorf tube-size disposable homogenizer, typically using 200 µl of 2 × TBE. Following the 2–3-min homogenization, the slurry was immediately applied on a microcentrifuge filter vial (Ultrafree-DA; Millipore), and the flow-through was aliquoted and stored at −70°C. Synthetic DNA oligonucleotides were either gel- or cartridge-purified. DNA oligonucleotides obtained from two independent vendors (Invitrogen and Sigma Genosys) were tested in the DNA–RNA binding experiments, with similar results. Key experiments (including the experiments described in Figure 7B, C and Figure S5) were carried out with synthetic DNA oligonucleotides obtained from Invitrogen. DNA and RNA oligonucleotides were incubated for 5 min at room temperature in the buffers specified in the Figure 7A legend, mixed with 1/5 volume of a loading buffer containing 50% glycerol (Sigma Ultrapure) and 0.01% bromphenol blue and analyzed by PAGE using 20 × 20 cm, 1-mm thick 12% polyacrylamide gels, which were cast and run using the buffers specified in the legend to Figure 7A. Typically, PAGE was performed at 20 milliamps/gel. Tris–Borate–EDTA (TBE) buffer from two independent vendors (KD Medical and MP Biomedicals)—obtained either as a 10× concentrate (KD Medical, MP Biomedicals) or a premixed powder (MP Biomedicals)—was tested, with similar results. The pH in different batches of commercially available 10× TBE was 7.8–8.3.


Escherichia coli RNA polymerase-associated SWI/SNF protein RapA: evidence for RNA-directed binding and remodeling activity.

McKinley BA, Sukhodolets MV - Nucleic Acids Res. (2007)

RapA promotes ATP-dependent separation of RNA from DNA in putative non-canonical DNA–RNA complexes. (A) PAGE-based demonstration of a non-canonical interaction between the double-stranded DNA probes (shown at the top) and (rA)20 RNA. The buffers used for the sample preparation, gel casting and running are indicated at the left. The 10 µl binding reactions in lanes 2 and 3, 5 and 6, 8 and 9, 11 and 12, 14 and 15 contained, respectively, 40 and 160 pmol DNA. Nucleic acid probes were purified as described in the Materials and Methods section. The overall design of nucleic acid probes used in this set of experiments mimics that described in Ref. (28). Note that the DNA probe shown in lanes 1–3 (5′-dA20dC4dT20) was utilized in the experiments with immobilized non-canonical DNA–RNA complexes described below. (B) RapA promotes destabilization of putative DNA–RNA triplexes in an ATP-dependent manner. A schematic of the experiment is illustrated in the top panel. RNA polymerase, S1, Hfq, NusA and RapA were isolated as described in the Materials and Methods section. Reaction components were present in the following concentrations: RNA polymerase and transcription factors, 200 nM; Tris–HCl (pH 7.5), 50 mM; NaCl, 200 mM; MgCl2, 5 mM; ATP, 0.2 mM. Kinetics of ATP-dependent (red bars) or ATP-independent (blue bars) disruption of DNA–RNA templates by RNA polymerase (lane 2), RapA (lane 5) or a 1:1 RNA polymerase–RapA complex (lane 6) in 10-min (open bars), 30-min (hatched bars) and 90-min (solid bars) reactions. Controls included: no proteins (lane 1); the polymerase-associated RNA-binding factor NusA (lane 3); NusA plus RNA polymerase (lane 4); the ribosomal protein S1 (lane 7); the Sm-like ATPase Hfq (lane 8) and S1 plus Hfq (lane 9). (C) Separation of RNA from DNA in putative triplexes by the RNA polymerase–RapA complex under conditions of excess RapA. Core RNA polymerase, 100 nM; RapA, 800 nM; Tris–HCl (pH 7.5), 50 mM; NaCl, 200 mM; MgCl2, 5 mM; ATP, 2 mM. Approximately 4000 c.p.m. of 32P-labeled DNA was used per reaction; data represent the results of four independent sets of experiments. Coomassie-stained samples of core RNA polymerase (5.6 µg) and RapA (1.4 µg) used in this experiment are shown below.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2175355&req=5

Figure 7: RapA promotes ATP-dependent separation of RNA from DNA in putative non-canonical DNA–RNA complexes. (A) PAGE-based demonstration of a non-canonical interaction between the double-stranded DNA probes (shown at the top) and (rA)20 RNA. The buffers used for the sample preparation, gel casting and running are indicated at the left. The 10 µl binding reactions in lanes 2 and 3, 5 and 6, 8 and 9, 11 and 12, 14 and 15 contained, respectively, 40 and 160 pmol DNA. Nucleic acid probes were purified as described in the Materials and Methods section. The overall design of nucleic acid probes used in this set of experiments mimics that described in Ref. (28). Note that the DNA probe shown in lanes 1–3 (5′-dA20dC4dT20) was utilized in the experiments with immobilized non-canonical DNA–RNA complexes described below. (B) RapA promotes destabilization of putative DNA–RNA triplexes in an ATP-dependent manner. A schematic of the experiment is illustrated in the top panel. RNA polymerase, S1, Hfq, NusA and RapA were isolated as described in the Materials and Methods section. Reaction components were present in the following concentrations: RNA polymerase and transcription factors, 200 nM; Tris–HCl (pH 7.5), 50 mM; NaCl, 200 mM; MgCl2, 5 mM; ATP, 0.2 mM. Kinetics of ATP-dependent (red bars) or ATP-independent (blue bars) disruption of DNA–RNA templates by RNA polymerase (lane 2), RapA (lane 5) or a 1:1 RNA polymerase–RapA complex (lane 6) in 10-min (open bars), 30-min (hatched bars) and 90-min (solid bars) reactions. Controls included: no proteins (lane 1); the polymerase-associated RNA-binding factor NusA (lane 3); NusA plus RNA polymerase (lane 4); the ribosomal protein S1 (lane 7); the Sm-like ATPase Hfq (lane 8) and S1 plus Hfq (lane 9). (C) Separation of RNA from DNA in putative triplexes by the RNA polymerase–RapA complex under conditions of excess RapA. Core RNA polymerase, 100 nM; RapA, 800 nM; Tris–HCl (pH 7.5), 50 mM; NaCl, 200 mM; MgCl2, 5 mM; ATP, 2 mM. Approximately 4000 c.p.m. of 32P-labeled DNA was used per reaction; data represent the results of four independent sets of experiments. Coomassie-stained samples of core RNA polymerase (5.6 µg) and RapA (1.4 µg) used in this experiment are shown below.
Mentions: Synthetic RNA oligonucleotides were obtained from Dharmacon. Nucleic acid probes were labeled at the 5′-end using T4 polynucleotide kinase (USB) and [Gamma 32P] ATP (MP Biomedicals), according to the USB protocol. Following the end-labeling procedure, the RNA probes were gel-purified on a denaturing 20% polyacrylamide gel (National Diagnostics). The gel-purification procedure for RNA probes was as follows. After PAGE, X-ray film was exposed to a ‘wet’ gel that had been covered with plastic wrap. Full-length RNA bands were visualized, marked and cut from the gel. The polyacrylamide gel slice containing the end-labeled RNA was then manually homogenized in an RNase-free, 1.5 ml Eppendorf tube-size disposable homogenizer, typically using 200 µl of 2 × TBE. Following the 2–3-min homogenization, the slurry was immediately applied on a microcentrifuge filter vial (Ultrafree-DA; Millipore), and the flow-through was aliquoted and stored at −70°C. Synthetic DNA oligonucleotides were either gel- or cartridge-purified. DNA oligonucleotides obtained from two independent vendors (Invitrogen and Sigma Genosys) were tested in the DNA–RNA binding experiments, with similar results. Key experiments (including the experiments described in Figure 7B, C and Figure S5) were carried out with synthetic DNA oligonucleotides obtained from Invitrogen. DNA and RNA oligonucleotides were incubated for 5 min at room temperature in the buffers specified in the Figure 7A legend, mixed with 1/5 volume of a loading buffer containing 50% glycerol (Sigma Ultrapure) and 0.01% bromphenol blue and analyzed by PAGE using 20 × 20 cm, 1-mm thick 12% polyacrylamide gels, which were cast and run using the buffers specified in the legend to Figure 7A. Typically, PAGE was performed at 20 milliamps/gel. Tris–Borate–EDTA (TBE) buffer from two independent vendors (KD Medical and MP Biomedicals)—obtained either as a 10× concentrate (KD Medical, MP Biomedicals) or a premixed powder (MP Biomedicals)—was tested, with similar results. The pH in different batches of commercially available 10× TBE was 7.8–8.3.

Bottom Line: Specifically, the formation of stable RapA-RNA intermediates in transcription and other, independent lines of evidence presented herein indicate that RapA binds and remodels RNA during transcription.Our results are consistent with RapA promoting RNA release from DNA-RNA polymerase-RNA ternary complexes; this process may be accompanied by the destabilization of non-canonical DNA-RNA complexes (putative DNA-RNA triplexes).Taken together, our data indicate a novel RNA remodeling activity for RapA, a representative of the SWI/SNF protein superfamily.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Biochemistry, Department of Chemistry, Lamar University, Beaumont, TX 77710, USA.

ABSTRACT
Helicase-like SWI/SNF proteins are present in organisms belonging to distant kingdoms from bacteria to humans, indicating that they perform a very basic and ubiquitous form of nucleic acid management; current studies associate the activity of SWI/SNF proteins with remodeling of DNA and DNA-protein complexes. The bacterial SWI/SNF homolog RapA-an integral part of the Escherichia coli RNA polymerase complex-has been implicated in remodeling post-termination DNA-RNA polymerase-RNA ternary complexes (PTC), however its explicit nucleic acid substrates and mechanism remain elusive. Our work presents evidence indicating that RNA is a key substrate of RapA. Specifically, the formation of stable RapA-RNA intermediates in transcription and other, independent lines of evidence presented herein indicate that RapA binds and remodels RNA during transcription. Our results are consistent with RapA promoting RNA release from DNA-RNA polymerase-RNA ternary complexes; this process may be accompanied by the destabilization of non-canonical DNA-RNA complexes (putative DNA-RNA triplexes). Taken together, our data indicate a novel RNA remodeling activity for RapA, a representative of the SWI/SNF protein superfamily.

Show MeSH
Related in: MedlinePlus