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

Formation of stable in vitro transcription reaction intermediates in the presence of RapA. In vitro transcription reactions with supercoiled DNA templates were carried out, in general, as previously described (23), except that 1 mol purified native RapA per mole of the RNA polymerase holoenzyme (0.05–0.06 mg/ml) was used throughout this study, unless indicated otherwise in the figure legends. Reaction products of 15 min in vitro transcription reactions (with or without 1-min boiling following the addition of a Stop solution) were fractionated on 6% PAA–urea gels. Buffer A: 50 mM MOPS (pH 7.0), 5 mM MgCl2; Buffer B: 50 mM Tris–HCl (pH 7.9), 10 mM MgCl2, 200 mM NaCl, 1 mM dithiothreitol. (A–D) Formation of stable RapA-specific transcription reaction intermediates does not depend on the nature of a supercoiled DNA template. Templates 1 and 3, which carry the T7A1 promoter and either lambda tr2 or t3te terminators are described in Ref. (27). Template 2, which carries the tac promoter and t1t2 terminator is described in Ref. (23). Template 4, which carries the lambda Pr promoter, but is otherwise similar to Template 2, was constructed by substituting the tac promoter in Template 2 samples were initiated by the addition of rNTP mix containing either [Alpha-32P] ATP (lanes 9–16) or [Gamma-32P] ATP plus T4 PNK (0.2 U/µl) (lanes 1–8). The stock solutions of both radiolabeled nucleotides (see Materials and Methods section) were diluted (typically, 120-fold and 9-fold, respectively) to obtain comparable incorporations of the label in the two sets of samples; the final rNTP concentrations remained the same ([ATP] = [UTP] = [GTP] = [CTP] = 0.2 mM). The formation of stable RapA-specific reaction intermediates with all four different DNA templates suggests that the effect is not template-specific.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 3: Formation of stable in vitro transcription reaction intermediates in the presence of RapA. In vitro transcription reactions with supercoiled DNA templates were carried out, in general, as previously described (23), except that 1 mol purified native RapA per mole of the RNA polymerase holoenzyme (0.05–0.06 mg/ml) was used throughout this study, unless indicated otherwise in the figure legends. Reaction products of 15 min in vitro transcription reactions (with or without 1-min boiling following the addition of a Stop solution) were fractionated on 6% PAA–urea gels. Buffer A: 50 mM MOPS (pH 7.0), 5 mM MgCl2; Buffer B: 50 mM Tris–HCl (pH 7.9), 10 mM MgCl2, 200 mM NaCl, 1 mM dithiothreitol. (A–D) Formation of stable RapA-specific transcription reaction intermediates does not depend on the nature of a supercoiled DNA template. Templates 1 and 3, which carry the T7A1 promoter and either lambda tr2 or t3te terminators are described in Ref. (27). Template 2, which carries the tac promoter and t1t2 terminator is described in Ref. (23). Template 4, which carries the lambda Pr promoter, but is otherwise similar to Template 2, was constructed by substituting the tac promoter in Template 2 samples were initiated by the addition of rNTP mix containing either [Alpha-32P] ATP (lanes 9–16) or [Gamma-32P] ATP plus T4 PNK (0.2 U/µl) (lanes 1–8). The stock solutions of both radiolabeled nucleotides (see Materials and Methods section) were diluted (typically, 120-fold and 9-fold, respectively) to obtain comparable incorporations of the label in the two sets of samples; the final rNTP concentrations remained the same ([ATP] = [UTP] = [GTP] = [CTP] = 0.2 mM). The formation of stable RapA-specific reaction intermediates with all four different DNA templates suggests that the effect is not template-specific.

Mentions: It is conceivable that if RapA were to promote the release of RNA from transcription complexes, RapA–RNA intermediates might be detected during fractionation of the components of in vitro transcription reactions by PAGE. The experiments described below were carried out to detect such hypothetical RapA–RNA and/or RNA polymerase–RapA–RNA intermediates. Typically, in vitro transcription reactions are denatured by boiling before their RNA content can be analyzed by PAGE. Bypassing the boiling step, we fractionated the content of entire in vitro transcription reactions by PAGE on 6% polyacrylamide–urea gels (Figure 3). This approach revealed unique protein–RNA complexes present only in the reactions containing RapA (Figure 3, complexes ‘B’; the sensitivity to boiling distinguishes these complexes from RNA transcripts). We tested whether these RapA-specific complexes were promoter- or terminator-specific and found that they were detected irrespective of the type of DNA template used in the reactions (Figure 3A–D).Figure 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)

Formation of stable in vitro transcription reaction intermediates in the presence of RapA. In vitro transcription reactions with supercoiled DNA templates were carried out, in general, as previously described (23), except that 1 mol purified native RapA per mole of the RNA polymerase holoenzyme (0.05–0.06 mg/ml) was used throughout this study, unless indicated otherwise in the figure legends. Reaction products of 15 min in vitro transcription reactions (with or without 1-min boiling following the addition of a Stop solution) were fractionated on 6% PAA–urea gels. Buffer A: 50 mM MOPS (pH 7.0), 5 mM MgCl2; Buffer B: 50 mM Tris–HCl (pH 7.9), 10 mM MgCl2, 200 mM NaCl, 1 mM dithiothreitol. (A–D) Formation of stable RapA-specific transcription reaction intermediates does not depend on the nature of a supercoiled DNA template. Templates 1 and 3, which carry the T7A1 promoter and either lambda tr2 or t3te terminators are described in Ref. (27). Template 2, which carries the tac promoter and t1t2 terminator is described in Ref. (23). Template 4, which carries the lambda Pr promoter, but is otherwise similar to Template 2, was constructed by substituting the tac promoter in Template 2 samples were initiated by the addition of rNTP mix containing either [Alpha-32P] ATP (lanes 9–16) or [Gamma-32P] ATP plus T4 PNK (0.2 U/µl) (lanes 1–8). The stock solutions of both radiolabeled nucleotides (see Materials and Methods section) were diluted (typically, 120-fold and 9-fold, respectively) to obtain comparable incorporations of the label in the two sets of samples; the final rNTP concentrations remained the same ([ATP] = [UTP] = [GTP] = [CTP] = 0.2 mM). The formation of stable RapA-specific reaction intermediates with all four different DNA templates suggests that the effect is not template-specific.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 3: Formation of stable in vitro transcription reaction intermediates in the presence of RapA. In vitro transcription reactions with supercoiled DNA templates were carried out, in general, as previously described (23), except that 1 mol purified native RapA per mole of the RNA polymerase holoenzyme (0.05–0.06 mg/ml) was used throughout this study, unless indicated otherwise in the figure legends. Reaction products of 15 min in vitro transcription reactions (with or without 1-min boiling following the addition of a Stop solution) were fractionated on 6% PAA–urea gels. Buffer A: 50 mM MOPS (pH 7.0), 5 mM MgCl2; Buffer B: 50 mM Tris–HCl (pH 7.9), 10 mM MgCl2, 200 mM NaCl, 1 mM dithiothreitol. (A–D) Formation of stable RapA-specific transcription reaction intermediates does not depend on the nature of a supercoiled DNA template. Templates 1 and 3, which carry the T7A1 promoter and either lambda tr2 or t3te terminators are described in Ref. (27). Template 2, which carries the tac promoter and t1t2 terminator is described in Ref. (23). Template 4, which carries the lambda Pr promoter, but is otherwise similar to Template 2, was constructed by substituting the tac promoter in Template 2 samples were initiated by the addition of rNTP mix containing either [Alpha-32P] ATP (lanes 9–16) or [Gamma-32P] ATP plus T4 PNK (0.2 U/µl) (lanes 1–8). The stock solutions of both radiolabeled nucleotides (see Materials and Methods section) were diluted (typically, 120-fold and 9-fold, respectively) to obtain comparable incorporations of the label in the two sets of samples; the final rNTP concentrations remained the same ([ATP] = [UTP] = [GTP] = [CTP] = 0.2 mM). The formation of stable RapA-specific reaction intermediates with all four different DNA templates suggests that the effect is not template-specific.
Mentions: It is conceivable that if RapA were to promote the release of RNA from transcription complexes, RapA–RNA intermediates might be detected during fractionation of the components of in vitro transcription reactions by PAGE. The experiments described below were carried out to detect such hypothetical RapA–RNA and/or RNA polymerase–RapA–RNA intermediates. Typically, in vitro transcription reactions are denatured by boiling before their RNA content can be analyzed by PAGE. Bypassing the boiling step, we fractionated the content of entire in vitro transcription reactions by PAGE on 6% polyacrylamide–urea gels (Figure 3). This approach revealed unique protein–RNA complexes present only in the reactions containing RapA (Figure 3, complexes ‘B’; the sensitivity to boiling distinguishes these complexes from RNA transcripts). We tested whether these RapA-specific complexes were promoter- or terminator-specific and found that they were detected irrespective of the type of DNA template used in the reactions (Figure 3A–D).Figure 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