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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.

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RapA promotes interaction of RNA polymerase with RNA. EMSA gel illustrating the effect of RapA on interaction of the core RNA polymerase (lanes 2–7) or the RNA polymerase holoenzyme (lanes 10–15) with end-labeled 55-nt RNA incorporating stem-loop structures and an rA18 tail. EMSA-binding experiments were carried out as described in Materials and Methods section. Other RNA probes of varied length and structure (see text for details) produced similar binding patterns, indicating a greatly increased RNA-binding affinity of the polymerase in the presence of RapA. Quantitation of the RNA polymerase (holoenzyme)-bound RNA in the presence or absence of RapA (lanes 15 and 12) indicated a >20-fold increase in RNA-binding affinity in the presence of RapA. Note that RapA abolishes the formation of multimeric RNA polymerase–RNA complexes formed by the core RNA polymerase (indicated with arrowheads).
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Figure 2: RapA promotes interaction of RNA polymerase with RNA. EMSA gel illustrating the effect of RapA on interaction of the core RNA polymerase (lanes 2–7) or the RNA polymerase holoenzyme (lanes 10–15) with end-labeled 55-nt RNA incorporating stem-loop structures and an rA18 tail. EMSA-binding experiments were carried out as described in Materials and Methods section. Other RNA probes of varied length and structure (see text for details) produced similar binding patterns, indicating a greatly increased RNA-binding affinity of the polymerase in the presence of RapA. Quantitation of the RNA polymerase (holoenzyme)-bound RNA in the presence or absence of RapA (lanes 15 and 12) indicated a >20-fold increase in RNA-binding affinity in the presence of RapA. Note that RapA abolishes the formation of multimeric RNA polymerase–RNA complexes formed by the core RNA polymerase (indicated with arrowheads).

Mentions: Proteins and the 32P-labeled purified RNA probe (typically present at 500–1000 c.p.m./20 µl binding reaction) were mixed in 50 mM Tris–HCl, pH 7.5, containing 5 mM MgCl2, 0.5 mg/ml BSA and 200 mM NaCl. The binding reactions were incubated for 5 min at room temperature, and 5 µl of loading buffer (50% glycerol supplemented with 0.05% bromphenol blue) was added to each binding reaction. Aliquots of 2–8 µl were then analyzed on 8% polyacrylamide gels containing 0.5× TBE or 2× TBE, as described in the legend to Figure 2. BioMax ML film was exposed to dried gels (typically 6–18 h at −70°C) with BioMax MS screens.


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 interaction of RNA polymerase with RNA. EMSA gel illustrating the effect of RapA on interaction of the core RNA polymerase (lanes 2–7) or the RNA polymerase holoenzyme (lanes 10–15) with end-labeled 55-nt RNA incorporating stem-loop structures and an rA18 tail. EMSA-binding experiments were carried out as described in Materials and Methods section. Other RNA probes of varied length and structure (see text for details) produced similar binding patterns, indicating a greatly increased RNA-binding affinity of the polymerase in the presence of RapA. Quantitation of the RNA polymerase (holoenzyme)-bound RNA in the presence or absence of RapA (lanes 15 and 12) indicated a >20-fold increase in RNA-binding affinity in the presence of RapA. Note that RapA abolishes the formation of multimeric RNA polymerase–RNA complexes formed by the core RNA polymerase (indicated with arrowheads).
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 2: RapA promotes interaction of RNA polymerase with RNA. EMSA gel illustrating the effect of RapA on interaction of the core RNA polymerase (lanes 2–7) or the RNA polymerase holoenzyme (lanes 10–15) with end-labeled 55-nt RNA incorporating stem-loop structures and an rA18 tail. EMSA-binding experiments were carried out as described in Materials and Methods section. Other RNA probes of varied length and structure (see text for details) produced similar binding patterns, indicating a greatly increased RNA-binding affinity of the polymerase in the presence of RapA. Quantitation of the RNA polymerase (holoenzyme)-bound RNA in the presence or absence of RapA (lanes 15 and 12) indicated a >20-fold increase in RNA-binding affinity in the presence of RapA. Note that RapA abolishes the formation of multimeric RNA polymerase–RNA complexes formed by the core RNA polymerase (indicated with arrowheads).
Mentions: Proteins and the 32P-labeled purified RNA probe (typically present at 500–1000 c.p.m./20 µl binding reaction) were mixed in 50 mM Tris–HCl, pH 7.5, containing 5 mM MgCl2, 0.5 mg/ml BSA and 200 mM NaCl. The binding reactions were incubated for 5 min at room temperature, and 5 µl of loading buffer (50% glycerol supplemented with 0.05% bromphenol blue) was added to each binding reaction. Aliquots of 2–8 µl were then analyzed on 8% polyacrylamide gels containing 0.5× TBE or 2× TBE, as described in the legend to Figure 2. BioMax ML film was exposed to dried gels (typically 6–18 h at −70°C) with BioMax MS screens.

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