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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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Immunological characterization of the composition of the RapA-specific in vitro transcription reaction intermediates. In vitro transcription reactions similar to those described in Figure 3 (Template 2) were carried out, and the entire reactions were fractionated on 6% polyacrylamide–6 M urea gels (National Diagnostics). Following the electrophoresis, X-ray films were exposed to ‘wet’ gels to visualize 32P-labeled RNA transcripts (left panels). Next, the gel contents were electroeluted onto Hybond P membranes (Amersham Pharmacia Biotech), and the membranes were immunostained with either RapA- or RNA polymerase-specific antibodies in independent, parallel reactions (right panels). Western blotting was performed as described (22). DNA-associated RNA polymerase (indicated with an arrowhead), free RNA polymerase, RNA polymerase–RNA and RapA–RNA complexes are indicated.
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Figure 4: Immunological characterization of the composition of the RapA-specific in vitro transcription reaction intermediates. In vitro transcription reactions similar to those described in Figure 3 (Template 2) were carried out, and the entire reactions were fractionated on 6% polyacrylamide–6 M urea gels (National Diagnostics). Following the electrophoresis, X-ray films were exposed to ‘wet’ gels to visualize 32P-labeled RNA transcripts (left panels). Next, the gel contents were electroeluted onto Hybond P membranes (Amersham Pharmacia Biotech), and the membranes were immunostained with either RapA- or RNA polymerase-specific antibodies in independent, parallel reactions (right panels). Western blotting was performed as described (22). DNA-associated RNA polymerase (indicated with an arrowhead), free RNA polymerase, RNA polymerase–RNA and RapA–RNA complexes are indicated.

Mentions: To determine the composition of these complexes, we used two independent experimental approaches. In the first approach (an immunoassay), proteins and protein–nucleic acid complexes were electroeluted from the gel onto a membrane which was subsequently incubated with either RNA polymerase-specific or RapA-specific polyclonal antibodies (in parallel reactions). In the second approach, RapA-specific complexes (‘B’ complexes in Figures 3 and 4), identified after exposure of X-ray films to ‘wet’ polyacrylamide gels, were excised from the gel; the gel slices were homogenized in Laemmli sample buffer and their content was analyzed on silver-stained SDS–polyacrylamide gels. Both approaches produced consistent results, indicating that the complexes in question contain a sole polypeptide—RapA—and RNA (the results of an immunoassay are shown in Figure 4). We also analyzed the nature of the RapA-associated RNA transcripts and determined that they are represented predominantly by a major promoter-specific RNA transcript, unique for each of the two sets of templates used (data not shown).Figure 4.


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)

Immunological characterization of the composition of the RapA-specific in vitro transcription reaction intermediates. In vitro transcription reactions similar to those described in Figure 3 (Template 2) were carried out, and the entire reactions were fractionated on 6% polyacrylamide–6 M urea gels (National Diagnostics). Following the electrophoresis, X-ray films were exposed to ‘wet’ gels to visualize 32P-labeled RNA transcripts (left panels). Next, the gel contents were electroeluted onto Hybond P membranes (Amersham Pharmacia Biotech), and the membranes were immunostained with either RapA- or RNA polymerase-specific antibodies in independent, parallel reactions (right panels). Western blotting was performed as described (22). DNA-associated RNA polymerase (indicated with an arrowhead), free RNA polymerase, RNA polymerase–RNA and RapA–RNA complexes are indicated.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

Figure 4: Immunological characterization of the composition of the RapA-specific in vitro transcription reaction intermediates. In vitro transcription reactions similar to those described in Figure 3 (Template 2) were carried out, and the entire reactions were fractionated on 6% polyacrylamide–6 M urea gels (National Diagnostics). Following the electrophoresis, X-ray films were exposed to ‘wet’ gels to visualize 32P-labeled RNA transcripts (left panels). Next, the gel contents were electroeluted onto Hybond P membranes (Amersham Pharmacia Biotech), and the membranes were immunostained with either RapA- or RNA polymerase-specific antibodies in independent, parallel reactions (right panels). Western blotting was performed as described (22). DNA-associated RNA polymerase (indicated with an arrowhead), free RNA polymerase, RNA polymerase–RNA and RapA–RNA complexes are indicated.
Mentions: To determine the composition of these complexes, we used two independent experimental approaches. In the first approach (an immunoassay), proteins and protein–nucleic acid complexes were electroeluted from the gel onto a membrane which was subsequently incubated with either RNA polymerase-specific or RapA-specific polyclonal antibodies (in parallel reactions). In the second approach, RapA-specific complexes (‘B’ complexes in Figures 3 and 4), identified after exposure of X-ray films to ‘wet’ polyacrylamide gels, were excised from the gel; the gel slices were homogenized in Laemmli sample buffer and their content was analyzed on silver-stained SDS–polyacrylamide gels. Both approaches produced consistent results, indicating that the complexes in question contain a sole polypeptide—RapA—and RNA (the results of an immunoassay are shown in Figure 4). We also analyzed the nature of the RapA-associated RNA transcripts and determined that they are represented predominantly by a major promoter-specific RNA transcript, unique for each of the two sets of templates used (data not shown).Figure 4.

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