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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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Lack of the RapA effect on the amount of DNA-bound RNA polymerase in in vitro transcription reactions with supercoiled DNA. In vitro transcription reactions carried out for 15 min at 37°C in the presence or absence of RapA were fractionated on non-denaturing 5% polyacrylamide gels, which were cast and run using 2× TBE, and the amounts of individual RNA polymerase subunits retained on supercoiled DNA were determined from quantitated SDS gels, as described in the text. Quantitated levels of RapA and sigma70 retained by the DNA-bound polymerase are shown in the right panel. Results of this experiment indicate that RapA does not reduce the fraction of DNA-bound RNA polymerase during in vitro transcription, contrary to the previously proposed model (23).
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Figure 6: Lack of the RapA effect on the amount of DNA-bound RNA polymerase in in vitro transcription reactions with supercoiled DNA. In vitro transcription reactions carried out for 15 min at 37°C in the presence or absence of RapA were fractionated on non-denaturing 5% polyacrylamide gels, which were cast and run using 2× TBE, and the amounts of individual RNA polymerase subunits retained on supercoiled DNA were determined from quantitated SDS gels, as described in the text. Quantitated levels of RapA and sigma70 retained by the DNA-bound polymerase are shown in the right panel. Results of this experiment indicate that RapA does not reduce the fraction of DNA-bound RNA polymerase during in vitro transcription, contrary to the previously proposed model (23).

Mentions: This model was considered in our earlier study (23), and it was based on the speculation that RNA polymerase ‘trapped’ on DNA cannot disengage from it in order to reinitiate new cycles of transcription efficiently. If RapA were indeed to promote transcriptional cycling by displacing RNA polymerase from DNA, under most circumstances [the exception being a nearly instantaneous transfer of the polymerase from the terminator to the promoter section in the DNA template, which, at least in theory, cannot be entirely ruled out due to possible co-alignment of these two sections in supercoiled DNA (see Ref. 23; Figure 8 therein)] this should be accompanied by a measurable increase in the fraction of free RNA polymerase in the system. We tested this possibility using PAGE- and ultracentrifugation-based techniques. In the first, PAGE-based assay, in vitro transcription reactions carried out to stationary phase (with or without RapA present) were fractionated on non-denaturing 5% polyacrylamide gels in the presence of magnesium, and the amounts and subunit composition of the DNA-bound polymerase were determined (Figure 6). These experiments showed no detectable reduction in the amount of DNA-associated RNA polymerase in the presence of RapA (Figure 6, densitogram; compare the levels of the large RNA polymerase subunits in reactions with or without RapA). Also, this set of experiments showed that >85% of RapA dissociated from the DNA-bound RNA polymerase, while >50% of the sigma70 subunit was retained by the DNA-bound enzyme (Figure 6, graph). In the second approach, in vitro transcription reactions were subjected to ultracentrifugation in order to determine the fractions of DNA-bound and free RNA polymerase in reactions with or without RapA. Similarly, these experiments showed no effect of excess RapA on the ratio of free and DNA-bound RNA polymerase in the system (data not shown). Furthermore, a number of other independent experiments, which assessed the amount of DNA-associated RNA polymerase in reactions with or without RapA consistently showed no effect of RapA on the amount of DNA-associated polymerase (for example, see Figure 4 above).Figure 6.


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)

Lack of the RapA effect on the amount of DNA-bound RNA polymerase in in vitro transcription reactions with supercoiled DNA. In vitro transcription reactions carried out for 15 min at 37°C in the presence or absence of RapA were fractionated on non-denaturing 5% polyacrylamide gels, which were cast and run using 2× TBE, and the amounts of individual RNA polymerase subunits retained on supercoiled DNA were determined from quantitated SDS gels, as described in the text. Quantitated levels of RapA and sigma70 retained by the DNA-bound polymerase are shown in the right panel. Results of this experiment indicate that RapA does not reduce the fraction of DNA-bound RNA polymerase during in vitro transcription, contrary to the previously proposed model (23).
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Figure 6: Lack of the RapA effect on the amount of DNA-bound RNA polymerase in in vitro transcription reactions with supercoiled DNA. In vitro transcription reactions carried out for 15 min at 37°C in the presence or absence of RapA were fractionated on non-denaturing 5% polyacrylamide gels, which were cast and run using 2× TBE, and the amounts of individual RNA polymerase subunits retained on supercoiled DNA were determined from quantitated SDS gels, as described in the text. Quantitated levels of RapA and sigma70 retained by the DNA-bound polymerase are shown in the right panel. Results of this experiment indicate that RapA does not reduce the fraction of DNA-bound RNA polymerase during in vitro transcription, contrary to the previously proposed model (23).
Mentions: This model was considered in our earlier study (23), and it was based on the speculation that RNA polymerase ‘trapped’ on DNA cannot disengage from it in order to reinitiate new cycles of transcription efficiently. If RapA were indeed to promote transcriptional cycling by displacing RNA polymerase from DNA, under most circumstances [the exception being a nearly instantaneous transfer of the polymerase from the terminator to the promoter section in the DNA template, which, at least in theory, cannot be entirely ruled out due to possible co-alignment of these two sections in supercoiled DNA (see Ref. 23; Figure 8 therein)] this should be accompanied by a measurable increase in the fraction of free RNA polymerase in the system. We tested this possibility using PAGE- and ultracentrifugation-based techniques. In the first, PAGE-based assay, in vitro transcription reactions carried out to stationary phase (with or without RapA present) were fractionated on non-denaturing 5% polyacrylamide gels in the presence of magnesium, and the amounts and subunit composition of the DNA-bound polymerase were determined (Figure 6). These experiments showed no detectable reduction in the amount of DNA-associated RNA polymerase in the presence of RapA (Figure 6, densitogram; compare the levels of the large RNA polymerase subunits in reactions with or without RapA). Also, this set of experiments showed that >85% of RapA dissociated from the DNA-bound RNA polymerase, while >50% of the sigma70 subunit was retained by the DNA-bound enzyme (Figure 6, graph). In the second approach, in vitro transcription reactions were subjected to ultracentrifugation in order to determine the fractions of DNA-bound and free RNA polymerase in reactions with or without RapA. Similarly, these experiments showed no effect of excess RapA on the ratio of free and DNA-bound RNA polymerase in the system (data not shown). Furthermore, a number of other independent experiments, which assessed the amount of DNA-associated RNA polymerase in reactions with or without RapA consistently showed no effect of RapA on the amount of DNA-associated polymerase (for example, see Figure 4 above).Figure 6.

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