The AdoCbl-riboswitch interaction investigated by in-line probing and surface plasmon resonance spectroscopy (SPR)
The unique feature of riboswitches is to control selected gene expression by specific recognition of a cognate ligand. AdoCbl (adenosyl cobalamin, coenzyme B12) riboswitches regulate the expression of enzymes and transporters involved in bacterial AdoCbl biosynthesis or uptake on a transcriptional a...
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| Veröffentlicht in: | Methods in enzymology 2014, Vol.549, p.467-488 |
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| creator | Schaffer, Michelle F Choudhary, Pallavi K Sigel, Roland K O |
| description | The unique feature of riboswitches is to control selected gene expression by specific recognition of a cognate ligand. AdoCbl (adenosyl cobalamin, coenzyme B12) riboswitches regulate the expression of enzymes and transporters involved in bacterial AdoCbl biosynthesis or uptake on a transcriptional and/or translational level. The analysis of ligand recognition and the induced conformational changes requires a detailed knowledge of the kinetics and thermodynamics of ligand binding and of the secondary structure rearrangements. This chapter describes the investigation of coenzyme B12 binding to the btuB riboswitch from Escherichia coli by in-line probing assays and surface plasmon resonance spectroscopy. The experimental conditions, requirements, and performance of both methods are presented together with the evaluation of the experimental data to determine the associated conformational changes of the RNA and the kinetic and thermodynamic parameters of ligand binding. Owing to the light sensitivity of the cobalt(I)'carbon bond, these methods were specifically modified to ensure the chemical integrity of AdoCbl. |
| doi_str_mv | 10.1016/B978-0-12-801122-5.00020-9 |
| format | Article |
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AdoCbl (adenosyl cobalamin, coenzyme B12) riboswitches regulate the expression of enzymes and transporters involved in bacterial AdoCbl biosynthesis or uptake on a transcriptional and/or translational level. The analysis of ligand recognition and the induced conformational changes requires a detailed knowledge of the kinetics and thermodynamics of ligand binding and of the secondary structure rearrangements. This chapter describes the investigation of coenzyme B12 binding to the btuB riboswitch from Escherichia coli by in-line probing assays and surface plasmon resonance spectroscopy. The experimental conditions, requirements, and performance of both methods are presented together with the evaluation of the experimental data to determine the associated conformational changes of the RNA and the kinetic and thermodynamic parameters of ligand binding. 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AdoCbl (adenosyl cobalamin, coenzyme B12) riboswitches regulate the expression of enzymes and transporters involved in bacterial AdoCbl biosynthesis or uptake on a transcriptional and/or translational level. The analysis of ligand recognition and the induced conformational changes requires a detailed knowledge of the kinetics and thermodynamics of ligand binding and of the secondary structure rearrangements. This chapter describes the investigation of coenzyme B12 binding to the btuB riboswitch from Escherichia coli by in-line probing assays and surface plasmon resonance spectroscopy. The experimental conditions, requirements, and performance of both methods are presented together with the evaluation of the experimental data to determine the associated conformational changes of the RNA and the kinetic and thermodynamic parameters of ligand binding. Owing to the light sensitivity of the cobalt(I)'carbon bond, these methods were specifically modified to ensure the chemical integrity of AdoCbl.</description><subject>Bacterial Outer Membrane Proteins - genetics</subject><subject>Base Sequence</subject><subject>Binding Sites</subject><subject>Cobamides - metabolism</subject><subject>Escherichia coli - genetics</subject><subject>Escherichia coli - metabolism</subject><subject>Escherichia coli Proteins - genetics</subject><subject>Membrane Transport Proteins - genetics</subject><subject>Molecular Sequence Data</subject><subject>Riboswitch</subject><subject>RNA, Bacterial - chemistry</subject><subject>RNA, Bacterial - genetics</subject><subject>RNA, Bacterial - metabolism</subject><subject>Surface Plasmon Resonance - methods</subject><issn>1557-7988</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>2014</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNo1kFtLAzEQhYMgtlb_giw-1YfUXJrbYy3eoKBo35dskm0ju8mabJX-ewPq05z5ODOcGQCuMVpghPntnRISIogJlAhjQiBbIIQIguoETDFjAgol5QSc5_xRuJAKn4EJYUtKBMdTkLZ7V61sXDcdTL6J-duPZl_5MLqkzehjKPrL5dHv9Ohs1RxLDzsfXDWk2Piwq3SwVT6kVpvCOp37MpNcjkGHQvLgzJhiNnE4VvP317ebC3Da6i67y786A9uH--36CW5eHp_Xqw00hMsRKkcttwQ3LbFOUkUVYZQwIqizZkkFYtwY1LaWM6VR0zohBLPSEkU5JY7OwPx3bcn5eSgX1L3PxnWdDi4eco05UUwuleLFevVnPTS9s_WQfK_Tsf5_E_0BLmprDA</recordid><startdate>2014</startdate><enddate>2014</enddate><creator>Schaffer, Michelle F</creator><creator>Choudhary, Pallavi K</creator><creator>Sigel, Roland K O</creator><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>7X8</scope></search><sort><creationdate>2014</creationdate><title>The AdoCbl-riboswitch interaction investigated by in-line probing and surface plasmon resonance spectroscopy (SPR)</title><author>Schaffer, Michelle F ; Choudhary, Pallavi K ; Sigel, Roland K O</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c268t-9e3d6d21bf2de8393925325273edc437056cc0ffd659a0bfe7775d8d293632e3</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>2014</creationdate><topic>Bacterial Outer Membrane Proteins - genetics</topic><topic>Base Sequence</topic><topic>Binding Sites</topic><topic>Cobamides - metabolism</topic><topic>Escherichia coli - genetics</topic><topic>Escherichia coli - metabolism</topic><topic>Escherichia coli Proteins - genetics</topic><topic>Membrane Transport Proteins - genetics</topic><topic>Molecular Sequence Data</topic><topic>Riboswitch</topic><topic>RNA, Bacterial - chemistry</topic><topic>RNA, Bacterial - genetics</topic><topic>RNA, Bacterial - metabolism</topic><topic>Surface Plasmon Resonance - methods</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Schaffer, Michelle F</creatorcontrib><creatorcontrib>Choudhary, Pallavi K</creatorcontrib><creatorcontrib>Sigel, Roland K O</creatorcontrib><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>MEDLINE - Academic</collection><jtitle>Methods in enzymology</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Schaffer, Michelle F</au><au>Choudhary, Pallavi K</au><au>Sigel, Roland K O</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>The AdoCbl-riboswitch interaction investigated by in-line probing and surface plasmon resonance spectroscopy (SPR)</atitle><jtitle>Methods in enzymology</jtitle><addtitle>Methods Enzymol</addtitle><date>2014</date><risdate>2014</risdate><volume>549</volume><spage>467</spage><epage>488</epage><pages>467-488</pages><eissn>1557-7988</eissn><abstract>The unique feature of riboswitches is to control selected gene expression by specific recognition of a cognate ligand. 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| ispartof | Methods in enzymology, 2014, Vol.549, p.467-488 |
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| subjects | Bacterial Outer Membrane Proteins - genetics Base Sequence Binding Sites Cobamides - metabolism Escherichia coli - genetics Escherichia coli - metabolism Escherichia coli Proteins - genetics Membrane Transport Proteins - genetics Molecular Sequence Data Riboswitch RNA, Bacterial - chemistry RNA, Bacterial - genetics RNA, Bacterial - metabolism Surface Plasmon Resonance - methods |
| title | The AdoCbl-riboswitch interaction investigated by in-line probing and surface plasmon resonance spectroscopy (SPR) |
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