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The AdoCbl–Riboswitch Interaction Investigated by In-Line Probing and Surface Plasmon Resonance Spectroscopy (SPR)


Schaffer, Michelle F; Choudhary, Pallavi K; Sigel, Roland K O (2014). The AdoCbl–Riboswitch Interaction Investigated by In-Line Probing and Surface Plasmon Resonance Spectroscopy (SPR). In: Burke-Aguero, Donald H. Riboswitch Discovery, Structure and Function. Amsterdam: Elsevier, 467-488.

Abstract

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)′single bondcarbon bond, these methods were specifically modified to ensure the chemical integrity of AdoCbl.

Abstract

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)′single bondcarbon bond, these methods were specifically modified to ensure the chemical integrity of AdoCbl.

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Additional indexing

Item Type:Book Section, refereed, further contribution
Communities & Collections:07 Faculty of Science > Department of Chemistry
Dewey Decimal Classification:540 Chemistry
Language:English
Date:2014
Deposited On:13 Jan 2016 16:24
Last Modified:30 Oct 2016 08:17
Publisher:Elsevier
Series Name:Methods in Enzymology
Number:549
ISSN:0076-6879
ISBN:978-0-12-801122-5
Publisher DOI:https://doi.org/10.1016/B978-0-12-801122-5.00020-9

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