Nucleotides Adjacent to the Ligand-Binding Pocket are Linked to Activity Tuning in the Purine Riboswitch

Stoddard, C.D.; Widmann, Jeremy; Trausch, J.J.; Marcano-Velázquez, Joan G.; Knight, Rob; Batey, Robert T.

doi:10.1016/j.jmb.2013.02.023

Cited by 48 publications

(66 citation statements)

References 72 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…A second conformational state with differing secondary structures, however, could not be detected, most probable due to the lack of competing structural nucleotides from the expression platform. Nevertheless, it could be derived that the conformation of this so called 'P2 tune box' leads to a modulation of the regulatory properties [43 ].…”

Section: Multiple Stable Long-lived Conformations For Apo-states Of Rmentioning

confidence: 99%

Multiple conformational states of riboswitches fine-tune gene regulation

Fürtig

Nozinovic

Reining

et al. 2015

Current Opinion in Structural Biology

View full text Add to dashboard Cite

Section: Multiple Stable Long-lived Conformations For Apo-states Of Rmentioning

confidence: 99%

Multiple conformational states of riboswitches fine-tune gene regulation

Fürtig

Nozinovic

Reining

et al. 2015

Current Opinion in Structural Biology

View full text Add to dashboard Cite

“…2-AP binding assays were performed in 50 mM HEPES (pH 7.5), 135 mM KCl, 15 mM NaCl, 10 mM MgCl 2 , and 50 nM 2-AP with a protocol used previously to monitor 2-AP binding to the purine riboswitch (19). Increasing concentrations of RNA were added to the reactions and incubated for 10 min prior taking fluorescence measurements in the Infinite M200 PRO plate reader.…”

Section: Generation Of Riboswitches In a Reportermentioning

confidence: 99%

“…This lower level of response for adenine may be the result of adenine catabolism in the cell or export by the endogenous PbuE efflux pump because adenine and 2-AP have similar properties of binding to the aptamer domain (10,22,23). In addition to the increased signal in vivo, several previous reports use 2-AP as a fluorescent probe of ligand binding to facilitate biochemical analysis (19,23,24). For these reasons, we conducted the mutagenic analysis of the pbuE riboswitch using 2-AP as a proxy for adenine-dependent binding and regulatory activity.…”

Section: Riboswitch Sequencementioning

confidence: 99%

Structure-guided Mutational Analysis of Gene Regulation by the Bacillus subtilis pbuE Adenine-responsive Riboswitch in a Cellular Context

Marcano-Velázquez

Batey

2015

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Background: Riboswitches regulate purine biosynthesis and transport in many bacteria. Results: Mutagenic analysis of an adenine-responsive riboswitch revealed features important for efficient co-transcriptional regulation. Conclusion: Adenine binding to the riboswitch results in a local barrier to strand exchange by the transcriptional terminator, whose formation is facilitated by a stem-loop element. Significance: This is the first study comprehensively analyzing an RNA structural switch in a cellular context.Riboswitches are a broadly distributed form of RNA-based gene regulation in Bacteria and, more rarely, Archaea and Eukarya. Most often found in the 5-leader sequence of bacterial mRNAs, they are generally composed of two functional domains: a receptor (aptamer) domain that binds an effector molecule and a regulatory domain (or expression platform) that instructs the expression machinery. One of the most studied riboswitches is the Bacillus subtilis adenine-responsive pbuE riboswitch, which regulates gene expression at the transcriptional level, up-regulating expression in response to increased intracellular effector concentrations. In this work, we analyzed sequence and structural elements that contribute to efficient ligand-dependent regulatory activity in a co-transcriptional and cellular context. Unexpectedly, we found that the P1 helix, which acts as the antitermination element of the switch in this RNA, supported ligand-dependent activation of a reporter gene over a broad spectrum of lengths from 3 to 10 bp. This same trend was also observed using a minimal in vitro single-turnover transcription assay, revealing that this behavior is intrinsic to the RNA sequence. We also found that the sequences at the distal tip of the terminator not directly involved in alternative secondary structure formation are highly important for efficient regulation. These data strongly support a model in which the switch is highly localized to the P1 helix adjacent to the ligandbinding pocket that likely presents a local kinetic block to invasion of the aptamer by the terminator.Since their discovery and structural characterization, purineresponsive riboswitches have emerged as an important model system for exploring small molecule-RNA interactions, RNA structure, in vitro and in vivo folding, and conformational dynamics (reviewed in Ref. 1). Purine riboswitches present themselves as an ideal model system to explore various aspects of RNA chemistry and biology because of their small size and simple architecture of the ligand-binding domain, very well behaved folding properties, and multiple activities (small-molecule recognition and regulation of gene expression). However, the majority of studies of riboswitches have focused solely upon the ligand-binding (aptamer) domain in isolation using in vitro biochemical, biophysical and structural approaches. Thus, the wealth of information about the structure and function of the aptamer domain is poorly linked to an understanding of regulatory activity, as is the case for most clas...

show abstract

“…For the eleven SAM-I riboswitches found in B. subtilis , this was hypothesized to “tune” the regulatory properties of the riboswitch to the needs of the transcriptional unit [29] a feature that has been proposed in other riboswitches as well [54, 55]. Despite these clear observations, the structural or mechanistic basis for this property of SAM-I riboswitches has remained unclear.…”

Section: Discussionmentioning

confidence: 99%

A Highly Coupled Network of Tertiary Interactions in the SAM-I Riboswitch and Their Role in Regulatory Tuning

Wostenberg

Ceres

Polaski

et al. 2015

Journal of Molecular Biology

Self Cite

View full text Add to dashboard Cite

RNA folding in vivo is significantly influenced by transcription, which is not necessarily recapitulated by Mg2+-induced folding of the corresponding full length RNA in vitro. Riboswitches that regulate gene expression at the transcriptional level are an ideal system for investigating this aspect of RNA folding as ligand-dependent termination is obligatorily co-transcriptional, providing a clear readout of the folding outcome. The folding of representative members of the SAM-I family of riboswitches have been extensively analyzed using approaches focusing almost exclusively upon Mg2+ and/or S-adenosylmethionine (SAM)-induced folding of full length transcripts of the ligand binding domain. To relate these findings to co-transcriptional regulatory activity, we have investigated a set of structure-guided mutations of conserved tertiary architectural elements of the ligand binding domain using an in vitro single-turnover transcriptional termination assay, complemented with phylogenetic analysis and isothermal titration calorimetry data. This analysis revealed a conserved internal loop adjacent to the SAM binding site that significantly affects ligand binding and regulatory activity. Conversely, most single point mutations throughout key conserved features in peripheral tertiary architecture supporting the SAM binding pocket have relatively little impact on riboswitch activity. Instead, a secondary structural element in the peripheral subdomain appears to be the key determinant in observed differences in regulatory properties across the SAM-I family. These data reveal a highly coupled network of tertiary interactions that promote high fidelity co-transcriptional folding of the riboswitch, but are only indirectly linked to regulatory tuning.

show abstract

Nucleotides Adjacent to the Ligand-Binding Pocket are Linked to Activity Tuning in the Purine Riboswitch

Cited by 48 publications

References 72 publications

Multiple conformational states of riboswitches fine-tune gene regulation

Multiple conformational states of riboswitches fine-tune gene regulation

Structure-guided Mutational Analysis of Gene Regulation by the Bacillus subtilis pbuE Adenine-responsive Riboswitch in a Cellular Context

A Highly Coupled Network of Tertiary Interactions in the SAM-I Riboswitch and Their Role in Regulatory Tuning

Contact Info

Product

Resources

About