Insights into the Regulatory Landscape of the Lysine Riboswitch

Garst, Andrew D.; Porter, Ely; Batey, Robert T.

doi:10.1016/j.jmb.2012.06.038

Cited by 37 publications

(43 citation statements)

References 84 publications

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“…As the concentration of NTPs was raised from 20 to 150 μM, the T 50 increases from 0.2 to 1.3 μM for 2,6-diaminopurine (an adenine analog). Similar results have been observed for the B. subtilis ribD FMN [88] and lysC lysine [90] riboswitches. Second, a long-lived transcriptional pause located downstream of the pbuE adenine riboswitch aptamer domain was observed at a stretch of uridine residues at positions 114-117 [86].…”

Section: Mechanisms: a Tale Of Two Adenine Riboswitchessupporting

confidence: 83%

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The purine riboswitch as a model system for exploring RNA biology and chemistry

Porter¹,

Marcano-Velázquez²,

Batey³

2014

Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanism

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Over the past decade the purine riboswitch, and in particular its nucleobase-binding aptamer domain, has emerged as an important model system for exploring various aspects of RNA structure and function. Its relatively small size, structural simplicity and readily observable activity enable application of a wide variety of experimental approaches towards the study of this RNA. These analyses have yielded important insights into small molecule recognition, co-transcriptional folding and secondary structural switching, and conformational dynamics that serve as a paradigm for other RNAs. In this article, the current state of understanding of the purine riboswitch family is examined and how this growing knowledge base is starting to be exploited in the creation of novel RNA devices.

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Section: Mechanisms: a Tale Of Two Adenine Riboswitchessupporting

confidence: 83%

“…Even more recently, an RNA pH sensor was characterized that represses expression of the alx locus at elevated extracellular pH [116]. Thus, the regulatory landscape of a specific riboswitch can be very complex allowing the RNA to respond in real time to variety of intra- and extracellular cues [90]. …”

Section: Why Riboswitches?mentioning

confidence: 99%

The purine riboswitch as a model system for exploring RNA biology and chemistry

Porter¹,

Marcano-Velázquez²,

Batey³

2014

Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanism

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“…Our data clearly show that while these groups do not contribute substantially to binding energy, the p ABA group significantly contributes to regulatory activity. Differences between binding and regulatory efficiency have also been observed in the lysine riboswitch (Garst et al, 2012). For example, the natural effector lysine has a T 50 to K D ratio of 1.0 (under transcription conditions of 50 μM NTPs) such that binding and regulation occur at the same concentration.…”

Section: Discussionmentioning

confidence: 95%

A Disconnect between High-Affinity Binding and Efficient Regulation by Antifolates and Purines in the Tetrahydrofolate Riboswitch

Trausch

Batey

2014

Chemistry & Biology

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SUMMARY The tetrahydrofolate (THF) riboswitch regulates folate transport and metabolism in a number of Firmicutes by cooperatively binding two molecules of THF. To further understand this riboswitch’s specificity for THF, binding and regulatory activity of a series of THF analogs and antifolates was examined. Our data reveal that while binding is dominated by the RNA’s interactions with the pterin moiety, the para-aminobenzoic acid (pABA) moiety plays a significant role in transcriptional regulation. Further, we find that adenine and several other analogs bind with high affinity by an alternative binding mechanism. Despite a similar affinity to THF, adenine is a poor regulator of transcriptional attenuation. These results demonstrate that binding alone does not determine a compound’s effectiveness in regulating the activity of the riboswitch—a complication in current efforts to develop antimicrobials that target these RNAs.

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“…The two primers used were 5Ј-GTGGTTGCTGGATAACTT-TACGGGC and 5Ј-CCCGGGGATCCTCTAGAGTCGAC at 1 M in a standard PCR amplification. These DNA templates were transcribed as described previously (12,16,17). Polymerase was bound to the promoter in a first reaction step by incubating 50 ng of DNA template at 37°C for 10 min in 12.5 l of 2ϫ transcription buffer (140 mM Tris-Cl (pH 8.0), 140 mM NaCl, 0.2 mM EDTA (pH 8.0), 28 mM ␤-mercaptoethanol, and 70 g/ml BSA), 2.5 l of 25 mM MgCl 2 , 0.5 Ci of [␣-…”

Section: Generation Of Riboswitches In a Reportermentioning

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

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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...

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Insights into the Regulatory Landscape of the Lysine Riboswitch

Cited by 37 publications

References 84 publications

The purine riboswitch as a model system for exploring RNA biology and chemistry

The purine riboswitch as a model system for exploring RNA biology and chemistry

A Disconnect between High-Affinity Binding and Efficient Regulation by Antifolates and Purines in the Tetrahydrofolate Riboswitch

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

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