The importance of helix P1 stability for structural pre-organization and ligand binding affinity of the adenine riboswitch aptamer domain

Nozinovic, Senada; Reining, Anke; Kim, Yong-Boum; Noeske, J.; Schlepckow, Kai; Wöhnert, Jens; Schwalbe, Harald

doi:10.4161/rna.29439

Cited by 32 publications

(37 citation statements)

References 31 publications

(67 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This again contrasts with a recent report suggesting that a pbuE riboswitch variant with an elongated P1 helix (9 bp stabilized with four additional G-C pairs) and stabilizing P1 have a negative impact on RNA folding and ligand affinity (32). We observed that the concentration of 2-AP in the growth medium required to elicit a half-maximal regulatory response was also very similar between the two RNAs (ϳ300 M for the wild type and ϳ100 M for (P1ϩ8),NUCL/GAAA), suggesting that P1 stability is not important for regulatory function.…”

Section: Volume 290 • Number 7 • February 13 2015contrasting

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

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

Section: Volume 290 • Number 7 • February 13 2015contrasting

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

View full text Add to dashboard Cite

show abstract

“…This difference in stemlength is conserved across tandem glycine riboswitches, with aptamer-1 always containing a 3-7 base P0 with an 8-9 base P1, while aptamer-2 has only a 5-6 base P1 helix Sherman et al 2012). Similar differences in P1 helix length and stability have been shown to cause pronounced differences in structure and ligand responsiveness for two adenine riboswitches (Nozinovic et al 2014).…”

Section: Discussionmentioning

confidence: 74%

“…Other riboswitches have been shown to use scaffolding to preform significant portions of the aptameric secondary structure prior to ligand binding, including prequeuosine class-II (Soulière et al 2013), S-adenosylmethionine (SAM)-I , SAM-II (Haller et al 2011), cyclic-di-guanosine monophosphate (c-di-GMP) (Wood et al 2012), and the purine riboswitches (Lemay et al 2006;Brenner et al 2010;Nozinovic et al 2014). Scaffolding can have important consequences for kinetically controlled riboswitches (Wickiser et al 2005a,b;Trausch and Batey 2014), allowing ligand binding to occur on transcriptionally relevant time scales.…”

Section: Discussionmentioning

confidence: 99%

Ligand binding by the tandem glycine riboswitch depends on aptamer dimerization but not double ligand occupancy

Ruff

Strobel

2014

RNA

View full text Add to dashboard Cite

The glycine riboswitch predominantly exists as a tandem structure, with two adjacent, homologous ligand-binding domains (aptamers), followed by a single expression platform. The recent identification of a leader helix, the inclusion of which eliminates cooperativity between the aptamers, has reopened the debate over the purpose of the tandem structure of the glycine riboswitch. An equilibrium dialysis-based assay was combined with binding-site mutations to monitor glycine binding in each ligand-binding site independently to understand the role of each aptamer in glycine binding and riboswitch tertiary interactions. A series of mutations disrupting the dimer interface was used to probe how dimerization impacts ligand binding by the tandem glycine riboswitch. While the wild-type tandem riboswitch binds two glycine equivalents, one for each aptamer, both individual aptamers are capable of binding glycine when the other aptamer is unoccupied. Intriguingly, glycine binding by aptamer-1 is more sensitive to dimerization than glycine binding by aptamer-2 in the context of the tandem riboswitch. However, monomeric aptamer-2 shows dramatically weakened glycine-binding affinity. In addition, dimerization of the two aptamers in trans is dependent on glycine binding in at least one aptamer. We propose a revised model for tandem riboswitch function that is consistent with these results, wherein ligand binding in aptamer-1 is linked to aptamer dimerization and stabilizes the P1 stem of aptamer-2, which controls the expression platform.

show abstract

“…In the holo-state, P1 is fully formed. Due to ligand-induced stabilization of helix P1 [42], formation of helix P4, which includes a long-range anti-SD/SD motif, is suppressed. Importantly, nucleotides A111 to U125 that contain the ribosome binding region (the Shine-Dalgarno sequence and the AUG-start codon) do not form base pairs and are therefore accessible to ribosome binding in the holo state.…”

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

Self Cite

View full text Add to dashboard Cite

The importance of helix P1 stability for structural pre-organization and ligand binding affinity of the adenine riboswitch aptamer domain

Cited by 32 publications

References 31 publications

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

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

Ligand binding by the tandem glycine riboswitch depends on aptamer dimerization but not double ligand occupancy

Multiple conformational states of riboswitches fine-tune gene regulation

Contact Info

Product

Resources

About