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

Abstract

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...

Linking aptamer‐ligand binding and expression platform folding in riboswitches: prospects for mechanistic modeling and design

“…Evidence that residues even further downstream play a role in the regulatory activity of the adenine‐responsive riboswitch comes from the P1 helix length dependence of transcription activation by the ligand . A minimal P1 helix (three base pairs) was sufficient to trigger the formation of the terminator helix, but mutations further downstream, not directly involved in strand switching, were also found to affect the outcome.…”

“…An extended nonoverlapping region will facilitate nucleation of the corresponding helix in the presence or absence of ligand. In the pbuE adenine‐responsive riboswitch extending the P1 helix to the point that it could compete with the closing base pair of a downstream helix (ordinarily not part of the competition region) disrupted the ligand sensitivity of the riboswitch …”

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

“…The single turnover in vitro transcription assay has been used to characterize other riboswitches including the lysine riboswitch [35, 36], the adenine riboswitch [37, 38], the magnesium riboswitch [39] and artificial riboswitches [32, 33]. This assay provides two important parameters: T 50 , the amount of ligand required to elicit half-maximal regulatory response, and dynamic range (DR), the amplitude of response as the difference between fraction terminated at low and high ligand concentration.…”

Regulatory effects of cotranscriptional RNA structure formation and transitions

“…In the adenine riboswitch cotranscriptional folding mechanism, the aptamer domain is formed prior to the expression domain by RNA polymerase, and the nascent aptamer domain is able to response to cellular cues and potentially bind to a cognate ligand before the transcription and folding of the expression domain . The binding of an adenine riboswitch ligand to an aptamer domain enables formation of an intrinsic terminator RNA hairpin stem structure, which inhibits RNA polymerase extension . An anti‐terminator stem‐loop of expression platform can be formed when the aptamer domain unbounded with ligand, thus causing the poly‐U stretch to be sequestered and mRNA synthesis to proceed successfully (Figure (a)) .…”