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

Abstract

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.

Gene Regulation Gets in Tune : How Riboswitch Tertiary-Structure Networks Adapt to Meet the Needs of Their Transcription Units

“…( a ) IL3a-IL3b cross-strand base stacking in the B. subtilis yitJ riboswitch (PDB code 4KQY) [9] exhibits bulged A47 in IL3a, whereas IL3b comprises two adenosines in one strand but no bulged bases in the opposing strand; by contrast, IL3b of metE possesses an adenosine and cytidine opposite a single bulged adenosine. Primary labels and the color scheme are derived from metE [17]; parenthetical labels are from crystal structures. Δ indicates a deletion that precludes alignment of the metE sequence to the yitJ structure.…”

“…Δ153) in metE compromised SAM binding and switching, despite its poor conservation and prior results suggesting IL3b is dispensable for activity in the context of the Tte metF riboswitch [10, 30, 31]. Using their established experimental conditions, the authors validated that a deletion within metF , comparable to metE Δ153, retains significant SAM binding [17]. The authors correctly hypothesized that IL3b ablation by Δ153 can be ameliorated by introduction of compensatory interactions in the distant PK1 subdomain.…”

“…In this issue, Batey and co-workers address this challenge by investigating the B. subtilis metE S-box riboswitch [17]. Their experimental design considers that RNA folding is dictated by kinetics rather than thermodynamics.…”

“…To quell nonnative structures, this ribozyme uses coupled, cooperative long-range interactions early in folding that help navigate the jagged folding-energy landscape on the path to a native conformation [25]. A telltale indication of such networks is that only the most harmful changes to tertiary structures compromise folding, and hence activity [17, 25, 26]. …”

“…Comparative sequence analysis revealed the nine base pair P2a stem of metE as atypical, and a preferred length of eight [17]. Deletion of one metE P2a base pair had little effect, whereas stems of six, seven or ten were intolerable, suggesting only a narrow window of helix lengths is permissible.…”