Folding of the lysine riboswitch: importance of peripheral elements for transcriptional regulation

Abstract

The Bacillus subtilis lysC lysine riboswitch modulates its own gene expression upon lysine binding through a transcription attenuation mechanism. The riboswitch aptamer is organized around a single five-way junction that provides the scaffold for two long-range tertiary interactions (loop L2–loop L3 and helix P2–loop L4)—all of this for the creation of a specific lysine binding site. We have determined that the interaction P2–L4 is particularly important for the organization of the ligand-binding site and for the riboswitch transcription attenuation control. Moreover, we have observed that a folding synergy between L2–L3 and P2–L4 allows both interactions to fold at lower magnesium ion concentrations. The P2–L4 interaction is also critical for the close juxtaposition involving stems P1 and P5. This is facilitated by the presence of lysine, suggesting an active role of the ligand in the folding transition. We also show that a previously uncharacterized stem–loop located in the expression platform is highly important for the riboswitch activity. Thus, folding elements located in the aptamer and the expression platform both influence the lysine riboswitch gene regulation.

A Kissing Loop Is Important for btuB Riboswitch Ligand Sensing and Regulatory Control

“…5, B and C). Such an unconventional role for a kissing loop interaction differs from previous studies reporting that kissing loop structures are usually involved in stabilizing the ligand-riboswitch complex as found for purine and lysine riboswitches (34,52). The presence of multiple transcriptional pause sites located across the expression platform indicates that the folding process is highly controlled in this region, consistent with the decreased folding response in the presence of ligand when the riboswitch is transcribed using T7 RNA polymerase (19).…”

“…We have recently shown for Bacillus subtilis riboswitches responding to adenine, lysine, and S-adenosylmethionine (27,34,35) and for the E. coli lysine-sensing riboswitch (32) that ligand-dependent gene regulation is highly dependent on the formation of P1 stem base pairs but not on the identity of residues involved. Because riboswitch crystal structures show that AdoCbl sensing is achieved through kissing loop tertiary interaction rather than using the P1 stem (8, 9), we investigated the role of the P1 stem in gene regulation by engineering riboswitches with various P1 stem mutations designed to disrupt or allow the formation of P1 stem base pairs ( Fig.…”

Constitutive Regulatory Activity of an Evolutionarily Excluded Riboswitch Variant

“…S-adenosylmethionine, thiamine pyrophosphate, and flavin mononucleotide), it seems unlikely that a single-point mutation, or even a combination of mutations within the aptamer domain, could replicate the network of interactions necessary to mimic the bond-like structure of these riboswitch classes. Obviously, additional riboswitch-activating mutations could be obtained through the stabilization of the P1 stem, as shown previously for riboswitches sensing adenine, lysine, and Sadenosylmethionine (19,20,41). Evidences in favor of alternative mechanisms involving the inhibition of ligand binding arise from the large body of existing data regarding riboswitch mutations induced by various antibiotics (42)(43)(44)(45).…”

“…SHAPE reactions were initiated by adding N-methylisatoic anhydride (Invitrogen) dissolved in dimethyl sulfoxide (DMSO), and the solution was allowed to react for 80 min at 37°C. Reverse transcriptions were performed and analyzed as described previously (20).…”

Basis for ligand discrimination between ON and OFF state riboswitch conformations: The case of the SAM-I riboswitch

“…On the other hand, our results show the minimal secondary structure requirements to form a SAM-binding competent tertiary fold, for SAM-I riboswitches. Other riboswitches, such as the purine (Stoddard and Batey 2009) and lysine (Blouin et al 2010), as well as simpler systems such as the quenosine (Kang et al 2009) and SAMIII-S MK (Lu et al 2011), also contain helixcompeting motifs. In some cases, including purine (Lemay and Lafontaine 2007;Greenleaf et al 2008) and TPP (Anthony et al 2012) riboswitches, there is evidence that the ligand stabilizes an analog to the SAM-I riboswitch P1 helix.…”