Transcriptional and Translational Riboswitches

Riboswitches are structured RNA elements that are most often present in the 5’ untranslated regions of some mRNAs and regulate gene expression via alterations in transcription termination or repression of translation initiation (Fig. 1). They achieve this modulation by changing RNA conformation upon binding of a specific ligand, which could be a metabolite, vitamin, metal ion, a nucleotide or small non-coding RNA, or an amino acid. Riboswitches are found in nearly all bacteria, including many pathogens, and thus they are actively pursued as potential antibiotic targets. The transcriptional and translational riboswitch subgroups of the Walter lab are currently focused on understanding the fundamental activities of a variety of riboswitches through cutting-edge single-molecule techniques.

The PreQ1 Riboswitch

The preQ1 riboswitch is the smallest known naturally occurring riboswitch with just 34 nucleotides required for substrate binding. It has a very high affinity for the ligand preQ1 (Kd ~ 50 nM) which is required for translational fidelity in many bacteria. The preQ1 riboswitch presents an interesting case of high affinity substrate recognition by a small RNA motif. Understanding the structural dynamics of the preQ1 riboswitch will give some insight into the mechanism of its conformational ‘switch’ and could also help in designing better drugs against pathogens that contain preQ1 riboswitch. We are using single molecule fluorescence resonance energy transfer (smFRET) microscopy to understand the conformational dynamics of the preQ1 riboswitch. (Widom et al. Molecular Cell. 2018)

The Mn2+ Riboswitch

The Mn2+ sensing yybP-ykoY riboswitch is one of the most commonly found riboswitches in bacteria, and it is primarily responsible for coordinating the response of Mn2+ homeostasis genes. A recent study by the Walter lab elucidated that Mn2+ binding induces small local RNA structural changes which cascade through the riboswitch structure to stabilize larger global RNA conformational changes in a [Mn2+]-dependent manner. These local to global Mn2+ signals are responsible for ultimately inducing readthrough of the transcription termination sequence.(Suddala et. al. Nature Communications. 2019)

The T-box Riboswitch

T-box riboswitches are tRNA-sensing riboswitches and are found primarily in gram-positive bacteria. They bind uncharged tRNAs to prevent premature transcription termination, essentially functioning as a feedback loop to control the expression levels of tRNA synthetase genes. Recent work by the Walter lab demonstrated that tRNAs bind to T-box riboswitches through a hierarchical mechanism, in which charged and uncharged tRNAs can both bind the riboswitch RNA, but only uncharged tRNAs are capable of inducing a “snap-lock-based trapping” that promotes transcription read-through.(Suddala et. al. Nature Communications. 2018)


Figure 1. Riboswitch-mediated gene regulation. (A) A simplified overview of gene regulation upon ligand binding to a typical riboswitch. Pausing of RNAP during transcription allows the time for ligand to bind the riboswitch in concentration dependent manner. ‘High’ ligand concentrations lead to downregulation of gene expression, whereas ‘low’ ligand concentrations allow gene expression to go forward. In the case of transcriptional riboswitches, ligand binding induces the formation of a typical terminator stem-loop followed by a polyuridine stretch that together dislodge the RNAP and thus terminate transcription. In the case of translational riboswitches, ligand binding promotes the formation of a sequester stem that prevents ribosome binding to the SD sequence. RNAP: RNA polymerase; SD: Shine-Dalgarno sequence; AUG: start codon. (B) The kinetic partitioning mechanism of mRNA selection by the ribosome. Cofactors including protein S1, the three initiation factors (IF1-3) and initiator fMet-tRNAfMet assist in loading an mRNA embedding a preQ1-sensing riboswitch onto the 30S ribosomal subunit (left). As the 30S and 70S initiation complexes (IC) form, commitment to translation increases while the vulnerability to mRNA decay decreases, as mediated by the bacterial degradosome, Hfq and Rho (bottom).