A mechanism for ligand gated strand displacement in ZTP riboswitch transcription regulation

Abstract

Cotranscriptional RNA folding forms structural intermediates that can be critically important for RNA biogenesis. This is especially true for transcriptional riboswitches that must undergo ligand-dependent structural changes during transcription to regulate the synthesis of downstream genes. Here, we systematically map the folding states traversed by the Clostridium beijerinckii pfl riboswitch as it controls transcription termination in response to the purine biosynthetic intermediate ZMP. We find that after rearrangement of a non-native hairpin to form the ZTP aptamer, cotranscriptional ZMP binding stabilizes two structural elements that lead to antitermination by tuning the efficiency of terminator hairpin nucleation and strand displacement. We also uncover biases within natural ZTP riboswitch sequences that could avoid misfolded intermediates that disrupt function. Our findings establish a mechanism for ZTP riboswitch control of transcription that has similarities to the mechanisms of diverse riboswitches and provide evidence of selective pressure at the level of cotranscriptional RNA folding pathways.superfolder GFP (SFGFP) coding sequence, listed as 'trailing sequence' below. Promoter sequences are blue. Mutation positions are highlighted with red. Deletion positions are shown as "-". Description Sequence Promoter GCTTGATTCTAAAGATCTTTGACAGCTAGCTCAGTCCTAGGTATAATACTAGT C. beijericnckii ZTP pfl riboswitch ATATTAGATATTAGTCATATGACTGACGGAAGTGGAGTTACCACATGAAGTATGACTAGGCATATTATCTTATATG CCACAAAAAGCCGACCGTCTGGGCAAAAAAAGCCTGGATTGCGTCGGCTTTTTTAT Cbe ZTP riboswitch PK4 ATATTAGATATTAGTCATATGACTGACGGAAGTGGAGTTACCACATGAAGTATGACTAGGCATATTATCTTATATG CCACAAAAAGCCGATCGCCTGGGCAAAAAAAGCCTGGGTTGCATCGGCTTTTTTAT Cbe ZTP riboswitch PK5 ATATTAGATATTAGTCATATGACTGGCGAAAGTGGAGTTACCACATGAAGTATGACTAGGCATATTATCTTATATG CCACAAAAAGCCGATCGCCTGGGCAAAAAAAGCCTGGGTTGCATCGGCTTTTTTAT Cbe ZTP riboswitch PKAT ATATTAGATATTAGTCATATGACTGACGAAAGTGGAGTTACCACATGAAGTATGACTAGGCATATTATCTTATATG CCACAAAAAGCCGATCGTCTGGGCAAAAAAAGCCTGGATTGCATCGGCTTTTTTAT Cbe ZTP riboswitch PKGC ATATTAGATATTAGTCATATGACTGGCGGAAGTGGAGTTACCACATGAAGTATGACTAGGCATATTATCTTATATG CCACAAAAAGCCGACCGCCTGGGCAAAAAAAGCCTGGGTTGCGTCGGCTTTTTTAT Cbe ZTP riboswitch PKMM ATATTAGATATTAGTCATATGACTGACGAAAGTGGAGTTACCACATGAAGTATGACTAGGCATATTATCTTATATG CCACAAAAAGCCGACCGCCTGGGCAAAAAAAGCCTGGGTTGCGTCGGCTTTTTTAT Cbe ZTP riboswitch T1 ATATTAGATATTAGTCATATGACTGACGGAAGTGGAGTTACCACATGAAGTATGACTAGGCATATTATCTTATATG CCACAAAAAGCCGACCGTCTGGGCAAAAAAAGCCCGGATTGCGTCGGCTTTTTTAT Cbe ZTP riboswitch T2 ATATTAGATATTAGTCATATGACTGACGGAAGTGGAGTTACCACATGAAGTATGACTAGGCATATTATCTTATATG CCACAAAAAGCCGACCGTCTGGGCAAAAAAAGCCTGGA-TG-GTCGGCTTTTTTAT Cbe ZTP riboswitch T3 ATATTAGATATTAGTCATATGACTGACGGAAGTGGAGTTACCACATGAAGTATGACTAGGCATATTATCTTATATG CCACAAAAAGCCGACCGTCTGGGCAAAAAAAGCCTGGA-CG-GTCGGCTTTTTTAT Cbe ZTP riboswitch H1 ATATTAGATATTAGTCATATGACTGACGGAAGTGGAGTTACCACATGAAGTATGACTAGGCATATTATCTTATATG CCACAAAAATCGCCGACCGTCTGGGCGCAAAAAAAGCGCCTGGATTGCGTCGGCTTTTTTAT Cbe ZTP riboswitch H2 ATATTAGATATTAGTCATATGACTGACGGAAGTGGAGTTACCACATGAAGTATGACTAGGCAT...

Minimizing amplification bias during reverse transcription for in vitro selections

“…SSIV RT reactions produced similar levels of full length product with library 1 and library 3A (Figure 2B) resulting in similar yield and processivity values between the two templates indicating less template related bias as compared to ImProm-II reactions. Although increasing the SSIV reaction temperature from 55°C to 65°C reduced template bias, the higher temperatures also negatively impacted the yield and processivity, consistent with previous reports of activity loss at higher temperatures (Strobel et al, 2019). Even under optimized conditions, SSIV reactions produced no significant improvement in yield or processivity compared to reactions using ImProm-II.…”

“…ImProm-II originates from Avian Myeloblastosis Virus (AMV) RT and has been used frequently in successful aptamer and ribozyme selections by our group (Alam et al, 2018;Gruenke et al, 2020Gruenke et al, , 2022 and others (Dua et al, 2018;Kang et al, 2009;Rahimi & Bitan, 2010). SSIV (and its predecessor SSIII) originated from Moloney murine leukemia virus (M-MLV) and is also commonly used in aptamer and ribozyme selections (Akoopie et al, 2021;Moretti & Müller, 2014;Ning et al, 2019;Pressman et al, 2019;Saha et al, 2018;Strobel et al, 2019). TGIRT-III, originating from a mobile type II intron, is reported to have higher processivity, fidelity, and thermostability than some retroviral RTs (Mohr et al, 2013).…”

Point-of-Use Detection of Environmental Fluoride via a Cell-Free Riboswitch-Based Biosensor

“…We imagine that these strategies could even be used to reengineer riboswitches to have novel function (27)(28)(29). As the rules of riboswitch mechanisms are deciphered at deeper levels (11,(30)(31)(32), we hope to reach a sufficient understanding to design their functional properties to meet the global needs for field-deployable environmental and health diagnostics.…”