A ligand-gated strand displacement mechanism for ZTP riboswitch transcription control

Abstract

Cotranscriptional folding is an obligate step of RNA biogenesis that can guide RNA structure formation and function through transient intermediate folds. This process is particularly important for transcriptional riboswitches in which the formation of ligand-dependent structures during transcription regulates downstream gene expression. However, the intermediate structures that comprise cotranscriptional RNA folding pathways and the mechanisms that enable transit between them remain largely unknown. Here we determine the series of cotranscriptional folds and rearrangements that mediate antitermination by the Clostridium beijerinckii pfl ZTP riboswitch in response to the purine biosynthetic intermediate ZMP. We uncover sequence and structural determinants that modulate an internal RNA strand displacement process and identify biases within natural ZTP riboswitch sequences that promote on-pathway folding. Our findings establish a mechanism for pfl riboswitch antitermination and suggest general strategies by which nascent RNA molecules navigate cotranscriptional folding pathways.

Chemical transcription roadblocking for nascent RNA display

“…Nascent RNA display is a powerful tool that has proven applications in mapping RNA structure and folding (1)(2)(3)(4) and systematic characterization of RNA function (5,6). Established approaches for halting RNAP have typically relied on protein roadblocks that are not easily navigable by transcribing polymerases.…”

“…Single-round in vitro transcription reactions were performed essentially as previously described (4,22). All reactions proceeded at 37C for 5 minutes before addition of 125 µL of stop solution.…”

“…The in vitro display of nascent RNA molecules from halted transcription elongation complexes is a powerful tool with proven applications in nascent RNA folding (1)(2)(3)(4) and systematic RNA aptamer characterization (5,6). Historically, transcription arrest at a defined DNA coordinate has been achieved by attaching a protein roadblock to a DNA template to halt the progression of RNA polymerases (RNAPs).…”

“…Preparation of internally modified double-stranded DNA (dsDNA) has been achieved for short DNA molecules by annealing and ligating modified oligonucleotides (11) and for long DNA molecules by enzymatically generating single-strand DNA gaps that can be filled with modified oligonucleotides (12)(13)(14). However, these approaches are sequence-dependent and therefore not suitable for internally modifying the complex DNA sequence libraries that are frequently used in nascent RNA display experiments (4)(5)(6). Second, some RNAPs have been shown to bypass non-bulky DNA lesions such as abasic sites (15)(16)(17)(18)(19); consequently, RNAP stalling at some chemical lesions is time-dependent.…”

Real-time monitoring of cotranscriptional riboswitch folding and switching

“…(Helmling et al, 2017; Wickiser et al, 2005a; Wickiser et al, 2005b) More recently, chemical probing approaches were combined with high-throughput sequencing and used to examine riboswitch structure during “roadblocked” transcription to obtain more complete maps of RNA folding for all transcript lengths. (Strobel et al, 2019; Watters et al, 2016) However, these approaches cannot measure real time folding dynamics because RNA conformations are measured long after transcription is stalled by a blockade.…”