Structural basis for control of bacterial RNA polymerase pausing by a riboswitch and its ligand

Abstract

Folding of nascent transcripts can be modulated by the proximal RNA polymerase (RNAP) that carries out their transcription, and vice versa. A pause of RNAP during transcription of a preQ1 riboswitch (que-ePEC) is stabilized by a previously characterized template consensus sequence and the ligand-free conformation of the nascent RNA. Ligand binding to the riboswitch induces RNAP pause release and downstream transcription termination, however, the mechanism by which riboswitch folding modulates pausing is unclear. Here, we report single-particle cryo-electron microscopy reconstructions of que-ePEC in ligand-free and ligand-bound states. In the absence of preQ1, the RNA transcript is in an unexpected hyper-translocated state, preventing downstream nucleotide incorporation. Strikingly, upon ligand binding the riboswitch rotates around its helical axis, expanding the surrounding RNAP exit channel and repositioning the transcript for elongation. Our study reveals the tight coupling by which small nascent RNA structures and their ligands can functionally regulate the macromolecular transcription machinery.

The Effect of Pseudoknot Base Pairing on Cotranscriptional Structural Switching of the Fluoride Riboswitch

“…This is different than what is observed in IVT conditions, where the T. pe WT fluoride riboswitch anti-terminated ∼50% of the time independent of fluoride conditions, showing that some structural switching can occur in this system, though it is not fluoride dependent (Figure 2D). This discrepancy could be attributed to unknown interactions between the riboswitch and RNA polymerase that are only beginning to be understood 50 and therefore cannot be captured by MD simulations.…”

Are non‐protein coding RNAs junk or treasure?

“…A second limitation of a sequence-based conservation analysis of function is illustrated by recent insights from the functional probing of riboswitches. RNA structure, and hence dynamics and function, is generally established co-transcriptionally, as evident from, for example, bacterial ncRNAs including riboswitches [85][86][87][88][89][90] and ribosomal RNAs, [91,92] as well as the co-transcriptional alternative splicing of eukaryotic pre-mRNAs, responsible for the important, vast diversification of the human proteome across ∼200 cell types by excision of F I G U R E 2 Riboswitch mechanism, function and conservation. (A) Riboswitches are highly structured RNA motifs embedded in the 5′ untranslated regions of many bacterial mRNAs, where they can enhance or suppress gene expression as they co-transcriptionally fold to bind a small-molecule or elemental ion ligand.…”

“…[93] In the case of riboswitches, the interactions of the ncRNA with its multiple protein effectors functionally engage essentially all of its nucleotides, sequence-conserved or not, including those responsible for affecting specific distances between other functional elements. [85,89] Consequently, the expression platformequally important for the gene regulatory function as the conserved aptamer domain-tends to be far less conserved, [83] because it interacts with the idiosyncratic gene expression machinery of the bacterium (Figure 2B). Consequently, taking a riboswitch out of this native environment into a different cell type for synthetic biology purposes has been notoriously challenging.…”

“…A second limitation of a sequence‐based conservation analysis of function is illustrated by recent insights from the functional probing of riboswitches. RNA structure, and hence dynamics and function, is generally established co‐transcriptionally, as evident from, for example, bacterial ncRNAs including riboswitches [ 85–90 ] and ribosomal RNAs, [ 91,92 ] as well as the co‐transcriptional alternative splicing of eukaryotic pre‐mRNAs, responsible for the important, vast diversification of the human proteome across ∼200 cell types by excision of varying ncRNA introns. [ 93 ] In the latter case, it is becoming increasingly clear that splicing regulation involves multiple layers synergistically controlled by the splicing machinery, transcription process, and chromatin structure.…”

A nascent riboswitch helix orchestrates robust transcriptional regulation through signal integration

“…This competition is expected to lead to kinetic control of gene regulation rather than the thermodynamic control invoked by the global free energy landscape of RNA folding 14,15 , rendering the true biological context challenging to capture. Recent studies of riboswitches in the context of a paused elongation complex (PEC), i.e., in the presence of the DNA template and paused RNAP, have revealed that conformational changes of riboswitches are heavily in uenced by the transcription machinery [16][17][18][19][20][21] , demonstrating the importance of the proximal RNAP for a mechanistic understanding of riboswitch-mediated gene regulation. While determinant, the kinetics of co-transcriptional riboswitch folding are di cult to observe at the short timescale dictated by transcription elongation 12,22 .…”