Biology subjects

Hertz, L. M.

Publications and source records attributed to Hertz, L. M..

3 recordsLinked to original sources

bioRxiv2025

High-throughput functional profiling and evolutionary covariation analysis of entire riboswitch sequences

Riboswitches are useful models for revealing how some RNA molecules undergo dynamic rearrangements of their structures to perform cellular functions. A great deal is known about the structure of riboswitch ligand-binding aptamer domains through evolutionary sequence covariation analysis. However, covariation analysis has been more difficult to apply to riboswitch expression platforms given their large range in cellular functions, and their large sequence diversity. Here, we develop an approach to identify whole transcriptional riboswitch sequences starting from their conserved aptamer domains. We then generate covariation models for the entire riboswitch including the aptamer domain and the expression platform. The method consists of first bioinformatically extending identified aptamer domains to include downstream sequence that could contain an expression platform. Filtering is then performed using either a computational prediction algorithm to identify bacterial intrinsic terminator sequences in the expression platform, or a high-throughput functional assay that uses massive parallel oligo synthesis and next generation sequencing to characterize transcriptional termination of riboswitch candidates as a function of ligand. Filtered sequences are then used to develop full riboswitch sequence covariation models. We developed this approach in the context of the fluoride riboswitch, characterizing 1901 fluoride riboswitch sequences using our high-throughput assay. We find that the prediction filtering approach results in few false positives to identify novel, highly functional fluoride riboswitch variants. Finally, we employ the computational approach to develop covariation models of the ZTP, lysine, and TPP riboswitches and find covariation support for previously published rearrangement mechanisms. Overall, our method represents a new hybrid computational and high throughput experimental approach to characterize large numbers of riboswitch sequences and to generate new covariation models of complete riboswitch sequences, which should expand our understanding of riboswitch mechanism and the evolution of RNA structure dynamics.

biochemistry↗

bioRxiv2023

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

A central question in biology is how RNA sequence changes influence dynamic conformational changes during cotranscriptional folding. Here we investigated this question through the study of transcriptional fluoride riboswitches, non-coding RNAs that sense the fluoride anion through the coordinated folding and rearrangement of a pseudoknotted aptamer domain and a downstream intrinsic terminator expression platform. Using a combination of E. coli RNA polymerase in vitro transcription and cellular gene expression assays, we characterized the function of mesophilic and thermophilic fluoride riboswitch variants. We showed that only variants containing the mesophilic pseudoknot function at 37 {degrees}C. We next systematically varied the pseudoknot sequence and found that a single wobble base pair is critical for function. Characterizing thermophilic variants at 65 {degrees}C through Thermus aquaticus RNA polymerase in vitro transcription showed the importance of this wobble pair for function even at elevated temperatures. Finally, we performed all-atom molecular dynamics simulations which supported the experimental findings, visualized the RNA structure switching process, and provided insight into the important role of magnesium ions. Together these studies provide deeper insights into the role of riboswitch sequence in influencing folding and function that will be important for understanding of RNA-based gene regulation and for synthetic biology applications.

molecular biology↗

bioRxiv2022

Cotranscriptional RNA strand displacement underlies the regulatory function of the E. coli thiB TPP translational riboswitch.

Riboswitches are cis-regulatory RNA elements that regulate gene expression in response to ligand through the coordinated action of a ligand-binding aptamer domain (AD) and a downstream expression platform (EP). Previous studies of transcriptional riboswitches have uncovered diverse examples that utilize cotranscriptional strand displacement to mediate the switching mechanism. The coupling of transcription and translation in bacteria motivates the intriguing question as to whether translational riboswitches can utilize the same mechanistic features. Here we investigate this question by studying the Escherichia coli thiB thiamine pyrophosphate (TPP) riboswitch. Using cellular gene expression assays, we first confirmed that the riboswitch acts at the level of translational regulation. Deletion mutagenesis showed the importance of the AD-EP linker sequence for riboswitch function, which based on sequence complementarity with the AD P1 stem suggested the possibility of an intermediate structure reminiscent of transcriptional riboswitches that exploit strand displacement. Point mutation analysis of this intermediate structure, followed by designed changes to P1, supported a strand displacement mechanism for E. coli thiB. This work provides an important new example of diverse riboswitch AD-EP combinations that exploit this switching mechanism.

molecular biology↗