Biology subjects

Szyjka, C. E.

Publications and source records attributed to Szyjka, C. E..

4 recordsLinked to original sources

bioRxiv2025

Co-transcriptional folding orchestrates sequential multi-effector sensing by a glycine tandem riboswitch

Riboswitches are non-coding RNA motifs that regulate gene expression in response to ligand binding. The glycine tandem riboswitch (GTR) is notable because it comprises two distinct glycine aptamers that interact extensively. These inter-aptamer contacts drive conformational changes in the downstream expression platform to control gene expression. Despite extensive studies, the role of glycine and RNA folding pathways in co-transcriptional regulation remains unclear. Here, we integrate single-molecule kinetic analysis, co-transcriptional RNA structure probing, and computational modeling to reveal that the GTR processes multiple molecular inputs sequentially, guided by polymerase pausing. Our findings elucidate its stepwise 5-to-3 folding pathway and demonstrate how sequential glycine binding to each aptamer, K+ binding to a kink-turn, non-native RNA folding intermediates, inter-aptamer docking that drives binding site pre-organization, and modulation by the transcription factor NusA collectively orchestrate co-transcriptional gene regulation. These results support a model in which glycine binding cooperativity arises through non-equilibrium mechanisms rather than a classical concerted model.

molecular biology↗

bioRxiv2024

Sequential structure probing of cotranscriptional RNA folding intermediates

Cotranscriptional RNA folding pathways typically involve the sequential formation of folding intermediates. Existing methods for cotranscriptional RNA structure probing map the structure of nascent RNA in the context of a terminally arrested transcription elongation complex. Consequently, the rearrangement of RNA structures as nucleotides are added to the transcript can be inferred but is not assessed directly. To address this limitation, we have developed linked-multipoint Transcription Elongation Complex RNA structure probing (TECprobe-LM), which assesses the cotranscriptional rearrangement of RNA structures by sequentially positioning E. coli RNAP at two or more points within a DNA template so that nascent RNA can be chemically probed. We validated TECprobe-LM by measuring known folding events that occur within the E. coli signal recognition particle RNA, Clostridium beijerinckii pfl ZTP riboswitch, and Bacillus cereus crcB fluoride riboswitch folding pathways. Our findings establish TECprobe-LM as a strategy for detecting cotranscriptional RNA folding events directly using chemical probing.

biochemistry↗

bioRxiv2023

Observation of coordinated cotranscriptional RNA folding events

RNA begins to fold as it is transcribed by an RNA polymerase. Consequently, RNA folding is constrained by the direction and rate of transcription. Understanding how RNA folds into secondary and tertiary structures therefore requires methods for determining the structure of cotranscriptional folding intermediates. Cotranscriptional RNA chemical probing methods accomplish this by systematically probing the structure of nascent RNA that is displayed from RNA polymerase. Here, we have developed a concise, high-resolution cotranscriptional RNA chemical probing procedure called Transcription Elongation Complex RNA structure probing-Multilength (TECprobe-ML). We validated TECprobe-ML by replicating and extending previous analyses of ZTP and fluoride riboswitch folding, and mapped the folding pathway of a ppGpp-sensing riboswitch. In each system, TECprobe-ML identified coordinated cotranscriptional folding events that mediate transcription antitermination. Our findings establish TECprobe-ML as an accessible method for mapping cotranscriptional RNA folding pathways.

molecular biology↗

bioRxiv2022

Purification of synchronized E. coli transcription elongation complexes by reversible immobilization on magnetic beads

Synchronized transcription elongation complexes (TECs) are a fundamental tool for in vitro studies of transcription and RNA folding. Transcription elongation can be synchronized by omitting one or more NTPs from an in vitro transcription reaction so that RNA polymerase can only transcribe to the first occurrence of the omitted nucleotide(s) in the coding DNA strand. This approach was developed over four decades ago and has been applied extensively in biochemical investigations of RNA polymerase enzymes, but has not been optimized for RNA-centric assays. In this work, we describe the development of a system for isolating synchronized TECs from an in vitro transcription reaction. Our approach uses a custom 5 leader sequence, called C3-SC1, to reversibly capture synchronized TECs on magnetic beads. We first show that complexes isolated by this procedure, called C3-SC1TECs, are >95% pure, >98% active, highly synchronous (94% of complexes chase in <15s upon addition of saturating NTPs), and compatible with solid-phase transcription; the yield of this purification is [~]8%. We then show that C3-SC1TECs perturb, but do not interfere with, the function of ZTP-sensing and ppGpp-sensing transcriptional riboswitches. For both riboswitches, transcription using C3-SC1TECs improved the efficiency of transcription termination in the absence of ligand but did not inhibit ligand-induced transcription antitermination. Given these properties, C3-SC1TECs will likely be useful for developing biochemical and biophysical RNA assays that require high-performance, quantitative bacterial in vitro transcription.

biochemistry↗