Distributed Biotin-Streptavidin Transcription Roadblocks for Mapping Cotranscriptional RNA Folding

Abstract

RNA molecules fold cotranscriptionally as they emerge from RNA polymerase. Cotranscriptional folding is an important process for proper RNA structure formation as the order of folding can determine an RNA molecules structure, and thus its functional properties. Despite its fundamental importance, the experimental study of RNA cotranscriptional folding has been limited by the lack of easily approachable methods that can interrogate nascent RNA structures at nucleotide resolution during transcription. We previously developed cotranscriptional selective 2-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-seq) to simultaneously probe all of the intermediate structures an RNA molecule transitions through during transcription elongation. Here, we improve the broad applicability of cotranscriptional SHAPE-Seq by developing a sequence-independent streptavidin roadblocking strategy to simplify the preparation of roadblocking transcription templates. We determine the fundamental properties of streptavidin roadblocks and show that randomly distributed streptavidin roadblocks can be used in cotranscriptional SHAPE-Seq experiments to measure the Bacillus cereus crcB fluoride riboswitch folding pathway. Comparison of EcoRIE111Q and streptavidin roadblocks in cotranscriptional SHAPE-Seq data shows that both strategies identify the same RNA structural transitions related to the riboswitch decision-making process. Finally, we propose guidelines to leverage the complementary strengths of each transcription roadblock for use in studying cotranscriptional folding.