TL;DR
This protocol describes the in vivo use of SHAPE chemistry in two evolutionary divergent model Archaea: Sulfolobus acidocaldarius and Haloferax volcanii.
This work explored how the guide RNA of CRISPR genome editing system folds and modulates its structural flexibility to carry out the roles required for each assembly state from its unbound apo form to the functional Cas9 RNP state for target DNA cleavage using ABEL-FRET spectroscopy.
High-resolution structure determination of RNA remains a major challenge due to its conformational flexibility, intrinsic heterogeneity, and susceptibility to degradation. Although RNA plays central roles in gene regulation, viral replication, and cellular homeostasis, RNA-only structures represent a small fraction of...
RNA molecules are inherently dynamic, forming complex structural ensembles of alternative conformations that direct their cellular functions. Current methods for resolving RNA structural heterogeneity and dynamics have been developed for short-read sequencing–based structure probing, limiting their sensitivity and reso...
This chapter describes a single-molecule fluorescence resonance energy transfer (smFRET) approach to monitor cotranscriptional RNA folding within native transcription complexes to study cotranscriptional regulatory mechanisms taking place either in bacterial or eukaryotic elongation complexes.
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