scholarly journals RNA polymerase pausing and nascent-RNA structure formation are linked through clamp-domain movement

2014 ◽  
Vol 21 (9) ◽  
pp. 794-802 ◽  
Author(s):  
Pyae P Hein ◽  
Kellie E Kolb ◽  
Tricia Windgassen ◽  
Michael J Bellecourt ◽  
Seth A Darst ◽  
...  
2019 ◽  
Author(s):  
Eric J. Strobel ◽  
John T. Lis ◽  
Julius B. Lucks

AbstractSite-specific arrest of RNA polymerase is fundamental to several technologies that measure RNA structure and function. Current in vitro transcription ‘roadblocking’ approaches inhibit transcription elongation using a protein blockade bound to the DNA template. One limitation of protein-mediated transcription roadblocking is that it requires the inclusion of a protein factor that is extrinsic to the minimal in vitro transcription reaction. In this work, we show that interrupting the transcribed DNA strand with an internal desthiobiotin-triethylene glycol modification efficiently and stably halts Escherichia coli RNA polymerase transcription. To facilitate diverse applications of chemical transcription roadblocking, we establish a simple and sequence-independent method for the preparation of internally modified double-stranded DNA templates by sequential PCR and translesion synthesis. By encoding an intrinsic stall site within the template DNA, our chemical transcription roadblocking approach enables nascent RNA molecules to be displayed from RNA polymerase in a minimal in vitro transcription reaction.


2021 ◽  
Author(s):  
Nils Walter ◽  
Adrien Chauvier ◽  
Jason Porta ◽  
Indrajit Deb ◽  
Emily Ellinger ◽  
...  

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.


2020 ◽  
Author(s):  
Adrián López Martín ◽  
Mohamed Mounir ◽  
Irmtraud M Meyer

Abstract RNA structure formation in vivo happens co-transcriptionally while the transcript is being made. The corresponding co-transcriptional folding pathway typically involves transient RNA structure features that are not part of the final, functional RNA structure. These transient features can play important functional roles of their own and also influence the formation of the final RNA structure in vivo. We here present CoBold, a computational method for identifying different functional classes of transient RNA structure features that can either aid or hinder the formation of a known reference RNA structure. Our method takes as input either a single RNA or a corresponding multiple-sequence alignment as well as a known reference RNA secondary structure and identifies different classes of transient RNA structure features that could aid or prevent the formation of the given RNA structure. We make CoBold available via a web-server which includes dedicated data visualisation.


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