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Co‐Transcriptional Folding of a Riboswitch Controls the Fate of the Transcriptional Machinery
Author(s) -
Walter Nils G.
Publication year - 2018
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2018.32.1_supplement.lb21
Subject(s) - riboswitch , transcription (linguistics) , terminator (solar) , rna , microbiology and biotechnology , biology , bacterial transcription , rna polymerase , chemistry , computational biology , biophysics , non coding rna , genetics , gene , ionosphere , linguistics , philosophy , physics , astronomy
Folding of nascent transcripts can be modulated by properties of the RNA polymerase (RNAP) that carries out the transcription process. For example, the site‐specific pausing of the RNAP and co‐transcriptional RNA‐protein interactions have been shown in several cases to be important for co‐transcriptional RNA folding. In bacteria, riboswitches are genetic elements frequently found in the 5′ untranslated regions of mRNAs, where they respond to cellular metabolites to regulate gene expression either at the level of transcription or translation. Since they change conformation “on‐the‐fly”, riboswitches are tractable models for studying the role of the transcription process in guiding the folding of nascent transcripts. The preQ1 riboswitch from Bacillus subtilis regulates gene expression by a transcriptional mechanism in which the binding of its ligand, the queuosine precursor preQ 1 , causes a conformational change that leads to the formation of a terminator hairpin. Previous studies have deciphered the structure and dynamics of the isolated riboswitch aptamer, but less is known about the impact of the transcriptional machinery on riboswitch folding and vice versa. We have identified a strong pause by RNAP during riboswitch transcription that is stabilized by a previously characterized consensus pause sequence and the ligand‐free conformation of the riboswitch. By using single molecule Förster resonance energy transfer (smFRET) analysis of the riboswitch in a paused elongation complex, we show that RNAP has a profound impact on RNA dynamics, stabilizing a tightly folded structure at this specific pause site. Molecular modeling and biochemical assays further support a specific functional interaction between the nascent transcript and the exit channel of RNAP through which ligand binding to the riboswitch unpauses the polymerase. We are currently using cryo‐electron microscopy of the paused elongation complex to further pinpoint the specific structural rearrangements in both riboswitch and RNAP that are induced first by pausing, then by ligand binding. Finally, we are evaluating the impact of transcription factors on pausing and riboswitch folding. Our study provides significant insights into the mechanisms by which small RNA structures can regulate the function of the macromolecular transcription machinery, and vice versa. Support or Funding Information NIH R01 grants GM062357, GM118524 and GM122803 This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .