Talk abstracts
Talk on Friday 04:00-04:15pm submitted by Elizabeth Franklin
Ligand and transcription factor NusA differentially modulate 30S subunit recruitment by a translational riboswitch-controlled mRNA
Elizabeth A. Franklin (Department of Chemistry, University of Michigan Ann Arbor), Adrien Chauvier (Department of Chemistry, University of Michigan Ann Arbor), Nils G. Walter (Department of Chemistry and Center for RNA Biomedicine, University of Michigan Ann Arbor)
Abstract:
Riboswitches are structured RNA elements found in the 5’ untranslated regions (UTRs) of mRNAs that fold into alternative conformations during transcription in response to a specific ligand that controls gene expression. Upon ligand binding, riboswitches typically function through either modulating transcription termination or translation initiation. The preQ1-sensing riboswitch of pathogenic Bacillus anthracis regulates gene expression through translation. Upon binding preQ1 the riboswitch undergoes a conformational change interfering with the recruitment of the 30S ribosomal subunit (30S). In bacteria, NusA is an essential transcription factor involved in the regulation of both transcription pausing and termination by RNA polymerase (RNAP). Previous studies have examined the role of transcription factors NusG and RfaH in 30S binding [1]; in contrast, the effects of factor NusA have yet to be elucidated.
We examined NusA recruitment on previously identified RNAP pauses. Single molecule fluorescence analyses of NusA binding indicate less recruitment and a decrease in bound time in the presence of ligand for a paused elongation complex (PEC) located within the 5’ UTR (PEC-63) compared to a PEC located in the ORF downstream of the riboswitch sequence (PEC-99). Addition of unlabeled 30S increases the dissociation rate of NusA only in the presence of ligand for PEC-99. This suggests that the ORF contains an RNA element that promotes NusA recruitment and stability, and that in PEC-99 NusA binding is shortened when both ligand and 30S are present. Additionally, SHAPE (Selective 2'-hydroxyl acylation analyzed by primer extension) probing of PEC-99 indicates that the riboswitch structure is altered differentially in the presence of ligand and 30S.
Our results lead to a model in which ligand binding effects a conformational rearrangement of the riboswitch-embedding mRNA, whereas NusA initiates a physical contact with RNAP modulated by the mRNA structure, thereby affecting translation initiation by the 30S differentially. These results provide additional insights on the role of transcription factors and mRNA folding on the fine-tuning of gene expression. Future work will involve combining NusA and the 30S into single molecule and further bulk probing assays.
References:
[1] S. Chatterjee, A. Chauvier, S. S. Dandpat, I. Artsimovitch, and N. G. Walter, (2021) “A translational riboswitch coordinates nascent transcription-translation coupling,” PNAS, vol. 118, no. 16. doi: 10.1073/pnas.2023426118
Keywords: Riboswitch, Translational Riboswitch