Poster abstracts
Poster number 1 submitted by Divyaa Bhagdikar
Identification and characterization of S-box riboswitches that regulate at the level of translation initiation
Divyaa Bhagdikar (Department of Microbiology and Center for RNA biology, The Ohio State University, Columbus OH), Frank J. Grundy (Department of Microbiology, The Ohio State University, Columbus OH), Tina M. Henkin (Department of Microbiology and Center for RNA biology, The Ohio State University, Columbus OH)
Abstract:
S-box riboswitches are found primarily in Gram-positive bacteria and regulate the expression of genes involved in methionine and cysteine metabolism in response to S-adenosylmethionine (SAM). Most S-box riboswitches operate at the level of transcription attenuation, such that a terminator helix is stabilized when SAM binds the aptamer domain of the riboswitch. In silico analyses suggested that a rarer class of S-box riboswitches regulate at the level of translation initiation; these include the metI gene from Desulfurispirilum indicum, which encodes cystathionine gamma-synthase. We performed binding assays to show that the aptamer domain of D. indicum metI RNA has SAM binding properties similar to those of the Bacillus subtilis transcriptional metI and other S-box riboswitches. Binding of SAM to the D. indicum metI RNA resulted in sequestration of the Shine-Dalgarno (SD) region and reduced binding of 30S ribosomal subunits to the RNA, demonstrating that the riboswitch regulates at the level of translation initiation. Binding studies showed that the half-life of the translational metI-SAM complex was significantly shorter than that of the transcriptional metI RNA. This suggests that in contrast to the transcriptional B. subtilis metI RNA, the D. indicum translational metI riboswitch can make multiple reversible regulatory decisions. Comparison of the variable regions in the aptamer domains of both RNAs showed that the second internal loop of helix P3, which predominantly has two A residues, consists of a rare single C residue in the translational metI RNA. This sequence is found only in two other S-box RNAs, both of which are predicted to regulate their genes translationally. Mutation of the translational metI RNA to resemble the predominant sequence led to a significant increase in the RNA-SAM complex stability, while an opposite mutation in the transcriptional RNA led to a significant decrease in the RNA-SAM complex half-life. These results indicate that the internal loop sequence plays an important role in determining the stability of the RNA-SAM complex in both transcriptional and translational metI RNAs, and therefore affects the molecular mechanism of riboswitch function.
Keywords: Riboswitch, S box, translation