Talk abstracts

Talk on Friday 01:10-01:30pm submitted by Ryan Fuchs

The Smk box riboswitch in Lactobacillales

Ryan T. Fuchs (Department of Microbiology, The Ohio State University), Frank J. Grundy (Department of Microbiology, The Ohio State University), Tina M. Henkin (Department of Microbiology, The Ohio State University)

Abstract:
The SMK box is a conserved riboswitch motif found in the 5’ untranslated region of metK genes (encoding S-adenosylmethionine (SAM) synthetase) in lactic acid bacteria, including Enterococcus, Streptococcus and Lactococcus species. This RNA element binds SAM in vitro, and SAM binding causes a structural rearrangement that sequesters the Shine-Dalgarno (SD) sequence. Mutations that disrupt pairing between the SD and a region complementary to the SD (designated the anti-SD, or ASD) block SAM binding, while compensatory mutations that restore pairing also restore SAM binding. The SMK box sequence from Enterococcus faecalis conferred translational repression of a lacZ reporter when cells were grown under conditions where SAM pools are elevated. Based on these studies, a model was developed in which SAM binding inhibits metK translation by sequestration of the SD region of the mRNA. In the current work, this model was tested using biochemical approaches. Addition of SAM was shown to inhibit binding of 30S ribosomal subunits to SMK box RNA; in contrast, addition of S-adenosylhomocysteine (SAH) had no effect. A mutant RNA defective in SAM binding showed no reduction of ribosome binding in the presence of SAM. Primer extension inhibition assays provided further evidence for SD-ASD pairing in the presence of SAM. These results strongly support the model that SMK box translational repression operates through occlusion of the ribosome binding site.

References:
Fuchs, R.T., Grundy, F.J., and Henkin, T.M. The Smk box is a new SAM-binding RNA for translational regulation of SAM synthetase. Nat. Struct. Mol. Biol. 13, 226-233 (2006).

Keywords: riboswitch, translation, SAM