Poster abstracts
Poster number 35 submitted by Kiel Kreuzer
tRNA binding specificity in the glyQS T box riboswitch
Kiel D. Kreuzer (MCDB, Center for RNA Biology, Department of Microbiology), Nicholas J. Green (MCDB, Department of Microbiology), Frank J. Grundy (Center for RNA biology, Department of Microbiology), Tina M. Henkin (Center for RNA biology, Department of Microbiology)
Abstract:
The T box riboswitch regulates expression of many amino acid-related genes in Gram-positive bacteria, primarily at the level of transcription attenuation. The transcript upstream of the coding region, or leader RNA, of T box family genes binds to a specific tRNA and detects its aminoacylation status, which determines if an intrinsic transcription terminator helix forms(1). Binding of uncharged tRNA stabilizes a competing antiterminator helix that allows RNA polymerase to transcribe the full length mRNA. The primary specificity determinant of the T box leader RNA-tRNA interaction is a three nucleotide Specifier Sequence that base pairs with the anticodon of the cognate tRNA. An additional base pairing interaction occurs between the acceptor arm of uncharged tRNA and a bulge within the antiterminator element. A third leader RNA-tRNA interaction described for the glyQS T box riboswitch, which regulates expression of glycyl-tRNA synthetase, consists of a stacking interaction between the Stem I terminal region and the D/T-loop elbow domain of tRNAGly(2,3). The terminal region of Stem I contains two highly conserved sequence motifs that interact to form the docking platform for the tRNA elbow. Glycyl T box genes contain a specific pattern of nucleotide identity and spacing in both Stem I motifs, including residues that comprise the core of the tRNA docking platform and interact with the tRNA, that differs from that found in the majority of T box RNAs. These differences in Stem I that are specific to glycyl T box genes suggest a potential base-pairing independent mechanism of tRNA binding specificity at this site of interaction. We demonstrated that changing the Bacillus subtilis glyQS Stem I terminal region to match the consensus pattern improved binding and in vitro antitermination for non-cognate tRNA. Additionally, this Stem I variant was less able to discriminate against non-cognate tRNA during transcription in vitro. We also uncovered structural changes in the mutant Stem I that may explain the differences in tRNA binding specificity. We propose that the wild-type glyQS Stem I terminal region pre-screens the cellular tRNA pools and discriminates against non-cognate tRNAs before the Specifier Sequence is transcribed to help facilitate proper leader RNA-tRNA interaction.
References:
1. Grundy FJ & Henkin TM. 1993. tRNA as a positive regulator of transcription antitermination in B. subtilis. Cell 74:475-482.
2. Grigg JC, Chen YJ, Grundy FJ, Henkin TM, Pollack L, Ke AL. 2013. T box RNA decodes both the information content and geometry of tRNA to affect gene expression. Proceedings of the National Academy of Sciences 110:7240-7245.
3. Zhang JW & Ferre-D'Amare AR. 2013. Co-crystal structure of a T-box riboswitch stem I domain in complex with its cognate tRNA. Nature 500:363-366.
Keywords: Riboswitch, tRNA, RNA-RNA interaction