T-box riboregulators are a class of cis-regulatory RNAs that govern the bacterial response to amino acid starvation by binding, decoding and reading the aminoacylation status of specific transfer RNAs. Here we provide a high-resolution crystal structure of a full-length T-box from Mycobacterium tuberculosis that explains tRNA decoding and aminoacylation sensing by this riboregulator. Overall, the T-box consists of decoding and aminoacylation sensing modules bridged by a rigid pseudoknot structure formed by the mid-region domains. Stem-I and the Stem-II S-turn assemble a claw-like decoding module, while the antiterminator, Stem-III, and the adjacent linker form a tightly interwoven aminoacylation sensing module. The uncharged tRNA is selectively recognized by an unexpected set of favorable contacts from the linker region in the aminoacylation sensing module. A complex structure with a charged tRNA mimic shows that the extra moiety dislodges the linker, which is indicative of the possible chain of events that lead to alternative base-pairing and altered expression output.
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Atomic coordinates and structure factors for Mtb-ileS_tRNA-OH_native and Mtb-ileS_tRNA-cP_native have been deposited in the Protein Data Bank with the accession codes 6UFG and 6UFH, respectively. Source data for Figs. 2b and 3f are available in Supplementary Table 3. Other data are available upon reasonable request.
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The authors thank A.J. Sachla and J.D. Helmann for advice on the β-galactosidase assay, the Bacillus Genetic Stock Center, the Nicholson Lab for the use of their spectrophotometer and the Fromme Lab for the use of their microscope. This work is supported by the National Institutes of Health (NIH) (grant nos. GM118174 and GM116632 to A.K.). This work is based on research conducted at the Northeastern Collaborative Access Team beamlines, which are funded by the National Institute of General Medical Sciences from the NIH (grant no. P30 GM124165). The Pilatus 6 M detector on the 24-ID-C beamline is funded by a NIH-ORIP HEI grant (no. S10 RR029205). This research used the resources of the Advanced Photon Source, a US Department of Energy Office of Science User Facility operated for the US Department of Energy Office of Science by Argonne National Laboratory (contract no. DE-AC02-06CH11357).
The authors declare no competing interests.
Peer review information Anke Sparmann was the primary editor on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.
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a, Size exclusion chromatogram of Mtb-ileS folded with (blue) and without (orange) tRNAIle. b, Urea denaturing PAGE gel of peak fractions from T-box + tRNA peak. c, Secondary structure model of tyrS T-box from Bacillus subtilis (Bsub-tyrS). Boxes highlight location and identity of each mutation. d, Conversion table for residue notation in Mtb-ileS and Bsub-tyrS. Fold difference from WT is the difference between the average of three induction measurements at 1 hour for WT versus mutation of indicated residue.
a, Detail of G132-U160 wobble pair in the aminoacylation sensing pocket showing AntiS (cyan), tRNA (pale blue) and linker (yellow). Hydrogen bonds represented by black dashes. Magnesium ion represented by green sphere. b, View of AntiS bound to tRNA showing tRNA 3′-end exposed in a large opening. c, Stem-III (pink) and AntiS minor groove interactions. d, Details of G98 interactions with AntiS. e, Details of A118 type-I A-minor interaction with AntiS.
a, Tertiary structure model of Mtb-ileS in complex with tRNAIle-cP. b, Detail of AntiS interaction with tRNAIle-cP NCCA sequence. Hydrogen bonds represented by black dashes. Asterisk indicates residues mutated from wildtype sequence. c, Size exclusion chromatograms of Mtb-ileS folded with tRNAIle-cP (blue) and with tRNAIle-OH (orange). d, Urea denaturing PAGE gel of peak fractions from T-box + tRNAIle-cP peak.
a, tRNA recruitment and decoding. b, Pseudoknot formation positions AntiS to interact with tRNA NCCA sequence. c, Transient intermediate where aminoacylation sensing module interacts with uncharged (top) and charged (bottom) tRNA. d, Favorable interactions between uncharged tRNA and linker locks T-box into ON conformation. Exposed Shine–Dalgarno (SDS) allows translation initiation. e, Steric clashing between charged tRNA and linker unravels the aminoacylation sensing module leading to alternative base pairing. Sequestrator formation prevents ribosome access to SDS and prevents translation.
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Battaglia, R.A., Grigg, J.C. & Ke, A. Structural basis for tRNA decoding and aminoacylation sensing by T-box riboregulators. Nat Struct Mol Biol 26, 1106–1113 (2019). https://doi.org/10.1038/s41594-019-0327-6
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