|
|
| Line 9: |
Line 9: |
| <div style="background-color:#fffaf0;"> | | <div style="background-color:#fffaf0;"> |
| == Publication Abstract from PubMed == | | == Publication Abstract from PubMed == |
| In Gram-positive bacteria, T-box riboswitches regulate the expression of aminoacyl-tRNA synthetases and other proteins in response to fluctuating transfer RNA aminoacylation levels under various nutritional states. T-boxes reside in the 5'-untranslated regions of the messenger RNAs they regulate, and consist of two conserved domains. Stem I contains the specifier trinucleotide that base pairs with the anticodon of cognate tRNA. 3' to stem I is the antiterminator domain, which base pairs with the tRNA acceptor end and evaluates its aminoacylation state. Despite high phylogenetic conservation and widespread occurrence in pathogens, the structural basis of tRNA recognition by this riboswitch remains ill defined. Here we demonstrate that the ~100-nucleotide T-box stem I is necessary and sufficient for specific, high-affinity (dissociation constant (Kd) ~150 nM) tRNA binding, and report the structure of Oceanobacillus iheyensis glyQ stem I in complex with its cognate tRNA at 3.2 A resolution. Stem I recognizes the overall architecture of tRNA in addition to its anticodon, something accomplished by large ribonucleoproteins such as the ribosome, or proteins such as aminoacyl-tRNA synthetases, but is unprecedented for a compact mRNA domain. The C-shaped stem I cradles the L-shaped tRNA, forming an extended (1,604 A(2)) intermolecular interface. In addition to the specifier-anticodon interaction, two interdigitated T-loops near the apex of stem I stack on the tRNA elbow in a manner analogous to those of the J11/12-J12/11 motif of RNase P and the L1 stalk of the ribosomal E-site. Because these ribonucleoproteins and T-boxes are unrelated, this strategy to recognize a universal tRNA feature probably evolved convergently. Mutually induced fit of stem I and the tRNA exploiting the intrinsic flexibility of tRNA and its conserved post-transcriptional modifications results in high shape complementarity, which in addition to providing specificity and affinity, globally organizes the T-box to orchestrate tRNA-dependent transcription regulation.
| | Compared to globular proteins, RNAs with complex 3D folds are characterized by poorly differentiated molecular surfaces dominated by backbone phosphates, sparse tertiary contacts stabilizing global architecture, and conformational flexibility. The resulting generally poor order of crystals of large RNAs and their complexes frequently hampers crystallographic structure determination. We describe and rationalize a postcrystallization treatment strategy that exploits the importance of solvation and counterions for RNA folding. Replacement of Li(+) and Mg(2+) needed for growth of crystals of a tRNA-riboswitch-protein complex with Sr(2+), coupled with dehydration, dramatically improved the resolution limit (8.5-3.2 A) and data quality, enabling structure determination. The soft Sr(2+) ion forms numerous stabilizing intermolecular contacts. Comparison of pre- and posttreatment structures reveals how RNA assemblies redistribute as quasi-rigid bodies to yield improved crystal packing. Cation exchange complements previously reported postcrystallization dehydration of protein crystals and represents a potentially general strategy for improving crystals of large RNAs. |
|
| |
|
| Co-crystal structure of a T-box riboswitch stem I domain in complex with its cognate tRNA.,Zhang J, Ferre-D'Amare AR Nature. 2013 Aug 15;500(7462):363-6. doi: 10.1038/nature12440. Epub 2013 Jul 28. PMID:23892783<ref>PMID:23892783</ref>
| | Dramatic Improvement of Crystals of Large RNAs by Cation Replacement and Dehydration.,Zhang J, Ferre-D'Amare AR Structure. 2014 Sep 2;22(9):1363-71. doi: 10.1016/j.str.2014.07.011. PMID:25185828<ref>PMID:25185828</ref> |
|
| |
|
| From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> | | From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.<br> |