Architecture and folding mechanism of the Azoarcus Group I Pre-tRNA.
Self-splicing RNAs must evolve to function in their specific exon context. The conformation of a group I pre-tRNA(ile) from the bacterium Azoarcus was probed by ribonuclease T(1) and hydroxyl radical cleavage, and by native gel electrophoresis. Biochemical data and three-dimensional models of the pre-tRNA showed that the tRNA is folded, and that the tRNA and intron sequences form separate tertiary domains. Models of the active site before steps 1 and 2 of the splicing reaction predict that exchange of the external G-cofactor and the 3'-terminal G is accomplished by a slight conformational change in P9.0 of the Azoarcus group I intron. Kinetic assays showed that the pre-tRNA folds in minutes, much more slowly than the intron alone. The dependence of the folding kinetics on Mg(2+) and the concentration of urea, and RNase T(1) experiments showed that formation of native pre-tRNA is delayed by misfolding of P3-P9, including mispairing between residues in P9 and the tRNA. Thus, although the intron and tRNA sequences form separate domains in the native pre-tRNA, their folding is coupled via metastable non-native base-pairs. This could help prevent premature processing of the 5' and 3' ends of unspliced pre-tRNA.
Rangan
Prashanth
P
T. C. Jenkins Department of Biophysics, Johns Hopkins University, 3400 N. Charles Street, Baltimore, MD 21218, USA.
Masquida
Benoît
B
Westhof
Eric
E
Woodson
Sarah A
SA
D020627:Azoarcus / Q000201:enzymology / Q000235:genetics*; D001483:Base Sequence; D001665:Binding Sites; D005095:Exoribonucleases / Q000378:metabolism; D017665:Hydroxyl Radical / Q000378:metabolism; D007438:Introns / Q000235:genetics*; D008274:Magnesium / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D012322:RNA Precursors / Q000235:genetics*; D022821:RNA Splice Sites / Q000235:genetics; D012326:RNA Splicing; D012329:RNA, Bacterial / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012355:RNA, Transfer, Ile / Q000737:chemistry / Q000235:genetics*; D013379:Substrate Specificity
D012322:RNA Precursors; D022821:RNA Splice Sites; D012329:RNA, Bacterial; D012355:RNA, Transfer, Ile; D017665:Hydroxyl Radical; D005095:Exoribonucleases; C042081:exoribonuclease T; D008274:Magnesium