U6 RNA is a key component of the catalytic core of the spliceosome. A metal ion essential for the first catalytic step of pre-mRNA splicing binds to the U80 Sp phosphate oxygen within the yeast U6 intramolecular stem-loop (ISL). Here we present the first structural data for U6 RNA, revealing the three-dimensional structure of the highly conserved U6 ISL. The ISL binds metal ion at the U80 site with the same stereo specificity as the intact spliceosome. The metal-binding site is adjacent to a readily protonated C.A wobble pair. Protonation of the C.A pair and metal binding are mutually antagonistic. These results support a ribozyme model for U6 RNA function and suggest a possible mechanism for the regulation of RNA splicing.

Peptide release on the ribosome is catalyzed in the large subunit peptidyl transferase center by release factors on recognition of stop codons in the small subunit decoding center. Here we examine the role of the decoding center in this process. Mutation of decoding center nucleotides or removal of 2'OH groups from the codon--deleterious in the related process of tRNA selection--has only mild effects on peptide release. The miscoding antibiotic paromomycin, which binds the decoding center and promotes the critical steps of tRNA selection, instead dramatically inhibits peptide release. Differences in the kinetic mechanism of paromomycin inhibition on stop and sense codons, paired with correlated structural changes monitored by chemical footprinting, suggest that recognition of stop codons by release factors induces specific structural rearrangements in the small subunit decoding center. We propose that, like other steps in translation, the specificity of peptide release is achieved through an induced-fit mechanism.

Phosphorothioate-substitution experiments are often used to elucidate functionally important metal ion-binding sites on RNA. All previous experiments with S P -phosphorothioate-substituted RNAs have been done in the absence of structural information for this particular diastereomer. Yeast U6 RNA contains a metal ion-binding site that is essential for spliceosome function and includes the pro-S P oxygen 5 of U 80 . S P -phosphorothioate substitution at this location creates spliceosomes dependent on thiophilic ions for the first step of splicing. We have determined the solution structure of the U 80 S P -phosphorothioatesubstituted U6 intramolecular stem-loop (ISL), and also report the refined NMR structure of the unmodified U6 ISL. Both structures were determined with inclusion of 1 H- 13C residual dipolar couplings. The precision of the structures with and without phosphorothioate (RMSD = 1.05 and 0.79 Å, respectively) allows comparison of the local and long-range structural effect of the modification. We find that the U6-ISL structure is unperturbed by the phosphorothioate. Additionally, the thermodynamic stability of the U6 ISL is dependent on the protonation state of the A 79 -C 67 wobble pair and is not affected by the adjacent phosphorothioate. These results indicate that a single S P -phosphorothioate substitution can be structurally benign, and further validate the metal ion rescue experiments used to identify the essential metal-binding site(s) in the spliceosome.