01 2&1-’0 345-& 263 · 01 2&1-’0 345-& 263 ... c

13
Structure of a Eukaryotic Decoding Region A-site RNA Stephen R. Lynch and Joseph D. Puglisi* Department of Structural Biology, Stanford University School of Medicine, Stanford CA 94305-5126, USA The aminoglycoside antibiotics target a region of highly conserved nucleotides in the aminoacyl-tRNA site (A site) of 16 S RNA on the 30 S subunit. The structures of a prokaryotic decoding region A-site oligonu- cleotide free in solution and bound to the aminoglycosides paromomycin and gentamicin C1A have been determined. Here, the structure of a eukaryotic decoding region A-site oligonucleotide has been determined using homonuclear and heteronuclear NMR spectroscopy, and compared to the unbound prokaryotic rRNA structure. The two structures are simi- lar, with a U1406-U1495 base-pair, a C1407-G1494 Watson-Crick base- pair, and a G1408-A1493 base-pair instead of the A1408-A1493 base-pair of the prokaryotic structure. The two structures differ in the orientation of the 1408 position with respect to A1493; G1408 is rotated toward the major groove, which is the binding pocket for aminoglycosides. The structures also differ in the stacking geometry of G1494 on A1493, which could have slight long-range conformational effects. # 2001 Academic Press Keywords: decoding; structure; oligonucleotide; NMR; eukaryotic 16 S rRNA *Corresponding author Introduction Decoding is the process in which the ribosome recognizes the correct interaction of the mRNA codon and the cognate tRNA. This interaction occurs at the decoding region aminoacyl-tRNA site (A site) on the small (30 S) ribosomal subunit near the 3 0 end of 16 S rRNA in a region of highly con- served nucleotides (Figure 1) (Noller, 1991). The decoding region A site (decoding site) probably contacts the codon-anticodon interaction to ensure accuracy during translation; consistent with this hypothesis, tRNA protects A1492 and A1493 from chemical probes (Moazed & Noller, 1986) and the activity of the deleterious mutations A1492G and A1493G can be recovered in vitro by replacing the mRNA with a 2 0 ¯uoro-mRNA (Yoshizawa et al., 1999). The three-dimensional X-ray crystal structures of the 70 S ribosome (Cate et al., 1999) and the 30 S (Clemons et al., 1999) subunit have con®rmed that the penultimate stem of the 30 S subunit, a few base-pairs below the decoding site, contacts the 900 region. Although little of the electron density had been ®t in the 70 S structure, the long penulti- mate stem and the 900 region were identi®ed and shown to be making an RNA-RNA contact, which agrees with the ®ndings from the structure of the 30 S subunit. The proximity of the decoding site to the 900 region is not surprising, considering that the 900 region, containing the switch helix, is criti- cal for ®delity of translation (Lodmell & Dahlberg, 1997). In the 30 S crystal structure, the penultimate stem is in a different conformation and oriented differently with respect to the 900 region, but the two regions of RNA still make the long-range RNA-RNA interaction. The difference in the con- formation of the penultimate stem in the two struc- tures suggests that the penultimate stem changes conformation during the translation cycle. Since the penultimate stem contacts other regions of 16 S RNA and is at the subunit interface, a local confor- mational difference in the decoding site could be transmitted to other regions of the ribosome. The mechanism of translation of mRNA to pro- tein is conserved in eukaryotes, although it is more complicated than that of prokaryotes. Many regions of rRNA, particularly those involved with important steps of translation, are conserved throughout all organisms. In the decoding site, six of the seven nucleotides in the internal loop are universally conserved, all except for position 1408, which is adenosine in all prokaryotic and mito- chondrial 16 S RNA sequences and guanosine in all eukaryotic cytosolic sequences (Gutell, 1994). Higher eukaryotes often have a mispair at pos- itions 1409-1491, but many lower eukaryotes including Giardia muris and Tetrahymena thermophila E-mail address of the corresponding author: [email protected] doi:10.1006/jmbi.2000.4419 available online at http://www.idealibrary.com on J. Mol. Biol. (2001) 306, 1023±1035 0022-2836/01/051023±13 $35.00/0 # 2001 Academic Press

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