Structure of 23S rRNA hairpin 35 and its interaction with the tylosin-resistance methyltransferase RlmAII

Abstract

The bacterial rRNA methyltransferase RlmA II (formerly TlrB) contributes to resistance against tylosinlike 16-membered ring macrolide antibiotics. RlmA II was originally discovered in the tylosin-producer Streptomyces fradiae, and members of this subclass of methyltransferases have subsequently been found in other Gram-positive bacteria, including Streptococcus pneumoniae. In all cases, RlmA II methylates 23S rRNA at nucleotide G748, which is situated in a stem±loop (hairpin 35) at the macrolide binding site of the ribosome. The conformation of hairpin 35 recognized by RlmA II is shown here by NMR spectroscopy to resemble the anticodon loop of tRNA. The loop folds independently of the rest of the 23S rRNA, and is stabilized by a non-canonical G±A pair and a U-turn motif, rendering G748 accessible. Binding of S.pneumoniae RlmA II induces changes in NMR signals at speci®c nucleotides that are involved in the methyltransferase±RNA interaction. The conformation of hairpin 35 that interacts with RlmA II is radically different from the structure this hairpin adopts within the 50S subunit. This indicates that the hairpin undergoes major structural rearrangement upon interaction with ribosomal proteins during 50S assembly.

Methylation of 23S rRNA nucleotide G745 is a secondary function of the RlmAI methyltransferase

“…The Acinetobacter rRNA has essentially the same sequence. (Right panel) Tertiary fold of same stem-loop determined by NMR (Lebars et al 2003); the structure is an average of the 17 coordinate sets in the database file. The sheared base pair between G745 and A752 is evident.…”

“…The RlmA I methyltransferase modifies its substrate before the rRNA associates with the r-proteins to assemble into functional 50S subunits (Hansen et al 2001). The structure of this RNA region, in the conformation that is recognized by the methyltransferase, has been solved by NMR (Lebars et al 2003) and shows that G745 and A752 stack onto helix 35 to form an irregular, noncanonical base pair, which does not involve the N1 position of G745 (Fig. 1).…”

Crystal structure of RlmA I : Implications for understanding the 23S rRNA G745/G748-methylation at the macrolide antibiotic-binding site

“…In ribosome structures, rRNA hairpin 35 interacts with the large ␤-sheet of the ribosomal protein L22 and adopts a complementary inverted ''U'' shape (Fig. 5B), which is different from the unbound structure of the hairpin determined by NMR (39). Docking of the L22-bound conformation of hairpin 35 from different ribosome structures (discussed in previous section) shows a reasonable match between the hairpin and the ridge of the W-shaped cleft of RlmA I ; in these modeled complexes, two nucleotides [U480 and A844 of H. marismortui (3), U760 and A764 of D. radiodurans (5), and A747 and A751 of T. thermophilus (38)], at equivalent E. coli positions 747 and 751, point to two SAM-binding pockets of the RlmA I dimer.…”

“…Docking of the rRNA substrate predicts that regions 6-8, 25, 38-52, 117-119, 138-141, 157-162, and 233-235 of molecule 1 and 6-8, 25, 38-52, 115-121, and 136-140 of molecule 2 of the RlmA I dimer are likely to be involved in protein͞RNA interactions. The length of the polypeptide linker (amino acids [35][36][37][38][39][40][41][42][43][44][45][46][47][48][49][50][51][52] between the two domains is 3-4 aa shorter in RlmA II than in RlmA I (Fig. 1 A).…”

“…Amino acid sequence comparison of RlmA I and RlmA II and mapping of the conserved regions onto the crystal structure of E. coli RlmA I indicate positioning of some of the key conserved amino acid residues at putative RNA-binding regions, at the SAM-binding pocket, and at the dimer interface. Docking the publicly available atomic coordinates for hairpin 35 of 23S rRNA and surrounding regions (3,5,(37)(38)(39) into the cleft of RlmA I dimer shows complementary RNA͞protein structural features. This crystal structure, along with earlier reported biochemical data, provides a basis for detailed investigations aimed at understanding structural features of the specific recognition of rRNA substrates, the role of this type of Zn-finger in RNA recognition, general aspects of RNA͞protein interactions, and the mechanism of RNA methylation by RlmA enzymes.…”