Water molecules participate in the binding specificity by exploiting the antibiotic hydration shell and the typical RNA water hydration patterns. The observed contacts rationalize the protection, mutation, and resistance data. The crystal packing mimics the intermolecular contacts induced by aminoglycoside binding in the ribosome.

The crystal structures of six complexes between aminoglycoside antibiotics (neamine, gentamicin C1A, kanamycin A, ribostamycin, lividomycin A and neomycin B) and oligonucleotides containing the decoding A site of bacterial ribosomes are reported at resolutions between 2.2 and 3.0 Å. Although the number of contacts between the RNA and the aminoglycosides varies between 20 and 31, up to eight direct hydrogen bonds between rings I and II of the neamine moiety are conserved in the observed complexes. The puckered sugar ring I is inserted into the A site helix by stacking against G1491 and forms a pseudo base pair with two H-bonds to the Watson–Crick sites of the universally conserved A1408. This central interaction helps to maintain A1492 and A1493 in a bulged-out conformation. All these structures of the minimal A site RNA complexed to various aminoglycosides display crystal packings with intermolecular contacts between the bulging A1492 and A1493 and the shallow/minor groove of Watson–Crick pairs in a neighbouring helix. In one crystal, one empty A site is observed. In two crystals, two aminoglycosides are bound to the same A site with one bound specifically and the other bound in various ways in the deep/major groove at the edge of the A sites.

Aminoglycoside antibiotics target the decoding aminoacyl site (A site) on the 16S ribosomal RNA and induce miscoding during translation. Here, we present the crystal structure, at 2.54 A resolution, of an RNA oligonucleotide containing the A site sequence complexed to the 4,6-disubstituted 2-deoxystreptamine aminoglycoside tobramycin. The three aminosugar rings making up tobramycin interact with the deep-groove atoms directly or via water molecules and stabilize a fully bulged-out conformation of adenines A(1492) and A(1493). The comparison between this structure and the one previously solved in the presence of paromomycin confirms the importance of the functional groups on the common neamine part of these two antibiotics for binding to RNA. Furthermore, the analysis of the present structure provides a molecular explanation to some of the resistance mechanisms that have spread among bacteria and rendered aminoglycoside antibiotics inefficient.

Circular RNAs are generated during splicing through various mechanisms. Ashwal-Fluss et al. demonstrate that exon circularization and linear splicing compete with each other in a tissue-specific fashion, and Zhang et al. show that exon circularization depends on flanking intronic complementary sequences. Both papers show that several types of circular RNA transcripts can be produced from a single gene.

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Aminoglycoside antibiotics target the 16S ribosomal RNA (rRNA) bacterial A site and induce misreading of the genetic code. Point mutations of the ribosomal A site may confer resistance to aminoglycoside antibiotics. The influence of bacterial mutations (introduced by site-directed mutagenesis) on ribosomal drug susceptibility was investigated in vivo by determination of minimal inhibitory concentrations. To determine the origin of the various resistance phenotypes at a molecular level, the in vivo results were compared with the previously published crystal structures of paromomycin, tobramycin, and geneticin bound to oligonucleotides containing the minimal A site. Two regions appear crucial for binding in the A site: the single adenine residue at position 1408 and the non-Watson-Crick U1406.U1495 pair. The effects of mutations at those positions are modulated by the nature of the substituent at position 6' (either hydroxy or ammonium group) on ring I, by the number of positive charges on the antibiotic, and by the linkage between rings I and III (either 4,5 or 4,6). In particular, the analysis demonstrates: 1) that the C1409-G1491 to A1409-U1491 polymorphism (observed in 15 % of bacteria) is not associated with resistance, which indicates that it does not affect the stacking of ring I on residue 1491, 2) that the high-level resistance to 6'-NH3+ aminoglycosides exhibited by the A1408G mutation most probably results from the inability of ring I forming a pseudo base pair with G1408, which prevents its insertion inside the A site helix, and 3) that mutations of the uracil residues forming the U1406.U1495 pair either to cytosine or to adenine residues mostly confer low to moderate levels of drug resistance, whereas the U1406C/U1495A double mutation confers high-level resistance (except for neomycin), which suggests that aminoglycoside binding to the wild-type A site and its functional consequences strongly depend on a particular geometry of the U1406.U1495 pair. The relationships between the resistance phenotypes observed in vivo and the interactions described at the molecular level define the biological importance of the different structural interactions observed by X-ray crystallography studies.

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