Distinct tRNA Accommodation Intermediates Observed on the Ribosome with the Antibiotics Hygromycin A and A201A

Abstract

SUMMARY The increase in multi-drug resistant bacteria is limiting the effectiveness of currently approved antibiotics, leading to a renewed interest in antibiotics with distinct chemical scaffolds. We have solved the structures of the Thermus thermophilus 70S ribosome with A-, P- and E-site tRNAs bound, and in complex with either the aminocyclitol-containing antibiotic hygromycin A (HygA) or the nucleoside antibiotic A201A. Both antibiotics bind at the peptidyl transferase center and sterically occlude the CCA-end of the A-tRNA from entering the A-site of the peptidyl transferase center. Single-molecule Förster resonance energy transfer (smFRET) experiments reveal that HygA and A201A specifically interfere with full accommodation of the A-tRNA, leading to the presence of tRNA accommodation intermediates, and thereby inhibiting peptide bond formation. Thus, our results provide not only insight into the mechanism of action of HygA and A201A, but also into the fundamental process of tRNA accommodation during protein synthesis.

Resistance mutations generate divergent antibiotic susceptibility profiles against translation inhibitors

“…Ribosomes were concentrated to ∼5 mg/mL. In vitro-coupled transcription-translation assays were carried out as described previously using purified components (27).…”

How EF-Tu can contribute to efficient proofreading of aa-tRNA by the ribosome

“…Computational3536 and experimental283738 studies have implicated the following sequence of conformational rearrangements during aa-tRNA accommodation: (1) aa-tRNA moves from an A/T to an elbow-accommodated (EA) conformation, (2) the aa-tRNA arm accommodates into the A-site and (3) the 3′-CCA end enters the peptidyl transferase centre (PTC). The first simulations of factor-free accommodation employed targeting techniques and showed that sequential movement of the elbow, arm and 3′-CCA end is sterically accessible35.…”

Characterization of the biosynthetic gene cluster (ata) for the A201A aminonucleoside antibiotic from Saccharothrix mutabilis subsp. capreolus

“…4 Its chemical structure includes a moiety derived from D-rhamnose, the N 6 , N 6 -dimethyl-3ʹ-amino-3ʹ-deoxyadenosyl (aminonucleoside) moiety of puromycin, an α-methyl-p-coumaric acid (a polyketide) and an unsaturated furanose moiety closely related to similar structures found in hygromycin A (Figure 1). 5 The structural basis for both Hygromycin A and A201A antibiotics binding and inhibition to ribosomes have been recently showed by Polikanov et al 6 The similarities of A201A structure with puromycin and hygromycin A antibiotics strongly suggest that certain enzymes, and hence the corresponding genes in the A201A biosynthetic pathway, may be related to their counterparts in the puromycin and hygromycin A biosynthetic pathways. [7][8][9][10][11] Homologs of the ata open reading frames (ORFs) have indeed been found with for at least 14 ORFs of the hygromycin A biosynthetic cluster in S. hygroscopicus 9 and as we showed previously a set of five consecutive genes involved in the biosynthesis of the aminonucleoside moiety of A201A 12 and their deduced products (AtaP3, AtaP4, AtaP5, AtaP7 and AtaP10) are similar to their counterparts from the pur cluster of S. alboniger (Pur3, Pur4, Pur5, Pur7 and Pur10, respectively), genes implicated in the biosynthesis of the aminonucleoside moiety of puromycin.…”