Direct crosslinking of the antitumor antibiotic sparsomycin, and its derivatives, to A2602 in the peptidyl transferase center of 23S-like rRNA within ribosome-tRNA complexes

Abstract

The antitumor antibiotic sparsomycin is a universal and potent inhibitor of peptide bond formation and selectively acts on several human tumors. It binds to the ribosome strongly, at an unknown site, in the presence of an N-blocked donor tRNA substrate, which it stabilizes on the ribosome. Its site of action was investigated by inducing a crosslink between sparsomycin and bacterial, archaeal, and eukaryotic ribosomes complexed with P-site-bound tRNA, on irradiating with low energy ultraviolet light (at 365 nm). The crosslink was localized exclusively to the universally conserved nucleotide A2602 within the peptidyl transferase loop region of 23S-like rRNA by using a combination of a primer extension approach, RNase H fragment analysis, and crosslinking with radioactive [ 125 I]phenol-alanine-sparsomycin. Crosslinking of several sparsomycin derivatives, modified near the sulfoxy group, implicated the modified uracil residue in the rRNA crosslink. The yield of the antibiotic crosslink was weak in the presence of deacylated tRNA and strong in the presence of an N-blocked P-site-bound tRNA, which, as was shown earlier, increases the accessibility of A2602 on the ribosome. We infer that both A2602 and its induced conformational switch are critically important both for the peptidyl transfer reaction and for antibiotic inhibition. This supposition is reinforced by the observation that other antibiotics that can prevent peptide bond formation in vitro inhibit, to different degrees, formation of the crosslink. Sparsomycin (Fig.

Peptidyl transferase antibiotics perturb the relative positioning of the 3′-terminal adenosine of P/P′-site-bound tRNA and 23S rRNA in the ribosome

“…In the presence of sparsomycin the cross-link yield to F19 increased, at least for the N-Ac-Phe-(2N 3 A76)tRNA (Table 3)+ This correlates with our recent finding that sparsomycin can cross-link directly to A2602, on irradiation at 365 nm, in the presence of P-site-bound tRNA, where N-Ac-Phe-tRNA stimulates the sparsomycin cross-link sixfold more than deacylated tRNA (Porse et al+, 1999b)+ The latter observation is consistent with sparsomycin stimulating the ribosomal binding of N-Ac-Phe-tRNA and vice versa (Lázaro et al+, 1991)+ Thus, in addition to interacting with A2602, sparsomycin may also interact directly with the 39-terminal adenosine of the tRNA+ For pristinamycin IA, a strong increase occurred in the cross-link with F49 (A2062/C2063) that correlates with the finding that this drug also generates a UVinduced modification (probably an internal rRNArRNA cross-link) at the same nucleotides in E. coli ribosomes (Porse et al+, 1999a)+ The concurrent decrease in the cross-link to F29 (U2506), exclusively with N-Ac-Phe-(2N 3 A76)tRNA, may also correlate with pristinamycin IA forming a putative direct cross-link to the neighboring positions m 2 A2503/x2504 on UV irradiation, an effect that is also strongly stimulated by the presence of P-site-bound deacylated or N-Ac-PhetRNA (Porse et al+, 1999a)+ Thus, we can conclude that pristinamycin IA and the 39-terminal adenosine of P9-site-bound tRNA are closely juxtapositioned on the 23S RNA+…”

Cross-linking in the Living Cell Locates the Site of Action of Oxazolidinone Antibiotics

“…Binding of several different ribosome-targeted antibiotics is known to be stimulated by simultaneous binding of co-ligands. For example, the affinity of sparsomycin significantly increases in the presence of peptidyl-tRNA (53), enhancing its ability to also cross-link to A-2602 (54). Moreover, erythromycin binding may be stimulated by specific nascent peptides (55).…”

RNA-based regulation of genes of tryptophan synthesis and degradation, in bacteria

“…Some peptide-ribosomal interactions believed to be responsible for tryptophan induction of tna operon expression. A region of the 50S ribosomal subunit is shown containing bound sparsomycin, puromycin, and presumably tryptophan, in the A site of the translating ribosome (Porse et al 1999;Hansen et al 2003;CruzVera et al 2005CruzVera et al , 2007. Also shown is the probable location of TnaCtRNA Pro in the ribosome exit tunnel; identified are likely locations of residues Trp12 and Pro24, residues essential for induction.…”