UV-induced modifications in the peptidyl transferase loop of 23S rRNA dependent on binding of the streptogramin B antibiotic, pristinamycin IA

Abstract

The naturally occurring streptogramin B antibiotic, pristinamycin IA, which inhibits peptide elongation, can produce two modifications in 23S rRNA when bound to the Escherichia coli 70S ribosome and irradiated at 365 nm. Both drug-induced effects map to highly conserved nucleotides within the functionally important peptidyl transferase loop of 23S rRNA at positions m 2 A2503/C2504 and G2061/A2062. The modification yields are influenced strongly, and differentially, by P-site-bound tRNA and strongly by some of the peptidyl transferase antibiotics tested, with chloramphenicol producing a shift in the latter modification to A2062/C2063. Pristinamycin IA can also produce a modification on binding to deproteinized, mature 23S rRNA, at position U2500/C2501. The same modification occurs on an ;37-nt fragment, encompassing positions ;2496-2532 of the peptidyl transferase loop that was excised from the mature rRNA using RNAse H. In contrast, no antibiotic-induced effects were observed on in vitro T7 transcripts of full-length 23S rRNA, domain V, or on a fragment extending from positions ;2496-2566, which indicates that one or more posttranscriptional modifications within the sequence Cm-C-U-C-G-m 2 A-C-G 2505 are important for pristinamycin IA binding and/or the antibiotic-dependent modification of 23S rRNA.

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+…”

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

“…Additional primer extension stops were observed in the peptidyl transferase loop with pristinamycin IA (2503͞2504), chloramphenicol (2611͞2612), and spiramycin (2608͞2609). Although the stops produced by pristinamycin IA also probably derive from a drug-rRNA crosslink (18), the spiramycin effect entirely depends on the presence of sparsomycin. This may reflect a small spiramycin-induced displacement of the crosslinking moiety of sparsomycin toward G2608͞ U2609, similar to the effects produced by the hydrophobic moieties of the sparsomycin derivatives (Fig.…”

Puromycin–rRNA interaction sites at the peptidyl transferase center

“…The streptogramin B drug pristinamycin IA produces modifications in E. coli 23S rRNA at m 2 A2503/⌿2504 and G2061/A2062 upon irradiation with 365 nM light, where the former sites, at least, are likely to involve a drug-rRNA crosslink (Porse et al+, 1999a)+ Given the proximity of the footprinting sites to m 2 A2503/⌿2504, the ability of puromycin to influence these pristinamycin IA-dependent rRNA modifications was investigated (Fig+ 8)+ Puromycin was added to a preformed complex of pristinamycin IA and 70S ribosome-poly (U) complexes before irradiating and analyzing the peptidyltransferase-loop region by primer extension+ Puromycin enhanced the pristinamycin IA-dependent reverse transcriptase stops at m 2 A2503/⌿2504 by 2+5-fold (Fig+ 8, lanes 2 and 3), but had no detectable effect on those at …”

“…(Porse et al+, 1999a)+ Moreover, the puromycin-induced enhancement of the PIA modification in both the presence and absence of a P-site bound tRNA suggests that puromycin resides in the A-site in both ribosomal complexes+…”

“…The antibiotic puromycin has played a central role in our understanding of the mechanism of peptide elongation in the ribosome+ This is because it is partially costructural with the 39 terminus of aminoacyl-tRNA (Harris & Symons, 1973) (Fig+ 1) and can, therefore, function as an amino acid acceptor substrate (Traut & Monro, 1964;Smith et al+, 1965)+ This property of the drug has been exploited in assays for peptide-bond formation and elongation+ These have yielded important, albeit superficial, insight into the mechanisms of inhibition of peptide-bond formation by many other antibiotics (reviewed in Pestka, 1977;Vázquez, 1979;Gale et al+, 1981)+ Several antibiotics have been footprinted on free ribosomes within the peptidyl-transferase loop of 23S rRNA, including some that bind competitively with puromycin (reviewed in + Moreover, for some of the antibiotics, but not puromycin, drug-resistant mutants have been isolated from bacteria and haloarchaea that carry single-site mutations in the peptidyl-transferase loop + This, together with recent UV-induced crosslinking data for sparsomycin (Porse et al+, 1999b) and for the streptogramin B, pristinamycin IA (Porse et al+, 1999a), within this rRNA region suggest that most, if not all, peptidyl-transferase antibiotics bind there (reviewed in Porse et al+, 2000)+ Localization of the puromycin-binding site is important for defining the position of the 39 end of aminoacyl-tRNA immediately prior to peptide-bond formation Kirillov et al+, 1997)+ However, these experiments are difficult to execute for several reasons+ First, puromycin binds very weakly to free ribosomes (Pestka, 1970;Fernandez-Muñoz & Vázquez, 1973)+ In addition, because the drug is an analog of the aminoacyl-acceptor substrate, it is also a potential analog of the donor substrate, and may therefore bind weakly in the P9 site (Kirillov et al+, 1997) of free ribosomes (Bourd et al+, 1983)+ Furthermore, probing in the presence of an active donor substrate will result in peptide bond formation, which will occur coincidentally with the probable movement of the puromycin-peptide complex (Odom et al+, 1990)+ Several crosslinking investigations with different puromycin derivatives have provided useful information on the location of the puromycin-binding site+ In one experiment, the derivative [ 3 H]-p-azidopuromycin was bound to free ribosomes and, upon irradiation with UV light, crosslinked to positions G2502 and U2504, at the base of the peptidyl-transferase loop+ The derivative was also crosslinked to protein L23, which binds to domain III of 23S rRNA (Nicholson et al+, 1982a(Nicholson et al+, , 1982bHall et al+, 1988)+ In another experiment, an attempt was made to locate the equivalent of C-74 of ...…”