Photoaffinity labeling of the pactamycin binding site on eubacterial ribosomes

Abstract

Pactamycin, an inhibitor of the initial steps of protein synthesis, has an acetophenone group in its chemical structure that makes the drug a potentially photoreactive molecule. In addition, the presence of a phenolic residue makes it easily susceptible to radioactive labeling. Through iodination, one radioactive derivative of pactamycin has been obtained with biological activities similar to the unmodified drug when tested on in vivo and cell-free systems. With the use of [125I]iodopactamycin, ribosomes of Escherichia coli have been photolabeled under conditions that preserve the activity of the particles and guarantee the specificity of the binding sites. Under these conditions, RNA is preferentially labeled when free, small ribosomal subunits are photolabeled, but proteins are the main target in the whole ribosome. This indicates that an important conformational change takes place in the binding site on association of the two subunits. The major labeled proteins are S2, S4, S18, S21, and L13. These proteins in the pactamycin binding site are probably related to the initiation step of protein synthesis.

Site-specific methylation of 16S rRNA caused by pct, a pactamycin resistance determinant from the producing organism, Streptomyces pactum

“…Moreover, at least one of the models (21) places residue 964 fairly close by on the opposite side of the cleft (i.e., on the proximal side of the head). These data may well imply that pactamycin binds into the cleft of the 30S particle and are consistent with the results of photoaffinity labelling studies (24) in which protein S21 was a major target for the drug, in addition to 16S rRNA. Protein S21 sits at the base of the cleft (9) and interacts with 16S RNA close to at least one of the sites that pactamycin protects (3).…”

“…Pactamycin (30 nmol) in 50 mM sodium phosphate (pH 7.5) plus 50% ethanol was iodinated with 0.5 mCi of Na1251 by a modification of the chloramine T method (to avoid direct contact of the drug with oxidant) as previously described (26) and then purified by thin-layer chromatography (24). Nonradioactive iodopactamycin was prepared similarly and added to the radiolabelled drug to give the desired specific radioactivity.…”

“…alone, we cannot conclude that pactamycin recognizes 16S RNA exclusively when binding to the 30S ribosomal subunit (although that may well be so) or that methylation of A-964 (E. coli numbering system) totally abolishes such binding. The filter binding assay used in the experiments summarized in Table 1 would have failed to detect weak residual drug binding, and pactamycin has been shown to photoincorporate into various proteins after binding to E. coli 30S ribosomal subunits (23)(24)(25). Nevertheless, ribosomes methylated on residue A-964 of 16S RNA are insensitive to pactamycin during in vitro protein synthesis (8), and that is the most sensitive assay available to detect meaningful drug binding.…”

Trichothecene Mycotoxins Trigger a Ribotoxic Stress Response That Activates c-Jun N-terminal Kinase and p38 Mitogen-activated Protein Kinase and Induces Apoptosis

“…Pactomycin, another compound that inhibits the ribotoxic stress response (11), also binds to the small ribosomal subunit. Although it is not known to interfere with the binding of anisomycin or trichothecenes, photoaffinity labeling experiments indicate that it also interacts with the large ribosomal subunit (54,55), suggesting that it could block access to the anisomycinand trichothecene-binding site. -12), and then treated with either Me 2 SO (control lanes), T-2 triol (10 M), anisomycin (3.8 M), or T-2 tetraol (10 M) as indicated; after 2 h, the JNKs activity was determined as described under "Experimental Procedures."…”

Resistance of lambda cI translation to antibiotics that inhibit translation initiation

“…Interestingly, alterations in S2 confer resistance to the antibiotic kasugamycin (16,27), an inhibitor of translation initiation (15,23). Furthermore, photoaffinity labeling studies show that S2 associates with the antibiotic pactamycin (24), which also inhibits translation initiation (2,23). If kasugamycin and pactamycin inactivate S2 or ribosomes containing S2, cI translation may be resistant to inhibition by these antibiotics.…”