On the use of the antibiotic chloramphenicol to target polypeptide chain mimics to the ribosomal exit tunnel.

The ribosomal exit tunnel had recently become the centre of many functional and structural studies. Accumulated evidence indicates that the tunnel is not simply a passive conduit for the nascent chain, but a rather functionally important compartment where nascent peptide sequences can interact with the ribosome to signal translation to slow down or even stop. To explore further this interaction, we have synthesized short peptides attached to the amino group of a chloramphenicol (CAM) base, such that when bound to the ribosome these compounds mimic a nascent peptidyl-tRNA chain bound to the A-site of the peptidyltransferase center (PTC). Here we show that these CAM-peptides interact with the PTC of the ribosome while their effectiveness can be modulated by the sequence of the peptide, suggesting a direct interaction of the peptide with the ribosomal tunnel. Indeed, chemical footprinting in the presence of CAM-P2, one of the tested CAM-peptides, reveals protection of 23S rRNA nucleotides located deep within the tunnel, indicating a potential interaction with specific components of the ribosomal tunnel. Collectively, our findings suggest that the CAM-based peptide derivatives will be useful tools for targeting polypeptide chain mimics to the ribosomal tunnel, allowing their conformation and interaction with the ribosomal tunnel to be explored using further biochemical and structural methods.

Interactions between subunits of Saccharomyces cerevisiae RNase MRP support a conserved eukaryotic RNase P/MRP architecture.

Ribonuclease MRP is an endonuclease, related to RNase P, which functions in eukaryotic pre-rRNA processing. In Saccharomyces cerevisiae, RNase MRP comprises an RNA subunit and ten proteins. To improve our understanding of subunit roles and enzyme architecture, we have examined protein-protein and protein-RNA interactions in vitro, complementing existing yeast two-hybrid data. In total, 31 direct protein-protein interactions were identified, each protein interacting with at least three others. Furthermore, seven proteins self-interact, four strongly, pointing to subunit multiplicity in the holoenzyme. Six protein subunits interact directly with MRP RNA and four with pre-rRNA. A comparative analysis with existing data for the yeast and human RNase P/MRP systems enables confident identification of Pop1p, Pop4p and Rpp1p as subunits that lie at the enzyme core, with probable addition of Pop5p and Pop3p. Rmp1p is confirmed as an integral subunit, presumably associating preferentially with RNase MRP, rather than RNase P, via interactions with Snm1p and MRP RNA. Snm1p and Rmp1p may act together to assist enzyme specificity, though roles in substrate binding are also indicated for Pop4p and Pop6p. The results provide further evidence of a conserved eukaryotic RNase P/MRP architecture and provide a strong basis for studies of enzyme assembly and subunit function.

On the mechanism of action of 9-O-arylalkyloxime derivatives of 6-O-mycaminosyltylonolide, a new class of 16-membered macrolide antibiotics.

New 16-membered 9-aryl-alkyl oxime derivatives of 5-O-mycaminosyl-tylonolid (OMT) have recently been prepared and were found to exhibit high activity against macrolide-resistant strains. In this study, we show that these compounds do not affect the binding of tRNAs to ribosomes in a cell-free system derived from Escherichia coli and that they cannot inhibit peptidyltransferase, peptidyl-tRNA translocation, or poly(U)-dependent poly(Phe) synthesis. However, they severely inhibit poly(A)-dependent poly(Lys) synthesis and compete with erythromycin or tylosin for binding to common or partially overlapping sites in the ribosome. According to footprinting analysis, the lactone ring of these compounds seems to occupy the classic binding site of macrolides that is located at the entrance of the exit tunnel, whereas the extending alkyl-aryl side chain seems to penetrate deeper in the tunnel, where it protects nucleoside A752 in domain II of 23S rRNA. In addition, this side chain causes an increased affinity for mutant ribosomes that may be responsible for their effectiveness against macrolide resistant strains. As revealed by detailed kinetic analysis, these compounds behave as slow-binding ligands interacting with functional ribosomal complexes through a one-step mechanism. This type of inhibitor has several attractive features and offers many chances in designing new potent drugs.