The antibiotic viomycin as a model peptide for the origin of the co-evolution of RNA and proteins.

Viomycin is an RNA-binding peptide antibiotic which inhibits prokaryotic protein synthesis and group I intron self-splicing. This antibiotic enhances the activity of the ribozyme derived from the Neurospora crassa VS RNA, and at sub-inhibitory concentrations it induces the formation of group I intron oligomers. Here, we address the question whether viomycin exerts specificity in the promotion of RNA-RNA interactions. In an in vitro selection experiment we tested the ability of viomycin to specifically select molecules out of an RNA pool. Group I intron RNA was incubated with a pool of random sequence RNA, or with a pool of RNA molecules which had previously been enriched for viomycin-binding RNAs. Viomycin was added in order to select viomycin-binding RNAs and to guide their interaction with the intron RNA resulting in recombinant molecules. Viomycin was indeed capable of specifically selecting RNA molecules which contain viomycin-binding sites promoting recombination. These results suggest that small peptides are able to play the role of selector molecules in a putative 'RNA World' launching the co-evolution of RNA and proteins into an 'RNA-protein World'.

Assaying RNA chaperone activity in vivo using a novel RNA folding trap.

In the absence of proteins, RNAs often misfold in vitro due to alternative base pairings which result from the molecule being trapped in inactive conformations. We identify an in vivo folding trap in the T4 phage td gene, caused by nine base pairs between a sequence element in the upstream exon of the td gene and another at the 3' end of the intron. During translation, the ribosome resolves this interaction; consequently the intron folds correctly and splicing occurs. The introduction of a stop codon upstream of this base pairing prevents resolution of the inactive structure so that splicing cannot proceed. We have used this folding trap to probe for RNA binding proteins which, when overexpressed, either resolve the misfolded structure or impede its formation in vivo. We distinguish between proteins which recognize the intron structure and those which bind non-specifically and apparently ignore the intron. The first class, e.g. Neurospora crassa CYT-18, can rescue the exonic trap and intron mutants which cause a structural defect. However, known RNA chaperones such as Escherichia coli StpA and S12 and the HIV protein NCp7, only resolve the exonic trap without suppressing intron mutations. Thus, this structural trap enables detection of RNA chaperone activity in vivo.

In vitro selection of a viomycin-binding RNA pseudoknot.

BACKGROUND: The peptide antibiotic viomycin inhibits ribosomal protein synthesis, group I intron self-splicing and self-cleavage of the human hepatitis delta virus ribozyme. To understand the molecular basis of RNA binding and recognition by viomycin, we isolated a variety of novel viomycin-binding RNA molecules using in vitro selection. RESULTS: More than 90% of the selected RNA molecules shared one continuous highly conserved region of 14 nucleotides. Mutational analyses, structural probing, together with footprinting experiments by chemical modification, and Pb2+-induced cleavage showed that this conserved sequence harbours the antibiotic-binding site and forms a stem-loop structure. Moreover, the loop is engaged in a long-range interaction forming a pseudoknot. CONCLUSIONS: A comparison between the novel viomycin-binding motif and the natural RNA target sites for viomycin showed that all these segments form a pseudoknot at the antibiotic-binding site. We therefore conclude that this peptide antibiotic has a strong selectivity for particular RNA pseudoknots.