MOLDING A PEPTIDE INTO AN RNA SITE BY IN-VIVO PEPTIDE EVOLUTION

Short peptides corresponding to the arginine-rich domains of several R NA-binding proteins are able to bind to their specific RNA sites with high affinities and specificities, In the case of the HIV-1 Rev-Rev re sponse element (RRE) complex, the peptide forms a single cu-helix that binds deeply in a widened, distorted RNA major groove and makes a sub stantial set of base-specific and backbone contacts, Using a reporter system based on antitermination by the bacteriophage lambda N protein, it has been possible to identify novel arginine-rich peptides from co mbinatorial libraries that recognize the RRE with affinities and speci ficities similar to Rev but that appear to bind in nonhelical conforma tions, Here we have used codon-based mutagenesis to evolve one of thes e peptides, RSG-1, into an even tighter binder, After two rounds of ev olution, RSG-1,2 bound the RRE with 7-fold higher affinity and 15-fold higher specificity than the wild-type Rev peptide, and in vitro compe tition experiments show that RSG-1.2 completely displaces the intact R ev protein from the RRE at low peptide concentrations. By fusing RRE-b inding peptides to the activation domain of HIV-1 Tat, we show that th e peptides can deliver Tat to the RRE site and activate transcription in mammalian cells, and more importantly, that the fusion proteins can inhibit the activity of Rev in chloramphenicol acetyltransferase repo rter assays, The evolved peptides contain proline and glutamic acid mu tations near the middle of their sequences and, despite the presence o f a proline, show partial alpha-helix formation in the absence of RNA, These directed evolution experiments illustrate how readily complex p eptide structures can be evolved within the context of an RNA framewor k, perhaps reflecting how early protein structures evolved in an ''RNA world.''