期刊名称：Proceedings of the National Academy of Sciences
印刷版ISSN：0027-8424
电子版ISSN：1091-6490
出版年度：1997
卷号：94
期号：22
页码：11887-11892
DOI：10.1073/pnas.94.22.11887
语种：English
出版社：The National Academy of Sciences of the United States of America
摘要：Short peptides corresponding to the arginine-rich domains of several RNA-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 response element (RRE) complex, the peptide forms a single -helix that binds deeply in a widened, distorted RNA major groove and makes a substantial 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 combinatorial libraries that recognize the RRE with affinities and specificities similar to Rev but that appear to bind in nonhelical conformations. Here we have used codon-based mutagenesis to evolve one of these peptides, RSG-1, into an even tighter binder. After two rounds of evolution, 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 competition experiments show that RSG-1.2 completely displaces the intact Rev protein from the RRE at low peptide concentrations. By fusing RRE-binding peptides to the activation domain of HIV-1 Tat, we show that the 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 reporter assays. The evolved peptides contain proline and glutamic acid mutations near the middle of their sequences and, despite the presence of a proline, show partial -helix formation in the absence of RNA. These directed evolution experiments illustrate how readily complex peptide structures can be evolved within the context of an RNA framework, perhaps reflecting how early protein structures evolved in an "RNA world."
关键词：RNA–protein recognition ; RNA structure ; arginine-rich motif ; HIV-1 Rev-Rev response element ; λ N antitermination
