title: Continuous in vitro evolution of bacteriophage RNA polymerase promoters.
abstract: Rapid in vitro evolution of bacteriophage T7, T3, and SP6 RNA polymerase promoters was achieved by a method that allows continuous enrichment of DNAs that contain functional promoter elements. This method exploits the ability of a special class of nucleic acid molecules to replicate continuously in the presence of both a reverse transcriptase and a DNA-dependent RNA polymerase. Replication involves the synthesis of both RNA and cDNA intermediates. The cDNA strand contains an embedded promoter sequence, which becomes converted to a functional double-stranded promoter element, leading to the production of RNA transcripts. Synthetic cDNAs, including those that contain randomized promoter sequences, can be used to initiate the amplification cycle. However, only those cDNAs that contain functional promoter sequences are able to produce RNA transcripts. Furthermore, each RNA transcript encodes the RNA polymerase promoter sequence that was responsible for initiation of its own transcription. Thus, the population of amplifying molecules quickly becomes enriched for those templates that encode functional promoters. Optimal promoter sequences for phage T7, T3, and SP6 RNA polymerase were identified after a 2-h amplification reaction, initiated in each case with a pool of synthetic cDNAs encoding greater than 10(10) promoter sequence variants.
authors.affiliation: Department of Chemistry, Scripps Research Institute, La Jolla, California 92037.
authors.forename: R R
authors.initials: RR
authors.lastname: Breaker
authors.forename: A
authors.initials: A
authors.lastname: Banerji
authors.forename: G F
authors.initials: GF
authors.lastname: Joyce
mesh_terms: D017903:Bacteriophage T3 / Q000201:enzymology; D017123:Bacteriophage T7 / Q000201:enzymology; D001435:Bacteriophages / Q000201:enzymology*; D001483:Base Sequence; D005075:Biological Evolution; D016384:Consensus Sequence; D004261:DNA Replication; D012321:DNA-Directed RNA Polymerases / Q000378:metabolism*; D008969:Molecular Sequence Data; D016296:Mutagenesis; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012194:RNA-Directed DNA Polymerase / Q000378:metabolism; D013379:Substrate Specificity; D014158:Transcription, Genetic
chemical_list: C070852:RNA polymerase SP6; D012194:RNA-Directed DNA Polymerase; D012321:DNA-Directed RNA Polymerases