Promoter analysis by saturation mutagenesis.
Gene expression and regulation are mediated by DNA sequences, in most instances, directly upstream to the coding sequences by recruiting transcription factors, regulators, and a RNA polymerase in a spatially defined fashion. Few nucleotides within a promoter make contact with the bound proteins. The minimal set of nucleotides that can recruit a protein factor is called a cis-acting element. This article addresses a powerful mutagenesis strategy that can be employed to define cis-acting elements at a molecular level. Technical details including primer design, saturation mutagenesis, construction of promoter libraries, phenotypic analysis, data analysis, and interpretation are discussed.
Baliga
Nitin S
NS
Institute for Systems Biology. 4225 Roosevelt way NE, Suite 200, Seattle, WA 98105. USA. nbaliga@systemsbiology.org