EXPERIMENTALLY DETERMINED WEIGHT MATRIX DEFINITIONS OF THE INITIATOR AND TBP BINDING-SITE ELEMENTS OF PROMOTERS

The basal elements of class II promoters are: (i) a-30 region, recogni zed by TATA binding protein (TBP); (ii) an initiator (Inr) surrounding the start site for transcription; (iii) frequently a downstream (+10 to +35) element, To determine the sequences that specify an Inr, we pe rformed a saturation mutagenesis of the Inr of the SV40 major late pro moter (SV40-MLP). The transcriptional activity of each mutant was dete rmined both in vivo and in vitro. An excellent correlation between tra nscriptional activity and closeness of fit to the optimal Inr sequence , 5'-CAG/TT-3', was found to exist both in vivo and in vitro, Employin g a neural network technique we generated from these data a weight mat rix definition of an Inr that can be used to predict the activity of a given sequence as an Inr, Using saturation mutagenesis data of TBP bi nding sites we likewise generated a weight matrix definition of the -3 0 region element, We conclude the following: (i) Inrs are defined by t he nucleotides immediately surrounding the transcriptional start site; (ii) most, if not all, Inrs are recognized by the same general transc ription factor(s), We propose that the mechanism of transcription init iation is fundamentally conserved, with the formation of pre-initiatio n complexes involving the concurrent binding of general transcription factors to the -30, Inr and, possibly, downstream elements of class II promoters.