Functional significance of sequences following the TATA box of an immunoglobulin promoter studied by random mutagenesis

Abstract

We have investigated the importance of sequences downstream to the TATA box or an immunoglobulin promoter by transfection and in vitro transcription assays. A sequence from −11 to +10 with respect to the transcriptional start site was synthsised by a procedure allowing for random misincorporation of nucleotides. The pool of mutant oligonucleotides was cloned into the respective position of a vector carrying a fusion of a synthetic immunoglobulin heavy chain promoter with the human growth hormone gene. From 200 clones sequenced, 115 were mutants with at least one nucleotide exchange in every position. Whereas most mutations are of minor functional importance, changes at or near the transcriptional start site reduce the promoter activity considerably.