POSITIVE TRANSCRIPTIONAL CONTROL ELEMENTS WITHIN THE SP6-KAPPA - PROMOTER DECAMER 3' FLANKING SEQUENCE

The SP6 kappa promoter decamer 3' flanking sequence was shown to contain several positive control elements for transcriptional stimulation which together increased transcriptional stimulation one order of magnitude. One was mapped to an A nucleotide immediately 3' of the decamer consensus motif. The mechanism was to increase the binding affinity of Oct2 for the decamer core motif by a direct interaction between the Oct2 protein and the template, since in vitro transcribed and translated Oct2 showed an identical binding preference when compared to the corresponding gene products in nuclear extracts. By mutation analysis it was shown that the functional activity of the SP6 kappa promoter 3' flanking sequence with regard to transcriptional stimulation was dependent on the presence of such a high affinity Oct binding site. Deletion analysis showed that the POU-Homeo domain of Oct2 sufficed for the flanking sequence mediated affinity increase for decamer elements and that amino or carboxy terminal variations in Oct2 did not influence this interaction. The flanking sequence element also increased Oct1 binding to the same decamer motif. The A 3' of the decamer increased affinity of Oct2 binding to a consensus as well as a mutated decamer. These data illustrate how flanking sequence elements can compensate mutations in the decamer core motif present in a majority of kappa promoters.