DIRECT REPEAT SEQUENCES IN THE STREPTOMYCES CHITINASE-63 PROMOTER DIRECT BOTH GLUCOSE REPRESSION AND CHITIN INDUCTION

The chi63 promoter directs glucose-sensitive, chitin-dependent transcr iption of a gene involved in the utilization of chitin as carbon sourc e, Analysis of 5' and 3' deletions of the promoter region revealed tha t a 350-bp segment is sufficient for wild-type levels of expression an d regulation, The analysis of single base changes throughout the promo ter region, introduced by random and site-directed mutagenesis, identi fied several sequences to be important for activity and regulation, Si ngle base changes at -10, -12, -32, -33, -35, and -37 upstream of the transcription start site resulted in loss of activity from the promote r, suggesting that bases in these positions are important for RNA poly merase interaction, The sequences centered around -10 (TATTCT) and -35 (TTGACC) in this promoter are, in fact, prototypical of eubacterial p romoters, Overlapping the RNA polymerase binding site is a perfect 12- bp direct repeat sequence, Some base changes within this direct repeat resulted in constitutive expression, suggesting that this sequence is an operator for negative regulation, Other base changes resulted in l oss of glucose repression while retaining the requirement for chitin i nduction, suggesting that this sequence is also involved in glucose re pression, The fact that cis-acting mutations resulted 0in glucose resi stance but not inducer independence rules out the possibility that glu cose repression acts exclusively by inducer exclusion, The fact that m utations that affect glucose repression and chitin induction fall with in the same direct repeat sequence module suggests that the direct rep eat sequence facilitates both chitin induction and glucose repression.