Functional analysis of different sequence elements in the Escherichia coli galactose operon P 2 promoter

Summary Starting with a DNA fragment containing the galactose operon P 2 promoter, we made a series of deletions that progressively replaced DNA sequences upstream of the transcription startpoint and determined their effects on P 2 activity. The results show that specific sequences upstream of −32 are not important. Removal of the sequence 5′‐CACA‐3′ from −32 to −28 reduces P 2 activity by 50%: longer deletions to −16 further reduce activity but do not remove the information specifying the transcription startpoint. DNA sequences between −32 and −16 at gal P 2 assist the isomerization of RNA polymerase from closed to open complexes rather than contributing to the initial binding of RNA polymerase. The activity of gal P 2 in the absence of −35 region sequences is dependent on the sequence TG just upstream of the − 10 hexamer, TATACT: a mutation at −14 changing the TG sequence to TT totally inactivates P 2. However, P 2 activity can be restored if the consensus −35 region sequence TTGACA is cloned 17 bp upstream of the −10 hexamer. Thus, for transcription initiation, the −10 hexamer, TATACT, must ‘cooperate’ with upstream sequences that may be located either around −35 or −14.