Segment-specific mutagenesis of the regulatory region in the Escherichia coli galactose operon: isolation of mutations reducing the initiation of transcription and translation.

Using hydroxylamine mutagenesis in vitro, mutations were introduced into a short DNA fragment containing the two overlapping promoters of the Escherichia coli galactose operon and the start of the first gal gene, galE. The mutagenised fragment was inserted into a lac expression plasmid. In such a vector, lac expression is controlled by the gal promoter region. Amongst eighteen candidates in which expression was reduced due to mutations in the gal fragment, twelve contained promoter mutations and six carried mutations that reduce the initiation of galE translation. The candidates in which promoter activity was reduced contained mutations affecting the promoter P1, which is dependent on the cyclic AMP-receptor protein complex (cAMP-CRP) for activation. All carried mutations in the sequence 5'GTGA3' at the CRP binding site. One of the twelve also contained a second mutation affecting the second promoter, P2, which normally functions in the absence of cAMP-CRP. Amongst the six candidates affecting galE translation, two contained a mutation that changes the initiator codon from AUG to AUA and almost completely suppresses galE expression. The mutations in the other four candidates affect the ribosome binding sequence, 5'GGAG3'. However, multiple mutations that abolish this sequence do not totally suppress galE expression, showing that there must be another way to guide ribosomes to the correct initiation site.