DNA regions essential for the function of a bacteriophage fd promoter.

The promoter for the major coat protein gene of bacteriophage fd contains a unique sequence. TATAAT, in the non-transcribed region corresponding to the Pribnow box. A R-Hha I cleavage site which destroys functions is located five pairs upstream from the TATAAT sequence (fifteen base pairs upstream from the RNA initiation site). The promoter was cleaved into two fragments by R-Hha I and each promoter fragment was joined to DNA fragments derived from other regions. Ligation of the TATAAT-containing fragment to any of the DNA fragments examined resulted in recovery of promoter function. The results suggest for this type of promoter that no unique sequence is necessary upstream from the R-Hha I cleavage site although a contiguous DNA chain must be present in this area.     

Use of lacZ fusions to delimit regulatory elements of the inducible divergent GAL1-GAL10 promoter in Saccharomyces cerevisiae.

We present the DNA sequence of a 914-base pair fragment from Saccharomyces cerevisiae that contains the GAL1-GAL10 divergent promoter, 140 base pairs of GAL10 coding sequence, and 87 base pairs of GAL1 coding sequence. From this fragment, we constructed four pairs of GAL1-lacZ and GAL10-lacZ fusions on various types of yeast plasmid vectors. On each type of vector, the fused genes were induced by galactose and repressed by glucose. The response of a GAL1-lacZ fusion to gal4 and gal80 regulatory mutations was similar to the response of intact chromosomal GAL1 and GAL10 genes. A set of deletions that removed various portions of the GAL10 regulatory sequences from a GAL10-CYC1-lacZ fusion was constructed in vitro. These deletions defined a relatively guanine-cytosine-rich region of 45 base pairs that contained sequences necessary for full-strength galactose induction and an adjacent guanine-cytosine rich 55 base pairs that contained sequences sufficient for weak induction.     

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.