Fig. 6. Mutational analysis of R2. (A) Band shift analysis of the
binding of DimB to point mutated forms of R2. The labelled probe is R2 and the
unlabelled competitors are R2 and the three R2 mutants illustrated at the base
of the figure. The underlined residues in R2 show the position of the scanning
mutation that reduced binding; the lower case letters in the mutant sequences
show the changes from the wild-type sequence. (B) In vivo analysis of
the effect of multiple point mutations in R2. This was determined by creating
the indicated fusion constructs and analysing their expression patterns in
Dictyostelium. All published ecmA promoter 5'-3'
deletion constructs (Fig. 1)
share the same proximal end point at nucleotide +251 (labelled relative to the
cap site of ecmA), four nucleotides upstream from the translation
initiation codon (Early et al.,
1993). All these constructs are fused to the vector
A15
bam-gal, which provides the basal transcription signals
(Pears and Williams, 1988). By
contrast, constructs S, R2S and R2pM1S:lacZ fuse immediately downstream of the
ATG initiation codon of ecmA to the lacZ-coding region,
hence using the basal promoter elements of ecmA
(Fig. 1). The distal end point
of S lies at nucleotide -493, just one nucleotide downstream of the cap-site
proximal end point of R2. The R2 oligonucleotide has a cap-site distal end
point just two nucleotides shorter than construct O
(Fig. 1). Hence, construct R2S
has a structure that, at its distal end, is very similar to that of construct
O. Construct R2pM1S has an identical structure to R2S, except that it contains
the four point mutations present in R2pM1.