The strong efficiency of the Escherichia coli gapA P1 promoter depends on a complex combination of functional determinants

摘要

pThe iEscherichia coli/i multi-promoter region of the igapA/i gene ensures a high level of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) production under various growth conditions. In the exponential phase of growth, igapA/i mRNAs are mainly initiated at the highly efficient igapA/i P1 promoter. In the present study, by using site-directed mutagenesis and chemical probing of the RPo (open complex) formed by Eσsup70/sup (holoenzyme associated with σsup70/sup) RNAP (RNA polymerase) at promoter igapA/i P1, we show that this promoter is an extended ?10 promoter that needs a ?35 sequence for activity. The ?35 sequence compensates for the presence of a suboptimal ?10 hexamer. A tract of thymine residues in the spacer region, which is responsible for a DNA distortion, is also required for efficient activity. We present the first chemical probing of an RPo formed at a promoter needing both a ?10 extension and a ?35 sequence. It reveals a complex array of RNAP–DNA interactions. In agreement with the fact that residue A-11 in the non-template strand is flipped out in a protein pocket in previously studied RPos, the corresponding A residue in igapA/i P1 promoter is protected in RPo and is essential for activity. However, in contrast with some of the previous findings on RPos formed at other promoters, the ?12 A:T pair is opened. Strong contacts with RNAP occur both with the ?35 sequence and the TG extension, so that the σ4 and σ2 domains may simultaneously contact the promoter DNA. RNAP–DNA interactions were also detected immediately downstream of the ?35 hexamer and in a more distal upstream segment, reflecting a wrapping of RNAP by the core and upstream promoter DNA. Altogether, the data reveal that promoter igapA/i P1 is a very efficient promoter sharing common properties with both extended ?10 and non-extended ?10 promoters./p