The activated Escherichia coli cAMP receptor protein, CRP, is capable of regulating the expression of more than 20 genes by binding to specific DNA sites. DNA bending is an important structural feature that has been observed in the regulatory mechanism of gene expression by CRP. On the basis of the results of the fluorescence energy transfer study of the gal P1 promoter, gal bends asymmetrically upon binding to CRP, although DNA bends symmetrically in the CRP-lac complex. The flanking sequence proximal to the TGTGA motif is involved in a sharper bend than the other side with an overall bending angle of ∼90-125°, without wrapping around the CRP molecule. To understand the factors that control the symmetry in DNA bending, a series of DNA sequences was tested to dissect the contribution of half-sites and flanking sequences, using the natural gal PI and lac P1 sequences as initial targets. The extent of DNA bending induced by CRP was monitored by the difference in fluorescence anisotropy between free DNA and the DNA-CRP complex. The extent of bending was sequence-dependent, and most importantly, the symmetry of bending was a function of the symmetry of the DNA sequence. For example, in the lac promoter the two binding half-sites (TGTGA and TCACT) were almost symmetric as an inverted repeat. The recognition F-helices of the two CRP subunits would bind to these half-sites with a 2-fold symmetry. The flanking sequences (ATAAA and CATTA) were almost identical mirror images. Thus, they are expected to bend in a similar manner. Finally, the sequence symmetry properties of a series of natural CRP promoters were analyzed. A strong tendency for symmetry sequence was encoded in class I promoter sites but not in class II promoter sites. Results from this analysis support the conclusion that the geometry of the CRP-DNA complex plays a major role in determining the molecular mechanism in gene transcription.
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