CRP-binding sites: evidence for two structural classes with 6-bp and 8-bp spacers.
While classifying protein binding DNA sequences of the type GTGNxCAC, based on the size of Nx [Shumilov, Mol. Biologya (Engl. Transl.) 21 (1987) 168-187], we had previously found that the cyclic AMP receptor protein (CRP)-binding sites found in the Escherichia coli genome are of at least two classes: (i) those with a conventional 6-bp spacer (N6) and (ii) those with a potential 8-bp spacer (N8) [Barber and Zhurkin, J. Biomol. Struct. Dyn. 8 (1990) 213-232]. In this paper, we present the first experimental evidence that CRP binds to DNA with an N8 spacer with relatively high affinity, as measured by gel electrophoresis of CRP-DNA complexes. We have tested two types of N8 spacers: A+T-rich and G+C-rich. Compared with the affinity of CRP for a reference site with an N6 spacer, the binding strength of CRP toward an A+T-rich N8 sequence is lower and that toward a G+C-rich N8 site is comparable. Just like DNA sites with N6 spacers, those with N8 spacers utilize both halves of the symmetrical protein recognition sequences, TGTGA and TCACA. Because of the increased number of nucleotides in the N8 spacer, the two recognition sequences in DNA will have an increased distance and a helical twist between them. These would cause displacement of the two recognition sequences with respect to the two symmetrically located alpha-helices of the CRP dimer, if there is no change in the DNA conformation.(ABSTRACT TRUNCATED AT 250 WORDS)
Laboratory of Mathematical Biology, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892.
A M
AM
Barber
V B
VB
Zhurkin
S
S
Adhya
D001483:Base Sequence; D001665:Binding Sites; D016384:Consensus Sequence; D002373:Cyclic AMP Receptor Protein / Q000235:genetics*; D004269:DNA, Bacterial / Q000737:chemistry*; D004274:DNA, Recombinant / Q000737:chemistry; D004268:DNA-Binding Proteins / Q000235:genetics; D003852:Deoxyribonucleoproteins / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D016680:Genome, Bacterial; D007763:Lac Operon / Q000235:genetics; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D017422:Sequence Analysis, DNA
D002373:Cyclic AMP Receptor Protein; D004269:DNA, Bacterial; D004274:DNA, Recombinant; D004268:DNA-Binding Proteins; D003852:Deoxyribonucleoproteins