E. coli Catabolite Activator Protein
Marla Fiorelli, '99
The Escherichia coli catabolite gene activator
protein (CAP) is a DNA binding protein involved with the transcription
of several genes, including those that code for enzymes involved in the
metabolism of certain sugars (i.e. lactose, maltose, and arabinose.)
Basically, CAP is responsible for the global regulation of carbon utilization.
Upon binding cAMP (adenosine 3', 5' monophosphate, or cyclic AMP), CAP
binds to a conserved DNA sequence from which it can either activate or
repress transcription initiation from various promoters. In some cases
clusters of several promoters are all controlled by a single cAMP-CAP complex
bound to the DNA.
Once CAP has bound cAMP, the protein exhibits
a higher affinity for a specific conserved DNA sequence. When the intracellular
level of cAMP increases, the second messenger is bound by CAP and the cAMP-CAP
complex binds to the DNA. Once bound, it is able to stimulate the transcription
of the aforementioned genes. DNA bound by the CAP-cAMP complex is bent
by ~90 degrees. This DNA bend, coupled with a protein-protein interaction
between CAP and RNA polymerase is thought to be the mechanism by which
CAP regluates transcription initiation on the chromosome.
II. General Structure
CAP is a dimer of 22, 500 molecular weight, composed
of two chemically identical polypeptide chains each 209 amino acids in
conformation in which the amino- and carboxy-termini
are closer together than in the more "open"
subunit. Each subunit is composed of two distinct
domains connected by a hinge region
<N-terminal domain is
responsible for dimerization and cAMP binding.
The carboxy-terminal domain
contains a helix-turn helix DNA
binding motif, <
III. cAMP Binding
An important recognition site for cAMP within
CAP is the ionic bond formed between the side chain of Arg-82
of cAMP. In the crystal structure, the two cAMP molecules are buried deep
within the beta roll
and the C-helix
Ser-128, Ser-83, and Glu-72
and 71 <
IV. DNA Binding
Once CAP has bound cAMP, it is ready to bind to
the DNA. Binding occurs at the conserved
sequence of 5'-AAATGTAGATCACATTT-3' <residue 139,
and the side chains of Thr-140, Ser-179,
and Thr-182 <Glu-181
both emanating from the recognition helix
and seven on
each side of the dyad axis, 5'-TG-3'
Lys-166, His-199 and the DNA sugar-phosphate
IV. Activating Regions
Transcription activation by CAP requires more
than merely the binding of cAMP and binding and bending of DNA. CAP contains
an "activating region" that has been proposed to participate in direct
protein-protein interactions with RNA polymerase and/or other basal transcription
factors. Specifically, amino acids 156,
159, and 162
Gunasekera, Angelo, Yon W. Ebright,
and Richard H. Ebright. 1992. DNA Sequence Determinants for Binding of
the Escherichia coli Catabolite Gene Activator Protein. The Journal
of Biological Chemistry 267:14713-14720.
Schultz, Steve C., George C.
Shields, and Thomas A. Steitz. 1991. Crystal Structure of a CAP-DNA complex:
The DNA Is Bent by 90 degrees Science 253: 1001-1007.
Vaney, Marie Christine, Gary
L. Gilliland, James G. Harman, Alan Peterkofsky, and Irene T. Weber. 1989.
Crystal Structure of a cAMP-Independent Form of Catabolite Gene Activator
Protein with Adenosine Substituted in One of Two cAMP-Binding Sites. Biochemistry
Weber, Irene T., Gary L. Gilliland,
James G. Harman, and Alan Peterkofsky. 1987. Crystal Structure of a Cyclic
AMP-independent Mutant of Catabolite Activator Protein. The Journal
of Biological Chemistry 262:5630-5636.
Zhou, Yuhong, Ziaoping Zhang,
and Richard H. Ebright. 1993. Identification of the activating region of
catabolite gene activator protein (CAP): Isolation and characterization
of mutants of CAP specifically defective in transcription activation. Proceedings
of the National Academy of Sciences of the United States of America