E. coli Catabolite Activator Protein
Marla Fiorelli '99 and Daniel Barich '05
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 length.
The overall structure of the dimer is assymetric; one subunit adopts a "closed"
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
The N-terminal domain is
responsible for dimerization and cAMP binding.
The carboxy-terminal domain
contains a helix-turn helix DNA
and is also responsible for DNA bending.
III. cAMP Binding
An important recognition site for cAMP within
CAP is the ionic bond formed between the side chain of Arg-82
and the negatively charged phosphate group of
cAMP. In the crystal structure, the two cAMP molecules are buried deep within
the beta roll and the C-helix.
It is unclear how cAMP enters or leaves the binding site, but this probably
requires the separation of the two subunits of the dimer, or the movement
of the beta roll and the C helix away from each other. Other side-chain interactions
between the protein and cAMP are hydrogen bonds occuring at Thr-127,
Ser-128, Ser-83, and Glu-72.
Additional hydrogen bonding between is seen between cAMP and the polypeptide
backbone at residues 83
IV. DNA Binding
Once CAP has bound cAMP, it is ready to bind to
. Binding occurs at the conserved
sequence of 5'-AAATGTAGATCACATTT-3'
Hydrogen bonds between the protein and the DNA phsophates occur at the
backbone amide of residue 139,
and the side chains of Thr-140, Ser-179,
In addition to these phosphate interactions, the side chains of Glu-181
both emanating from the recognition helix
directly contact the bases within the major groove of the DNA. Because
of the way that the protein binds to the DNA, a kink of ~40 degrees occurs
between nucleotide base pairs six
and seven on
each side of the dyad axis, 5'-TG-3'
This sequence has been shown to favor DNA flexibility and bending in other
systems as well. Because of this kink, an additional five ionic interactions
and four hydrogen bonds are able to occur between the protein and the DNA
strand. Examples of these new interactions occur between Lys-26,
Lys-166, His-199 and the DNA sugar-phosphate
The DNA bend is integral to the mechanism of transcription activation.
Not only does it place CAP in the proper orientation for interaction with
RNA polymerase, but wrapping the DNA around the protein may result in direct
contacts between upstream DNA and RNA polymerase.
V. 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
have been proposed to be critical for transcription activation by CAP.
These amino acids are part of a surface loop composed of residues 152-166
Researchers have concluded that the third and final step in transcription
activation is this direct protein-protein contact between amino acids 156-162
of CAP, and RNA polymerase.
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 90:6081-6085.
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