The Arabinose Operon

Arabinose is a five-carbon sugar that can serve as an energy and carbon source for E.
coli. Arabinose must first be converted into ribulose-5-phosphate before it can be
metabolized. The arabinose operon has three genes,araB, araA and araD that encode
for three enzymes to carry out this conversion. A fourth gene, araC, which has its own
promoter, encodes a regulatory factor called the C protein.

The regulatory sites of the ara operon include four sites that bind the C protein and one
CAP binding site. The araO1 and araO2 sites are upstream of the promoter and CAP
binding sites. The other two C protein binding sites called araI1 and araI2 are located
between the CAP binding site and the promoter.

Negative Control of the araC Operon

In the absence of arabinose, dimers of the C protein bind to araO2, araO1 and araI1. The
C proteins bound to araO2 and araI1 associate with one another causing the DNA
between them to form a loop effectively blocking transcription of the operon.

Positive Control of the araC Operon

The C protein binds arabinose and undergoes a conformational change that enables it to
also bind the araO2 and araI2 sites. This results in the generation of a different DNA
loop that is formed by the interaction of C proteins bound to the araO1 and araO2 sites.
The formation of this loop stimulates transcription of the araC gene resulting in
additional C protein synthesis, thus the C protein autoregulates its own synthesis. In the
absence of glucose, cAMP-CAP is formed which binds to the CAP site. C protein
bound at the araI1 and araI2 sites interacts with the bound CAP enabling RNA
polymerase to initiate transcription from the ara operon promoter.
The araBAD Operon: Positive and Negative Control by AraC
 Figure
31.22 ·

Regulation of the araBAD operon by the combined action of CAP and AraC protein.

E. coli can use the plant pentose L-arabinose as sole source of carbon and
energy. Arabinose is metabolized via conversion to D-xylulose-5-P (a pentose
phosphate pathway intermediate and transketolase substrate [Chapter 23]) by
three enzymes encoded in the araBAD operon. Transcription of this operon is
regulated by both catabolite repression and arabinose-mediated induction.
CAP functions in catabolite repression; arabinose induction is achieved via the
product of the araC gene, which lies next to the araBAD operon on the E. coli
chromosome. The araC gene product, the protein AraC,5 is a 292-residue
protein consisting of an N-terminal domain (residues 1-170) that binds
arabinose and acts as a dimerization motif and a C-terminal (residues 178-
292) DNA-binding domain. Regulation of araBAD by AraC is novel in that it
acts both negatively and positively. The ara operon has three binding sites for
AraC: araO1, located at nucleotides -106 to -144 relative to the araBAD
transcription start site; araO2 (spanning positions -265 to -294); and araI, the
araBAD promoter. The araI site consists of two “half-sites”; araI1
(nucleotides -56 to -78) and araI2 (-35 to -51). (The araO1 site contributes
minimally to ara operon regulation.)
        The details of araBAD regulation are as follows: When AraC protein
levels are low, the araC gene is transcribed from its promoter pc (adjacent to
araO1) by RNA polymerase (Figure 31.22). araC is transcribed in the
direction away from araBAD. When cAMP levels are low and arabinose is
absent, an AraC protein dimer binds to two sites, araO2 and the araI1 half-site,
forming a DNA loop between them and restricting transcription of araBAD
(Figure 31.22). In the presence of L-arabinose, the monomer of AraC bound
to the araO2 site is released from that site; it then associates with the
unoccupied araI half-site, araI2. L-Arabinose thus behaves as an allosteric
effector that alters the conformation of AraC. In the arabinose-liganded
conformation, the AraC dimer interacts with CAP-(cAMP)2 to activate
transcription by RNA polymerase. Thus, AraC protein is both a repressor and
an activator.
 Figure 31.23 ·
Introduction or
removal of half a
helical turn in the
DNA between araO2
and araI prevents
AraC protein from
interacting with both
sites and achieving
araBAD repression.
(Adapted from
Schleif, R., 1987. The
L-arabinose operon.
In Escherichia coli
and Salmonella typhimurium, vol. 2. Edited by Neidhardt, F. C., et al. Washington, DC: American
Society for Microbiology.)

       Deletion studies reveal that both araO2 and araI must be present on the
chromosome in order for AraC protein to repress araBAD. The DNA loop
created when AraC binds both araO2 and araI1 consists of some 210 bp. If 5
bp of DNA (one-half a helical turn) are added or deleted in this intervening
region, the two AraC-binding sites are rotated away from each other, so that
interaction of AraC with both sites is not possible, and repression is no longer
observed (Figure 31.23). The creation of DNA loops by sequence-specific,
DNA-binding proteins is a mechanism common to many regulatory
phenomena involving DNA. (DNA looping is considered in greater detail later
in this chapter.)
        Positive control of the araBAD operon occurs in the presence of L-
arabinose and cAMP. Arabinose binding by AraC protein causes the release of
araO2, opening of the DNA loop, and association of AraC with araI2. CAP-
(cAMP)2 binds at a site between araO1 and araI, and together the AraC-
(arabinose)2 and CAP-(cAMP)2 complexes influence RNA polymerase,
through protein:protein interactions, to create an active transcription initiation
complex. Supercoiling-induced DNA looping may promote protein:protein
interactions between DNA-binding proteins by bringing them into
juxtaposition.