Dual control of the lac operon.
Glucose and lactose levels control the
initiation of transcription of the lac
operon through their effects on the lac
repressor protein and CAP. Lactose
addition increases the concentration of
allolactose, which binds to the repressor
protein and removes it from the DNA.
Glucose addition decreases the
concentration of cyclic AMP; because
cyclic AMP no longer binds to CAP, this
gene activator protein dissociates from
the DNA, turning off the operon. As
shown in, CAP is known to induce a
bend in the DNA when it binds; for
simplicity, the bend is not shown here.
LacZ, the first gene of the lac operon,
encodes the enzyme β-galactosidase,
which breaks down the disaccharide
lactose to galactose and glucose. The
essential features of the lac operon are
summarized in the figure, but in reality
the situation is more complex. For one
thing, there are several lac repressor
binding sites located at different
positions along the DNA. Although the
one illustrated exerts the greatest effect,
the others are required for full
repression. In addition, expression of the
lac operon is never completely shut
down. A small amount of the enzyme βgalactosidase is required to convert
lactose to allolactose thereby permitting
the lac repressor to be inactivated when
lactose is added to the growth medium.
Induction of the LAC Operon. (A) In the absence of lactose, the lac repressor
binds DNA and represses transcription from the lac operon. (B) Allolactose or
another inducer binds to the lac repressor, leading to its dissociation from DNA
and to the production of lac mRNA.
Positive control of the lac operon by glucose Low levels of glucose activate
adenylyl cyclase, which converts ATP to cyclic AMP (cAMP). Cyclic AMP then
binds to the catabolite activator protein (CAP) and stimulates its binding to
regulatory sequences of various operons concerned with the metabolism of
alternative sugars, such as lactose. CAP interacts with the α subunit of RNA
polymerase to activate transcription
Negative control of the lac operon The i gene encodes a repressor which,
in the absence of lactose (top), binds to the operator (o) and blocks
transcription of the three structural genes (z, β-galactosidase; y, permease;
and a, transacetylase). Lactose induces expression of the operon by binding
to the repressor (bottom), which prevents the repressor from binding to the
operator. P = promoter; Pol = polymerase
Catabolite control of the lac operon. The operon is inducible by lactose to the maximal levels when
cAMP and CAP form a complex. (a) Under conditions of high glucose, a glucose breakdown
product inhibits the enzyme adenylate cyclase, preventing the conversion of ATP into cAMP. (b)
Under conditions of low glucose, there is no breakdown product, and therefore adenylate cyclase is
active and cAMP is formed. (c) When cAMP is present, it acts as an allosteric effector, complexing
with CAP. (d) The cAMP–CAP complex acts as an activator of lac operon transcription by binding to
a region within the lac promoter. (CAP = catabolite activator protein; cAMP = cyclic adenosine
monophosphate.)
Negative and positive control of the lac operon by the Lac repressor and catabolite activator protein (CAP), respec-tively.
(a) In the absence of lactose to serve as an inducer, the Lac repressor is able to bind the operator; regardless of the levels
of cAMP and the presence of CAP, mRNA production is repressed. (b) With lactose present to bind the repressor, the
repressor is unable to bind the operator; however, only small amounts of mRNA are produced because the presence of
glucose keeps the levels of cAMP low, and thus the cAMP–CAP complex does not form and bind the promoter. (c) With
the repressor inactivated by lactose and with high levels of cAMP present (owing to the absence of glucose), cAMP binds
CAP. The cAMP–CAP complex is then able to bind the promoter; the lac operon is thus activated, and large amounts of
mRNA are produced. (d) When CAP binds the promoter, it creates a bend greater than 90° in the DNA. Apparently, RNA
polymerase binds more effectively when the promoter is in this bent configuration. (e) CAP bound to its DNA recognition
site. This part is derived from the structural analysis of the CAP–DNA complex
Regulation of the lac operon. The I gene continually makes repressor. The repressor
binds to the O (operator) region, blocking the RNA polymerase bound to P (the
promoter region) from transcribing the adjacent structural genes. When lactose is
present, it binds to the repressor and changes its shape so that the repressor no
longer binds to O. The RNA polymerase is then able to transcribe the Z, Y, and A
structural genes, so the three enzymes are produced.
A simplified lac operon model. The three genes Z, Y, and A are coordinately
expressed. The product of the I gene, the repressor, blocks the expression of the
Z, Y, and A genes by interacting with the operator (O). The inducer can inactivate
the repressor, thereby preventing interaction with the operator. When this
happens, the operon is fully expressed.
The base sequence and the genetic boundaries of the control region of the lac
operon, with partial sequences for the structural genes.
Structure of the inducer of the lac operon, IPTG. The β-d-thiogalactoside
linkage is not cleaved by β-galactosidase, allowing manipulation of the
intracellular concentration of this inducer
Heterodimerization of leucine zipper proteins can alter their DNA-binding
specificity. Leucine zipper homodimers bind to symmetric DNA sequences, as
shown in the left-hand and center drawings. These two proteins recognize
different DNA sequences, as indicated by the red and blue regions in the DNA.
The two different monomers can combine to form a heterodimer, which now
recognizes a hybrid DNA sequence, composed from one red and one blue region
A leucine zipper dimer bound to DNA. Two α-helical DNA-binding domains
(bottom) dimerize through their α-helical leucine zipper region (top) to form an
inverted Y-shaped structure. Each arm of the Y is formed by a single α helix,
one from each monomer, that mediates binding to a specific DNA sequence in
the major groove of DNA. Each α helix binds to one-half of a symmetric DNA
structure. The structure shown is of the yeast Gcn4 protein, which regulates
transcription in response to the availability of amino acids in the environment.
One type of zinc finger protein. This protein belongs to the Cys-Cys-His-His
family of zinc finger proteins, named after the amino acids that grasp the zinc. (A)
Schematic drawing of the amino acid sequence of a zinc finger from a frog protein
of this class. (B) The three-dimensional structure of this type of zinc finger is
constructed from an antiparallel β sheet (amino acids 1 to 10) followed by an α
helix (amino acids 12 to 24). The four amino acids that bind the zinc (Cys 3, Cys 6,
His 19, and His 23) hold one end of the α helix firmly to one end of the β sheet.
Summary of sequence-specific interactions
between different six zinc fingers and their
DNA recognition sequences. Even though all
six Zn fingers have the same overall structure
(see Figure 7-17), each binds to a different DNA
sequence. The numbered amino acids form the
α helix that recognizes DNA (Figures 7-17 and
7-18), and those that make sequence-specific
DNA contacts are colored green. Bases
contacted by protein are orange. Although
arginine-guanine contacts are common (see
Figure 7-27), guanine can also be recognized
by serine, histidine, and lysine, as shown.
Moreover, the same amino acid (serine, in this
example) can recognize more than one base.
Two of the Zn fingers depicted are from the TTK
protein (a Drosophila protein that functions in
development); two are from the mouse protein
(Zif268) that was shown in Figure 7-18; and two
are from a human protein (GL1), whose
aberrant forms can cause certain types of
cancers
DNA binding by a zinc finger protein. (A) The structure of a fragment of a mouse
gene regulatory protein bound to a specific DNA site. This protein recognizes DNA
using three zinc fingers of the Cys-Cys-His-His type (see Figure 7-17) arranged as
direct repeats. (B) The three fingers have similar amino acid sequences and
contact the DNA in similar ways. In both (A) and (B) the zinc atom in each finger is
represented by a small sphere
A dimer of the zinc finger domain of the intracellular receptor family bound to its specific DNA
sequence. Each zinc finger domain contains two atoms of Zn (indicated by the small gray spheres);
one stabilizes the DNA recognition helix (shown in brown in one subunit and red in the other), and one
stabilizes a loop (shown in purple) involved in dimer formation. Each Zn atom is coordinated by four
appropriately spaced cysteine residues. Like the helix-turn-helix proteins shown in Figure 7-14, the
two recognition helices of the dimer are held apart by a distance corresponding to one turn of the DNA
double helix. The specific example shown is a fragment of the glucocorticoid receptor. This is the
protein through which cells detect and respond transcriptionally to the glucocorticoid hormones
produced in the adrenal gland in response to stress
Families of DNA-binding domains (A) Zinc finger domains consist of loops in which an α helix and a β sheet
coordinately bind a zinc ion. (B) Helix-turn-helix domains consist of three (or in some cases four) helical regions. One
helix (helix 3) makes most of the contacts with DNA, while helices 1 and 2 lie on top and stabilize the interaction. (C)
The DNA-binding domains of leucine zipper proteins are formed from two distinct polypeptide chains. Interactions
between the hydrophobic side chains of leucine residues exposed on one side of a helical region (the leucine zipper)
are responsible for dimerization. Immediately following the leucine zipper is a DNA-binding helix, which is rich in basic
amino acids. (D) Helix-loop-helix domains are similar to leucine zippers, except that the dimerization domains of these
proteins each consist of two helical regions separated by a loop.