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43
CONTROL OF GENE EXPRESSION
C. HELIX-LOOP-HELIX PROTEIN (HLH; Figure
7-4). The HLH proteins consist of a short al-
pha helix connected by a loop to a longer al-
pha helix. The loop allows for dimerization of
two HLH proteins to occur and form a Y-
shaped dimer. Dimerization may occur be-
tween two of the same proteins (homodimers)
or two different proteins (heterodimers). The
diagram shows the three-dimensional struc-
ture of an HLH protein forming an HLH ho-
modimer. Specific examples of HLH proteins
are
1.
MyoD protein
which regulates various
genes involved in muscle development.
2.
MYC protein
which regulates various
genes involved in the cell cycle. The MYC
protein is encoded by the MYC gene (a
proto-oncogene; v-myc myelocytomatosis
viral oncogene homolog) on chromosome
8q24.
D. ZINC FINGER PROTEINS (Figure 7-5). The
zinc finger proteins consist of one alpha helix
with a zinc (Zn) atom bound to four cysteine
amino acids. The zinc finger proteins contain
both a hormone-binding region and a 70
amino acid long region near the zinc atom that
binds specifically to DNA segments. The dia-
gram shows the three-dimensional structure of
a specific zinc finger protein (i.e., the gluco-
corticoid receptor) which behaves as a gene
regulatory protein. The glucocorticoid recep-
tor has a DNA-binding region and a steroid
hormone-binding region. In the presence of
● Figure 7-4 Helix-Loop-Helix Protein.
DNA
binding
region
DNA
binding
region
HLH
protein
HLH
protein
COOH
COOH
NH
2
NH
2
HLH homodimer
COOH
COOH
NH
2
NH
2
● Figure 7-5 Zinc Finger Protein.
glucocorticoid hormone, the glucocorticoid receptor will bind to a gene regulatory se-
quence known as the GRE which loops to interact with the TI complex and allows the
start of gene transcription. Specific examples of zinc finger proteins are
1.
Glucocorticoid receptor
2.
Estrogen receptor
3.
Progesterone receptor
4.
Thyroid hormone receptor
5.
Retinoic acid receptor
6.
Vitamin D3 receptor
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44
CHAPTER 7
Other Mechanisms of Gene Expression
A. MICRO RNA (miRNA; Figure 7-6)
1.
The miRNA genes are first transcribed
into a
70-bp RNA precursor which con-
tains an inverted repeat. This permits
double-stranded hairpin RNA formation.
2.
This
70-bp RNA precursor is cleaved by
a dsRNA-specific endonuclease called
Dicer which produces
25-bp RNA prod-
uct called small interfering RNA (siRNA)
or microRNA (miRNA).
● Figure 7-6 Micro RNA.
3.
The double-stranded miRNA unwinds to form a single-stranded miRNA which
then hunts for a matching sequence on some mRNA encoding for some protein.
4.
When the miRNA binds to the mRNA, an RNA-induced silencing complex is
formed which either cleaves the mRNA or physically blocks translation. In either
case, the expression of the gene that encoded the mRNA is blocked. Therefore,
miRNAs seem to be very potent blockers of gene expression.
B. ANTISENSE RNA (Figure 7-7)
1.
The antisense RNA genes encode for antisense RNA that binds to mRNA and phys-
ically blocks translation.
2.
During protein synthesis, the DNA template
strand is transcribed into mRNA (or “sense”
RNA) from which a protein is translated.
3.
The DNA nontemplate strand is normally
not transcribed. However, there are
1600
genes in which the DNA nontemplate
strand is also transcribed, thereby produc-
ing “antisense” RNA.
● Figure 7-7 Antisense RNA.
mRNA
Ribosome
Nucleus
Antisense gene
Antisense RNA
Antisense RNA
DNA
4.
The antisense RNA then hunts for a matching sequence on the mRNA (or sense
RNA) encoding for some protein.
5.
When the antisense RNA binds to the sense RNA, the expression of the gene that
encoded the sense RNA (or mRNA) is blocked. Therefore, antisense RNAs seem
to be very potent blockers of gene expression.
C. RIBOSWITCH RNA (Figure 7-8)
1.
The riboswitch genes encode for ri-
boswitch RNA which binds to a target
molecule, changes shape, and then switches
on protein synthesis.
2.
Riboswitch RNA folds into a complex
three-dimensional shape where one por-
tion recognizes a target molecule and the
other portion contains a protein-coding
RNA sequence.
● Figure 7-8 Riboswitch RNA.
3.
When the riboswitch RNA binds to the target molecule, the “switch” is turned on
and the protein-coding RNA sequence is translated into a protein product.
4.
Note that a protein product is only formed if the riboswitch RNA binds to the tar-
get molecule.
D. ALTERNATIVE PROMOTERS AND ALTERNATIVE INTERNAL PROMOTERS
1.
Alternative promoters.
Alternative promoters start transcription from alter-
native versions of the first exon, which is then spliced into a common set of
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45
CONTROL OF GENE EXPRESSION
downstream exons which produces an isoform of the same molecular weight.
There are several human genes that have two or more alternative promoters which
can result in the expression of a protein isomer.
2.
Alternative internal promoters.
Alternative internal promoters start transcrip-
tion from different exons located within the gene which produces a truncated pro-
tein with a different molecular weight.
E.
RNA-BINDING PROTEINS. There are a number of RNA-binding proteins that bind
specifically to the 3
UTR (untranslated region) of mRNA and seem to be potent block-
ers of gene expression.
F.
ALTERNATIVE RNA SPLICING
1.
RNA splicing
is a process whereby all introns (noncoding regions; intervening
sequences) are removed from the RNA transcript and all exons (coding regions;
expression sequences) are joined together within the RNA transcript.
2.
RNA splicing is carried out by a large RNA–protein complex called the spliceosome
which consists of five types of small nuclear RNA and
50 different proteins.
3.
Alternative RNA splicing is a process whereby different exon combinations are rep-
resented in the RNA transcript producing protein isoforms.
G. X CHROMOSOME INACTIVATION
1.
X chromosome inactivation is a process whereby either the maternal X chromo-
some (X
M
) or paternal X chromosome (X
P
) is inactivated resulting in a hete-
rochromatin structures called the Barr body which is located along the inside of
the nuclear envelope in female cells. This inactivation process overcomes the sex
difference in X gene dosage.
2.
Males have one X chromosome and are therefore constitutively hemizygous, but
females have two X chromosomes. Gene dosage is important because many X-
linked proteins interact with autosomal proteins in a variety of metabolic and
developmental pathways, so there needs to be a tight regulation in the amount of
protein for key dosage-sensitive genes. X chromosome inactivation makes females
functionally hemizygous.
3.
X chromosome inactivation begins early in embryological development at about
the late blastula stage.
4.
Whether the X
M
or the X
P
becomes inactivated is a random and irreversible event.
However, once a progenitor cell inactivates the X
M
, for example, all the daughter
cells within that cell lineage will also inactivate the X
M
(the same is true for the
X
P
). This is called clonal selection and means that all females are mosaics com-
prising mixtures of cells in which either the X
M
or X
P
is inactivated.
5.
X chromosome inactivation does not inactivate all the genes;
20% of the total genes
on the X chromosome escape inactivation. These
20% inactivated genes include
those genes that have a functional homolog on the Y chromosome (gene dosage is
not affected in this case) or those genes where gene dosage is not important.
6.
The mechanism of X chromosome inactivation involves
a.
Xic (X-inactivation center) is a cis-acting DNA sequence located on the X chro-
mosome (Xq13) which controls the initiation and propagation of inactivation.
b.
Xce (X-controlling element) is a cis-acting DNA sequence located on the X
chromosome which affects the choice of whether X
M
or X
P
is inactivated.
c.
XIST gene (X-inactive specific transcript) encodes for a 17-kb RNA that is
the primary signal for spreading the inactivation along the X chromosome.
H. MATERNAL MRNA
1.
In the adult, genes are regulated at the level of transcription-initiation.
2.
In the early embryo, genes are regulated at the level of translation.
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46
CHAPTER 7
3.
Immediately after fertilization of the secondary oocyte by the sperm, protein syn-
thesis is specified by maternal mRNAs present within the oocyte cytoplasm.
4.
These maternal mRNAs are stored in the oocyte cytoplasm in an inactive form due
to shortened poly A-tails.
5.
At fertilization, the stored inactive maternal mRNAs are activated by polyadenyla-
tion which restores the poly-A tail to its normal length.
6.
This situation remains until the four or eight cell stage when transcription from
the genome of zygote (called zygotic transcription) begins.
An operon is a set of genes adjacent to one another in
the genome that are transcribed from a single promoter as one long mRNA. The lac operon
involved in lactose metabolism is classic in the annals of molecular biology because the
details of gene regulation were first discovered using the lac operon in Escherichia coli bac-
teria. Upstream of the lac operon lies the lac operator, lac promoter, lac I gene, lac I pro-
moter, and the CAP-binding site. The diagram shows the four culture conditions involved
in the lac operon.
● Figure 7-9 Lac Operon.
IacI
promoter
Glucose +
Lactose +
Iac operon OFF
IacI
CAP
binding site
Iac
promoter
Iac
operator
IacZ
IacY
IacA
IacI
promoter
Glucose +
Lactose –
Iac operon OFF
IacI
CAP
binding site
Iac
promoter
Iac
operator
IacZ
IacY
IacA
IacI
promoter
Glucose –
Lactose –
Iac operon OFF
IacI
CAP
binding site
Iac
promoter
Iac
operator
IacZ
IacY
IacA
IacI
promoter
Glucose –
Lactose +
Iac operon ON
IacI
CAP
binding site
Iac
promoter
Iac
operator
IacZ
IacY
IacA
IacI
promoter
IacI
IacI mRNA
Iac repressor
Iac mRNA
β
-galactosidase
lactose permease
β
-galactoside transacetylase
CAP
binding site
Iac
promoter
Iac
operator
IacZ
IacY
IacA
CAP
CAP
A. The lac operon consists of three genes positioned in sequence:
1.
lac Z gene:
encodes
-galactosidase which splits (hydrolyzes) lactose into glu-
cose and galactose.
2.
lac Y gene:
encodes lactose permease which pumps lactose into the cell.
3.
lac A gene:
encodes
-galactoside transacetylase which also splits (hydrolyzes)
lactose into glucose and galactose.
B. The lac I gene lies upstream of the lac operon and is expressed separately using its own
lac I promoter. The lac I gene encodes for a protein called the lac repressor which
blocks the transcription of lac Z, lac Y, and lac A genes of the lac operon.
C. CAP (catabolite activator protein; inducer) is a gene regulatory protein that binds
to a cis-acting DNA sequence (called the CAP binding site) upstream of the lac
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47
CONTROL OF GENE EXPRESSION
promoter when cAMP levels are high (c cAMP) and increases the transcription of lac
Z, lac Y, and lac A genes of the lac operon.
D. Consequently, the lac operon is under the control of the lac repressor and CAP
(inducer). This is highlighted by the response of E. coli to four culture conditions as
indicated below:
1.
Glucose
and lactose
culture medium S lac operon OFF.
When E. coli is cul-
tured in glucose
and lactose
culture medium, there is glucose available for me-
tabolism. Therefore, the lac operon is switched off because the lac repressor is not
bound to the lac operator and CAP is not bound to the CAP binding site due to
T
cAMP levels.
2.
Glucose
and lactose
culture medium S lac operon OFF.
When E. coli is cul-
tured in glucose
and lactose
culture medium, there is glucose available for me-
tabolism. Therefore, the lac operon is switched off because the lac repressor is
bound to the lac operator and CAP is not bound to the CAP binding site due to
T
cAMP levels.
3.
Glucose
and lactose
culture medium S lac operon OFF.
When E. coli is cul-
tured in glucose
and lactose
culture medium, there is no glucose available for
metabolism. Therefore, the lac operon is switched off because the lac repressor is
bound to the lac operator and CAP is bound to the CAP binding site due to
c
cAMP levels.
4.
Glucose
and lactose
culture medium S lac operon ON.
When E. coli is cul-
tured in glucose
and lactose
culture medium, there is no glucose available for
metabolism. Therefore, the lac operon is switched on because the lac repressor is
not bound to the lac operator and CAP is bound to the CAP binding site due to
c
cAMP levels.
An operon is a set of genes adjacent to one another in
the genome that are transcribed from a single promoter as one long mRNA. The trp operon
involved in tryptophan biosynthesis is classic in the annals of molecular biology because
the details of gene regulation were first discovered using the trp operon in E. coli bacteria.
Upstream of the trp operon lies the trp operator, trp promoter, trp repressor gene, and trp
repressor promoter. The diagram shows the two culture conditions involved in the trp
operon.
● Figure 7-10 trp Operon.
trp operon OFF
Tryptophan +
trp repressor
promoter
trp
repressor
trp repressor mRNA
trp repressor
tryp
tryp
trp mRNA
5 Proteins
trp
promoter
trp
operator
E
D
C
B
A
trp repressor
promoter
trp
repressor
trp
promoter
trp
operator
E
D
C
B
A
trp operon ON
Tryptophan –
trp repressor
promoter
trp
repressor
trp
promoter
trp
operator
E
D
C
B
A
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