Chapter 18
REGULATION OF GENE EXPRESION
Only certain portion of the genetic information is expressed in a cell.
Genes are regulated in several ways:
1. By controlling the amount of mRNA that is available
2. By controlling the rate of translation of the m RNA.
3. By controlling the activity of the protein product.
BACTERIA REGULATES TRANSCRIPTION
Cells can adjust the activities of enzymes already present.
Cells can adjust the production levels of certain enzymes by controlling the amount of mRNA that is
transcribed.
The basic mechanism for this control of gene expression in bacteria is called the operon model.
The French scientists Francois Jacob and Jacques Monod at the Pasteur Institute in Paris discovered the
operon model.
OPERON, THE BASIC CONCEPT
Genes of related functions can be grouped into one transcription unit.
A single on-off switch can control the whole cluster of functionally related genes.
The switch is a segment of DNA called the operator.
The operator is a sequence of bases that overlaps the promoter and serves as the regulatory switch
responsible for transcriptional level control of the operon.
Most regulated genes in bacteria are organized into operons.
Operons permit coordinated control of functionally related genes.
An operon may encode for several proteins.
Each protein encoding sequence of an operon is called a structural gene.
Each operon has a single promoter region upstream from the protein coding regions.
The promoter is the DNA sequence to which the RNA polymerases attach.
The operator is positioned within the promoter or in between the promoter and genes it controls.
All together, the operator, the promoter, and the genes they control constitute an operon.
The entire stretch of DNA required for enzyme production.
Repressor genes encode repressor proteins.
Repressor proteins bind specifically to the operator sequence and block transcription by preventing
RNA polymerase from binding to the promoter.
Some repressor may always be “on” thus repressing the expression of a gene in a cell.
The repressor is the product of a regulatory gene, which is located some distance from the operon it
controls and has its own promoter.
Regulatory genes are expressed continuously.
The binding of repressors to operators is reversible.
A repressible operon is usually turned on.
They are turned off under special conditions, when a repressor-corepressor complex is bound to the DNA
operator.
The repressor protein is synthesized in an inactive form that cannot bind to the operator.
A metabolite, often an end product, acts as a corepressor that binds to the allosteric site of the
repressor protein.
When the supply of the end product (corepressor) is low, all enzymes in the pathway are actively
synthesized and the repressor cannot bind to the operator.
The metabolite (corepressor) and the protein (repressor) must come together to repress the gene.
REPRESSIBLE AND INDUCIBLE OPERONS: TWO TYPES OF NEGATIVE GENE
REGULATION
Repressible operons are usually on but can be inhibited (repressed) when a specific small molecule binds
allosterically to a regulatory protein. .
Inducible operons are usually turned off but can induced or stimulated when a small molecule interacts
with a regulatory protein.
1. Inducible genes:
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Repressor protein alone turns off regulated genes.
Repressor protein plus inducer inactivate the repressor and the gene continuous to transcribe.
The inducer represses the repressor.
2. Repressible genes:
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Repressor protein alone cannot turn off regulated genes.
Repressor protein and corepressor turn off the regulated gene.
Inducible enzymes are synthesized (induced) by chemical signals.
Repressible enzymes function in anabolic pathways, which synthesize end products from raw materials.
The production of the end product is suspended when there is enough material produced.
POSITIVE REGULATORS
Some inducible operons are under positive control.
Positive controls operate through an activator protein, catabolite gene activator protein or CAP.
CAP increases the affinity of the promoter for RNA polymerases. It allows the enzyme to recognize the
promoter efficiently and to bind tightly to the DNA.
The active form of CAP has cAMP bound to an allosteric site. cAMP is a coactivator.
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cAMP: cyclic adenosine monophosphate
derived from ATP
involved in signal transduction, e.g. activation of kinases
Activator protein (CAP) alone cannot stimulate transcription or regulated genes.
Activator protein and coactivator (CAP + cAMP) stimulate transcription of regulated gene.
EUKARYOTIC GENE EXPRESSION
All cells in the body have the same genome.
Human cells express about 20% of its genes.
Highly differentiated cells like nerve and muscle cells express an even smaller fraction of its genes.
The genes expressed in each cell type are unique.
The differences between cell types are due not to different genes being present but to differential gene
expression.
1. REGULATION OF CHROMATIN STRUCTURE
The structural organization of chromatin not only packs a cell’s DNA into a compact form that fits inside
the nucleus but also helps regulate gene expression in several ways.
The location of a gene’s promoter relative to nucleosomes and to the sites where the DNA attaches to the
chromosome scaffold or nuclear lamina can affect whether the gene is transcribed.
Genes with heterochromatin, which is highly condensed are usually not expressed.
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Tightly coiled or condensed chromatin.
Genes in the heterochromatin do not expressed
2. HISTONE MODIFICATIONS
There is evidence that chemical modifications to histones, the proteins around which the DNA is wrapped
in nucleosomes, play a direct role in the regulation of gene transcription.
The N-terminus of each histone molecule in a nucleosome protrudes outward from the nucleosome. These
histone tails are accessible to various modifying enzymes.
In histone acetylation, acetyl groups (-COCH3) are attached to lysine’s in histone tails. When lysine’s are
acetylated, their positive charges are neutralized and the histone tails not longer bind to neighboring
nucleosomes.
Acetylation of histone tails promotes loose chromatin structure that permits transcription.
Methyl group and phosphate groups can be reversibly attached to amino acids in histone tails.
Methyl groups attached to histone tails can promoted condensation of the chromatin, and the attachment
of a phosphate group next to a methylated amino acid can have the opposite effect.
The histone code hypothesis proposes that specific combinations of modifications help determine the
chromatin configuration, which in turn influences transcription.
3. DNA METHYLATION
Certain enzymes can methylate certain bases in the DNA itself. Usually genes that are heavily methylated
are not expressed.
Removal of methyl groups can turn on some genes.
A dual mechanism that involves both methylation of the DNA and deacetylation of histones can repress
certain transcription.
In some species, DNA methylation seems to be essential for the long-term inactivation of genes that
occurs during normal cell differentiation in the embryo. Deficient DNA methylation due to lack of
methylating enzyme leads to abnormal embryonic development in mice and Arabidopsis.
Once methylated, genes usually remain that way through successive cell divisions in a given individual,
e.g. genomic imprinting in mammals.
4. EPIGENIC INHERITANCE
Mutations in the DNA are permanent while changes in the chromatin can be reversed.
Chromatin modification already discussed may be passed along to future generations of cells.
Inheritance of traits transmitted by mechanisms not directly involving the nucleotide sequence is called
epigenic inheritance.
Enzymes that modify chromatin structure are integral parts of the eukaryotic cell’s machinery for
regulating transcription.
Examples of epigenic inheritance: imprinting, X-chromosome silencing, maternal effects, and others.
REGULATION OF TRANSCRIPTION INITIATION
Transcription in both eukaryotic and prokaryotic cells requires an initiation site and a promoter to which
RNA polymerase binds.
1. ORGANIZATION OF A TYPICAL EUKARYOTIC GENE
A cluster of proteins called the transcription initiation complex assembles on the promoter sequence at the
upstream end of the gene.
Then RNA polymerase II proceeds to transcribe the gene synthesizing the pre-mRNA.
Associated with most eukaryotic genes are multiple control elements, segments of noncoding DNA that
help regulate transcription by binding certain proteins.
These control elements and the proteins they bind are critical to the precise regulation of gene expression
seen in different cell types.
2. THE ROLES OF TRANCRIPTION FACTORS.
RNA polymerase needs the assistance of proteins called transcription factors in order to initiate
transcription.
Some transcription factors are essential for the transcription of all protein-coding genes; they are often
called general transcription factors.
Only a few general transcription factors bind to DNA sequences such the TATA box within the promoter.
Others bind to proteins, including each other and RNA polymerase II.
Protein-to-protein interactions are crucial to the initiation of eukaryotic transcription.
The interaction of general transcription factors and RNA polymerase II with a promoter usually leads to
only a low rate of initiation and production of few RNA transcripts.
High levels of transcription of particular genes at the appropriate time and place depend on the interaction
of control elements with another set of proteins, which ca be thought of a specific transcription factors
3. ENHANCERS AND SPECIFIC TRANSCRIPTION FACTORS
Proximal control elements are located closer to the promoter.
More distant control elements are called enhancers.
Enhancers are DNA sequences that increase the rate of transcription.
- An enhancer can regulate a gene thousands of base pairs away.
- They may be located upstream or downstream of the promoters they control, or even within an intron.
A given gene may have multiple enhancers, each active at a different time or in a different cell type or
location in the organism.
Each enhancer is associated with only one gene and no other.
Activator proteins bind to distal control elements grouped as an enhancer in the DNA.
A DNA-bending protein brings the bound activators closer to the promoter.
The activators bind to certain mediator proteins and general transcription factors helping them for an
active transcription initiation complex on the promoter.
These multiple protein-protein interactions help assemble and position the initiation complex on the
promoter.
Researchers have identified two common structural elements in a large number of activator proteins: a
DNA-binding domain and one or more activation domains.
Repressors can bind to the DNA-binding site of the activator proteins or to proteins that allow the
activators to bind to DNA.
Some activators and repressors act indirectly on transcription by affecting chromatin structure.
Some activators recruit proteins that acetylate histones near the promoters of specific genes thus
promoting transcription.
Others recruit proteins that deacetylate histones, leading to reduced transcription; this is called silencing.
4. COMBINATORIAL CONTROL OF GENE ACTIVATION
The different nucleotide sequences found in control elements are small when considering the large
number of genes that have to be regulated.
On the average, each enhancer is composed of about ten control elements, each of which can bind only
one or two specific transcription factors.
A particular combination of control elements will be able to activate transcription only when the
appropriate activator proteins are preset, which may occur at a precise time during development or in a
particular cell type.
5. COORDINATELY CONTROLLED GENES IN EUKARYOTES
In bacteria, genes are often clustered together into an operon, which is regulated by a single promoter and
transcribed into a single mRNA molecule. These types of operons have not been found in eukaryotic
cells.
In eukaryotic cells, some genes are clustered but each gene has its own promoter and is individually
transcribed.
The coordinate regulation of these clustered genes is thought to involve changes in chromatin structure
that make the entire group of genes either available or unavailable for transcription.
In nematodes, some genes share an promoter but the RNA transcript is processed into separate mRNAs.
Co-expressed eukaryotic genes are most commonly found scattered in different chromosomes.
The coordinate gene expression seems to depend on the association of a specific combination of control
elements with every gene of a dispersed group.
Copies of the activators that recognize the control elements bind to them, promoting simultaneous
transcription of the genes, no matter where they are in the genome.
Coordinate control of dispersed gene often occurs in response to chemical signals from outside the cells,
e.g. steroid hormones.
MECHANISMS OF POST-TRANSCRIPTIONAL REGULATION
Prokaryotic mRNA is transcribed in a form that can be translated immediately.
Eukaryotic mRNA requires further modifications before it can be translated.
1. Multiple splicing patterns of exons (alternative RNA splicing) can yield different proteins. The splicing
pattern depends on the tissue.
2. Degradation of mRNA is due to enzymatic action that begins by shortening the polyA tail.
Enzymes degrade bacterial mRNA within a few minutes of their synthesis.
In multicellular eukaryotes, m RNA can survive for hours, days or weeks.
Increasing the life of mRNA molecules allows more proteins to be formed.
3. The initiation of translation of some mRNAs can be blocked by regulatory proteins that bind to specific
sequences or structures within the untranslated region a the 5’ end, preventing the attachment of
ribosomes.
Translation of all the mRNAs in a cell may be regulated simultaneously. It usually involves the activation
or inactivation of one or more of the protein factors required to initiate translation, e.g. the activation of
translation initiation factors right after fertilization of the egg.
4. Protein processing and degradation
Often, eukaryotic polypeptides must be processed in order to yield functional proteins.
Regulation may occur at any step involved in modifying or transporting a protein,.
Regulatory proteins are commonly activated or deactivated by the reversible addition of phosphate
groups, or by the addition of sugars to proteins destined to the surface of animal cells.
To mark a protein for destruction, the cell attaches molecules of a small protein called ubiquitin to the
protein.
Giant protein complexes called proteasomes then recognize the ubiquitin-tagged protein and degrade
them
NONCODING RNA PLAY MULTIPLE ROLES IN CONTROLLING GENE EXPRESSION
About 1.5% of the human genome codes for proteins.
Most of the rest of the DNA was thought until recently to contain no meaningful genetic information.
A major part of the genome may be transcribed into non-protein-coding RNA, also called noncoding
RNA.
Regulation by noncoding RNAs occurs at two points in the pathway of gene expression: mRNA
translation and chromatin configuration.
MICRO-RNAS AND SMALL INTERFERING RNAS.
Micro RNA (miRNA) binds to complementary strands of mRNA.
RNA precursor folds back into itself forming a short double stranded hairpin structure held together by H
bonds.
An enzyme cuts each hairpin from the primary miRNA transcript.
A second enzyme called dicer, trims the loop and the single stranded ends from the hairpin.
On strand of the double RNA is degraded.
The other strand then forms a complex with one or more proteins.
The miRNA in the complex can bind to any target mRNA that contains at least six bases of
complementary sequence.
The miRNA complex either degrades the target mRNA or blocks its translation.
It is estimated that up to one third of human genes are regulated by miRNA.
Small interfering RNA is similar in size and function as miRNA. It can turn off genes.
The difference between the two is based on the nature of the precursor molecule for each
siRNA is formed from a much longer double-stranded RNA.
CHROMATIN REMODELING AND SILENCING OF TRANSCRIPTION BY SMALL RNA
Small RNAs appear to be crucial in the formation of heterochromatin at the centromeres of chromosomes.
An RNA transcript produced from DNA in the centromeric region of the chromosome is copied into
double-stranded RNA by an enzyme and then processed into siRNAs.
These siRNAs associated with proteins move to the centromeric region and recruit enzymes that modify
the chromatin turning it into a heterochromatin.
Chromatin remodeling blocks the expression of large regions of the chromosome.
Noncoding RNA can regulate gene expression at multiple steps.
DIFFERENTIAL GENE EXPRESSION LEAD TO THE DIFFERENT CELL TYPES IN
MULTICELLULAR ORGANISMS
Embryonic development involves cell division, cell differentiation, and morphogenesis.
During embryonic development, cells increase in number.
Cells also become specialized in structure and function. This is called differentiation.
As cells differentiate, they become arranged in tissues and organs. The physical processes that give an
organism its shape constitute morphogenesis.
GENETIC PROGRAM OF EMBRYONIC DEVELOPMENT
Development, or ontogeny, is an orderly, predictable sequence of events beginning with fertilization and
ending with death.
It includes fertilization, embryogenesis, birth, infancy, childhood, adolescence, adulthood, senescence,
and death.
Morphogenesis is the process by which an animal takes shape and the differentiated cells end up in the
appropriate locations.
During differentiation, cells become specialized in structure and function.
CYTOPLASMIC DETERMINANTS AND INDUCTIVE SIGNALS
Genes that are expressed in cells control differentiation of cells.
Development requires the timely differentiation of many kinds of cells in specific locations.
In many animals, the uneven distribution of cytoplasmic determinants in the unfertilized egg leads to
regional differences in the early embryo.
Maternal substances in the cytoplasm of the egg.
 RNAs and proteins encoded by the mother’s genome.
 Substances in the cytoplasm are not evenly distributed.
 Distribution of substances influences the development of the future embryo.
Interaction among embryonic cells brings about changes in gene expression, which in turn, bring about
the differentiation of specialized cell types. This is called induction.
The environment of the cell.
 Embryonic cells send signals to other embryonic cells, including contact with cell surface
molecules.
 Signal molecules send cells down a specific path of differentiation.
SEQUENTIAL REGULATION OF GENE EXPRESSION DURING CELLULAR
DIFFERENTIATION
Determination refers to the events that lead to the observable differentiation of a cell.
At the end of determination, the cell is irreversibly committed to its final fate, and it is said to be
determined.
Observable cell differentiation is marked by the expression of genes for tissue-specific proteins.
Differentiated cells are specialists at making these tissue-specific proteins.
Liver cells make albumins; lens cells make crystallins, muscle cells make actin and myosin.
The first evidence of differentiation is the appearance of mRNAs for these proteins.
These proteins are found only in a specific cells type and give the cells its characteristic structure and
function.
The appearance of mRNA for these proteins is the first evidence of differentiation.
Beginning with the first mitotic division, the cells set on a path to specialization.
At a certain moment, the cells become irreversibly committed to its final fate. This is called
determination.
Determination is the outcome of molecular changes in the cell caused by the expression of genes for
tissue specific proteins.
On the molecular level, different sets of genes are sequentially expressed in a regulated manner as new
cells arise from division of their precursors.
PATTERN FORMATION: SETTING THE BODY PLAN
Before morphogenesis can shape an animal or plant, the organism’s body plan — its overall threedimensional arrangement — must be established.
Pattern formation is the development of a spatial organization in which the tissues and organs of an
organism are all in their characteristic places.
The axes of an animal are established very early: head and tail, right and left, front and back.
The molecular cues that control pattern formation, collectively called positional information, tell a cell its
location relative to the body axes and to neighboring cells and determine how the cell and its progeny will
respond to future molecular signals.
Cytoplasmic determinants and inductive signals are the substances that initially establish the axes of the
animal body’s.
GENETIC STUDIES ON DROSOPHILA
Key developmental events in the life cycle of Drosophila on page 370-371.
1. GENETIC ANALYSIS OF EARLY DEVELOPMENT: SCIENTIFIC INQUIRY
Edward Lewis was the first to use a genetic approach to study embryonic development in Drosophila.
Antennae, legs and wings develop on the appropriate segments. The anatomical identity of the segments
is set by master regulatory genes called homeotic genes.
Homeotic genes specify what kind of appendages and other structures are going to develop in each
segment.
Mutations of the homeotic genes cause the development of appendages on the wrong segments, e.g. legs
on the head.
Embryonic lethals are mutations with phenotypes causing death at the embryonic stage or larval stage.
All these genes encode transcription factors.
These regulatory proteins control the expression of the genes responsible for specific anatomical
structures.
Homeotic proteins activate genes specifying the proteins that actually build the fly structures.
The homeotic genes of Drosophila directly correspond to similar gene complexes in animals, all the way
up to man.
Many of the proteins found on fly pattern formation have been found to have close counterparts
throughout the animal kingdom.
“The homeotic genes (here called "HOM" genes) in Drosophila are clustered close to each other in the DNA, and they set a
ground plan for embryonic development in the fly. Vertebrates also contain homeotic (HOX) genes. Here, we find four clusters,
or complexes, of homeotic genes. These genes are closely related to the insect genes, their order in the DNA is the same, and
their action during embryonal development follows the same order in time and space in the fly. Thus, the effects on the
embryonic head-to-tail axis of these HOX genes largely follow the principles, which Lewis' set up for the fly.
It is actually possible to transfer a HOX gene from man to the embryo of a fruit fly, where it is can perform some of the functions
that the corresponding Drosophila gene normally executes. The location of our shoulders and hips on the vertebral column, for
example, may be controlled by specific homeotic genes.”
Source: http://nobelprize.org/medicine/laureates/1995/illpres/more-l-simgenes.html
http://nobelprize.org/medicine/laureates/1995/illpres/index.html
2. AXIS ESTABLISHMENT
Genetic analysis of Drosophila reveals how genes control development.
Cytoplasmic determinants are already present in the unfertilized egg and are coded by genes of the mother
called maternal effect genes.
Mutations in these maternal effect genes result in defective phenotypes in the offspring or the fertilized
egg fails to develop properly, regardless of the offspring’s own genome.
Maternal effects genes are also called egg-polarity genes because they control the orientation (polarity) of
the egg.
Messenger RNA produced by the mother and concentrated at one end of the egg act as pattern setter for
the anterior—posterior end of the embryo (bicoid gene, bcd); another group of maternal effect genes
establishes the dorsal-ventral axis.
An embryo whose mother has mutant bcd gene lacks the front half of its body and has posterior structures
at both ends.
The bcd mRNA is concentrated at the anterior end of the mature egg. After fertilization, the mRNA is
translated into protein. The bicoid protein diffuses from the anterior end toward the posterior, resulting in
a gradient of protein within the early embryo.
The bcd is a maternal morphogen, a substance that establishes the embryo’s axes.
Morphogens are the products of egg-polarity genes. They are transcription factors, proteins that regulate
the activity (transcription) of some of the embryo’s own genes.
The morphogen gradient hypothesis was first proposed a century ago: gradients of substances called
morphogens establish an embryo’s axes and other features of its form.
Segmentation pattern
Gradients of these morphogens bring about regional differences in the expression of segmentation genes,
the genes of the embryo that direct the actual formation of segments after the embryo’s major axes are
defined.
A cascade of gene activations sets up the segmentation pattern in Drosophila.
There are three sets of segmentation genes. These sets of genes are activated sequentially and provide the
positional information for increasingly fine details of the animal’ body plan.
They produce transcription factors that directly activate the next set of genes in the hierarchical scheme of
pattern formation.
Gap genes map out the basic subdivisions along the anterior-posterior axis of the embryo.
Mutations in the map genes cause an embryo to miss a given segment.
Pair-rule genes are the second set of segmentation genes to act. They subdivide these broad bands into
pairs of segments.
Mutations in the pair-rule genes causes to have half the normal number of segments because every other
segment fails to develop.
The segment polarity genes set the anterior-posterior axis of each segment.
Mutations in the segment polarity genes cause each segment to have a mirror image repetition of one side
on the other side.
Genes in each set not only activate the next set of genes but also maintain their own expression in most
cases.
Other segmentation genes operate indirectly, supporting the functioning of the transcription factors in
various ways, e.g. components of cell-signaling pathways, signal molecules and receptor molecules.
The ultimate difference between cells is transcriptional regulation; the turning on and off of certain genes.
TYPES OF GENES ASSOCIATED WITH CANCER
Genes for growth factors, their receptors, and the intracellular molecules of signaling pathways can
mutate and lead to cancer.
1. ONCOGENES AND PROTO-ONCOGENES
Proto-oncogenes code for proteins that stimulate normal cell growth and division.
Oncogenes are cancer-causing genes.
The products of proto-oncogenes and tumor-suppressor genes control cell division.
A DNA change that makes a proto-oncogene or its protein product excessively active converts it to an
oncogene, which may promote excessive cell division and cancer.
Cancer cells often contain chromosomes that have broken and rejoined incorrectly, translocating
fragments from one chromosome to another.
The proto-oncogene may end up near an especially active promoter; its transcription may increase making
it an oncogene.
2. TUMOR-SUPPRESSOR CELLS
A tumor-suppressor gene encodes a protein that inhibits abnormal cell division.
A mutation in such a gene that reduces the activity of its protein product may also lead to excessive cell
division and possibly cancer.
Tumor suppressor proteins are involved in…
Repair DNA and help prevent accumulation of cancer causing mutations.
Control adhesion of cells to each other or to the extracellular matrix; normal adhesion is absent in cancer
cells.
Cell-signaling proteins that inhibit the cell cycle
3. INTERFERENCE WITH NORMAL CELL-SIGNALING PATHWAYS
Many proto-oncogenes and tumor-suppressor genes encode components of growth-stimulating and
growth-inhibiting signaling pathways, respectively.
A hyperactive version of a protein in a stimulatory pathway, such as RAS (a G protein), functions as an
oncogene protein.
A defective version of a protein in an inhibitory pathway, such as p53 ( a transcription activator), fail to
function as a tumor suppressor.
4. THE MULTISTEP MODEL OF CANCER DEVELOPMENT
Normal cells are converted to cancer cells by the accumulation of mutations affecting proto-oncogenes
and tumor-suppressor genes.
More than one mutation is needed to produce all the changes characteristics of a cancer cell.
This may explain why the longer we live the more likely we are to develop cancer.
About a half dozen changes must occur at the DNA level for a cell to become fully cancerous.
These mutations usually include one active oncogene and the mutation or loss of several tumorsuppressor genes.
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Tumor-suppressor alleles are usually recessive.
Oncogenes are dominant.
In many malignant tumors, the gene for telomerase is activated. This enzyme reverses the shortening of
chromosome ends during DNA replication.
Production of telomerase in cancer cells removes a natural limit on the number of times the cells can
divide.
5. INHERITED PREDISPOSITION AND OTHER FACTORS COTRIBUTING TO CANCER.
Individuals who inherit a mutant oncogene or tumor-suppressor allele have an increased risk of
developing cancer.
Certain viruses promote cancer by integration of viral DNA into a cell’s genome.