Chapter 9
Molecular Mechanisms of
Gene Regulation
Transcription Regulation: Prokaryotes
• In prokaryotes, on–off gene activity is often
controlled through transcription
• In negative regulation, the default state of
transcription is “on” until it is turned “off” by a
repressor protein that binds to the DNA upstream
from the transcriptional start site
2
Transcription Regulation: Prokaryotes
• In inducible transcription, a repressor keeps
transcription in the “off” state (repressor +
inducer  on)
• In repressible transcription, the default state is
“on” until an active repressor is formed to turn it
“off” (aporepressor + co-repressor  off)
3
Figure 9.1: Negative regulation
4
Transcription Regulation: Prokaryotes
• In positive regulation, the default state of transcription
is “off” and binding with a transcriptional activator
protein is necessary to turn it “on”
• Many systems are both positively and negatively
regulated
• Negative regulation is more common in prokaryotes,
positive regulation in eukaryotes
• Some genes exhibit autoregulation—the protein
product of a gene regulates its own transcription
5
Figure 9.2: Transcription
6
Operon Model of Regulation
• In bacterial systems, when several enzymes act
in sequence in a single metabolic pathway,
usually either all or none of the enzymes are
produced
• This coordinate regulation results from control
of the synthesis of one or more mRNA
molecules that are polycistronic
• Polycistronic mRNAs are transcribed from an
operon: a collection of adjacent structural
genes regulated by an operator
7
lac Operon
• The genetic regulatory mechanism in bacteria was
first explained by the operon model of François
Jacob and Jacques Monod
• They studied lactose-utilization system in E.coli
• The lactose-utilization system consists of two kinds
of components: structural genes (lacZ and lacY),
which encode proteins needed for the transport and
metabolism of lactose, and regulatory elements (the
repressor gene lacI, the promoter lacP, and the
operator lacO)
8
lac Operon
• The products of the lacZ (enzyme b-galactosidase)
and lacY (transporter lactose permease) genes are
coded by a single polycistronic mRNA
• The linked structural genes, together with lacP and
lacO, constitute the lac operon
• The promoter mutations (lacP–) eliminate the ability
to synthesize lac mRNA
9
lac Operon
• The product of the lacI gene is a repressor,
which binds to a unique sequence of DNA
bases constituting the operator
• When the repressor is bound to the operator,
initiation of transcription of lac mRNA by RNA
polymerase is prevented
• Because the repressor is necessary to shut off
mRNA synthesis, regulation by the repressor is
negative regulation
10
lac Operon
• Lactose (inducer) stimulate mRNA synthesis by
binding to and inactivating the repressor
Figure 9.4a/b: lac operon
11
lac Operon
• In the presence of an
inducer, the operator is not
bound with the repressor,
and the promoter is available
for the initiation of mRNA
synthesis
Figure 9.4c: lac operon
12
lac Operon
• lac operon is also subject to positive regulation
• Positive regulation of the lac operon involves
cAMP-CRP (cyclic AMP receptor protein), which
binds to the promoter to activate transcription by
RNA polymerase
• In the absence of the cAMP–CRP complex, RNA
polymerase binds only weakly to the promoter
13
Figure 9.6: Four regulatory states of the lac operon
14
trp Operon
• The trp operon contains the structural genes that
encode enzymes required for the synthesis of the
amino acid tryptophan
• The trp operon is transcriptionally active unless
tryptophan is present
• The trp operon is an example of a repressible system
regulated by a negative feedback loop
15
Figure 9.9: The trp operon in E. coli
16
trp Operon
• The trp operon is shut off when tryptophan binds
to inactive aporepressor
• Tryptophan-repressor complex binds to operator
to block transcription when tryptophan levels are
high
• If tryptophan levels fall, trp-repressor complex
dissociates from operator
17
Figure 9.10: Regulation of the E. coli trp operon
18
Attenuation
• Attenuation is a very sensitive form of translational
regulation of the trp operon
• The trp attenuator consists of
5' base sequence in mRNA
that is complementary and can
base-pair to form a stem and
loop structure
Figure 9.11: The terminal region of the trp attenuator sequence
19
Attenuation
• Attenuation results in the premature termination
of mRNA synthesis due to stem and loop
formation in the 5' region of mRNA
• If tRNATrp is present , synthesis of the leader
peptide results in pairing of mRNA, which
blocks the action of RNA polymerase
Figure 9.12: The sequence of bases in the trp leader mRNA
20
Figure 9.13: Mechanism of attenuation in the E. coli trp operon
21
Attenuation
• At low concentrations of tRNATrp, the ribosome
stalls, and the mRNA opens so that transcription
continues
• Attenuation permits the cell to respond to
tryptophan levels by expressing the genes needed
for its synthesis when needed
• Attenuation is possible because in bacteria
transcription is coupled with translation
22
Riboswitches
• Transcription termination can also be triggered by
direct binding of a small molecule (vitamin B12 or
flavin mononucleotide) to a 5' untranslated leader
mRNA
• An RNA leader sequence able to switch between
an antiterminator conformation and a terminator
conformation is known as a riboswitch
• Comparison of genomic sequences indicates that
riboswitches are present in archaea, eubacteria,
and eukarya
23
Gene Regulation: Eukaryotes
Gene regulation in eukaryotes differs from
prokaryotes:
• The processes of transcription and translation
are uncoupled
• The primary transcripts of many genes are
alternatively spliced to yield different products
• Each gene is regulated by a separate, often
multiple promoters
24
Gene Regulation: Eukaryotes
Genome organization in eukaryotes:
• DNA in complex with histones forms chromatin
• Significant fraction of DNA is moderately or highly
repetitive
• Large fraction of DNA does not code for proteins
• Many coding sequences are interrupted by introns
25
Transcriptional Regulation
• Many eukaryotic genes differ in their expression
according to cell type or stage of the cell cycle.
These genes are often regulated at the level of
transcription.
• Galactose metabolism in yeast illustrates
transcriptional regulation
26
Figure 9.15: Metabolic pathway
27
Galactose Metabolism in Yeast
• Despite the tight linkage of the three genes
involved in galactose metabolism, they are not
part of an operon. The mRNAs are monocistronic.
• The GAL1 and GAL10 mRNAs are synthesized
from divergent promoters lying between the
genes, and GAL7 mRNA is synthesized from its
own promoter.
• These genes are inducible because the mRNAs are
synthesized only when galactose is present.
28
Figure 9.16: The linked GAL genes of Saccharomyces cerevisiae
29
Galactose Metabolism in Yeast
• Constitutive and uninducible mutants have been
observed.
• In two types of mutants, gal80 and gal81c, the
mutants synthesize GAL1, GAL7, and GAL10
mRNAs constitutively.
• Another type of mutant, gal4, is uninducible: It
does not synthesize the mRNAs whether or not
galactose is present.
30
Table 9.3: Characteristics of Diploids Containing Various Combinations of
gal80, gal4, and gal81c Alleles
31
Enhancers
• Enhancer sequences are typically rather short
(~ 20 bp) and are found at a variety of
locations around the gene they regulate
• They may be located many kb from a gene or
inside introns and may are able to function as
enhancers irrespective of their orientation
• Enhancers are essential components of gene
organization in eukaryotes because they
enable genes to be transcribed only when
proper transcriptional activators are present
32
Silencers
• Some genes are also subjected to regulation by
transcriptional silencers: short nucleotide
sequences that are targets for DNA-binding proteins
that promote the assembly of large protein
complexes that prevent transcription of the silenced
genes
• PcG (Polycomb group) proteins in D. melanogaster
silence certain genes during development.
33
Chromatin-remodeling Complexes
• Several different multiprotein complexes, known as
chromatin-remodeling complexes (CRCs), can
restructure chromatin and enable it to be
transcribed
• CRCs can disrupt nucleosome structure without
displacing them or reposition the nucleosomes
making key DNA-binding sites accessible
• CRCs use energy derived from ATP to restructure
chromatin
• The molecular mechanism of chromatin remodeling
is unknown
34
Figure 9.22a/b: Function of chromatin-remodeling complexes
35
Figure 9.22c/d: Function of chromatin-remodeling complexes
36
Alternative Promoters
• Some eukaryotic genes have multiple promoters that
are active in different cell types
• The different promoters result in different primary
transcripts that contain the same protein-coding
regions
• Alternative promoters make possible the independent
regulation of transcription in different tissues or at
different stages of development
37
Figure 9.23: Use of alternative promoters
38
Transcription Activation
• Transcription complex is an aggregate of protein factors that
combines with the promoter to initiate transcription
• The basal transcription factors are proteins in the complex
that are used in the transcription of many different genes
• The basal transcription factors in eukaryotes have been
highly conserved in evolution
• Transcriptional activation occurs by a mechanism called
recruitment—the interaction of transcription factors with
promoter and enhancers
39
Figure 9.20: Transcriptional activation by recruitment
40
Transcription Regulation
• Combinatorial control means that a few genes can
control many others
• Strategically placed enhancers can act as genetic
switches
• One gene can have two or more promoters that
are regulated differently
41
Epigenetic Regulation
• Epigenetic: heritable changes in gene expression
that are due to either chemical modification of the
DNA bases, or protein factors bound with the DNA
• In most higher eukaryotes, a proportion of the
cytosine base are modified by the addition of a
methyl (CH3) group to the number-5 carbon atom
• The cytosines are incorporated unmodified during
DNA replication, and then the methyl group is added
by an enzyme called DNA methylase
42
DNA Methylation
• In mammals, cytosines are modified preferentially
in 5'-CG-3' dinucleotides
• Many mammalian genes have CG-rich regions
upstream of the coding region that provide
multiple sites for methylation—CpG islands
• Heavy methylation is associated with genes for
which the rate of transcription is low
• One example is the inactive X chromosome in
mammalian cells, which is extensively methylated.
43
Imprinting
• Mammals feature an unusual type of epigenetic
silencing known as genomic imprinting, a process
with the following characteristics:
 Imprinting occurs in the germ line and affects at
most a few hundred genes
 It is accompanied by heavy methylation
 Imprinted genes are differentially methylated in the
female and male germ lines
 Once imprinted, a silenced gene remains inactive
during embryogenesis
 Imprints are erased early in germ-line
development, then later reestablished according to
sex-specific patterns
44
Imprinting
• The epigenetic, sex-specific gene silencing
associated with imprinting is dramatically evident in
Prader–Willi syndrome and Angelman syndrome
characterized by neuromuscular defects, mental
retardation, and other abnormalities
45
Figure 9.24: Imprinting of genes and varying results
46
Alternative Splicing
• Regulation also takes place at the levels of RNA
processing
• Alternative splicing is a form of gene regulation
that results in the generation of alternative mRNAs
from a single gene
• Different splice patterns may occur in different
tissues resulting in tissue-specific gene expression
47
Alternative Splicing
• Alternative splicing is an important source of
human genetic complexity
• Although the number of human genes exceeds
that in worms or flies by a factor of about two, the
number of different human proteins may be
greater than that in worms or flies by a factor of
about five
48
Figure 9.25: Alternative splicing of the primary transcript
49
RNA Interference
• Different mRNAs can differ in their persistence in
the cell
• An important mechanism regulating the stability of
RNA transcripts is a phenomenon of RNA
interference (RNAi), in which the introduction of a
few hundred nucleotide pairs of double-stranded
RNA triggers degradation of RNA transcripts
containing homologous sequences
50
Figure 9.27: Mechanisms of gene silencing by the
siRNA and miRNA pathways
51
Translation Regulation
• In eukaryotes, gene expression regulated at the
level of translation separately from transcription
• The principle types of translational control are:
 Inability of an mRNA to be translated except under
certain conditions
 Regulation of the overall rate of protein synthesis
 Inhibition or activation of translation by small
regulatory RNAs that undergo base pairing with the
mRNA
52
Figure 9.28: Regulation of translation of target mRNAs
Adapted from S. Altuvia and E. G. H. Wagner, Proc. Natl. Acad. Sci. USA
97 (2000): 9824-9826.
53
Translation Regulation
• An RNA sequence complementary to an mRNA is
called an antisense RNA
• The antisense regulatory RNAs act by pairing with
the mRNA to either inhibit or activate translation
• Regulatory RNAs that control developmental timing
in the nematode C. elegans were the first examples
of small regulatory RNAs described in eukaryotes.
54
DNA Rearrangements
• Some developmental processes are controlled by
programmed DNA rearrangements
• A permanent change in DNA sequence implies that
the genotype of a cell lineage becomes permanently
altered
• Such irreversible changes take place only in somatic
cells, so they are not genetically transmitted
• Programmed DNA rearrangement take place in the
bone-marrow–derived (B) cells and thymus-derived
(T) cells that play key roles in the vertebrate immune
system
55
Figure 9.30: Formation of a gene for the light chain of an antibody molecule
56
DNA Rearrangements
• Programmed transpositions take place in the
regulation of yeast mating type
• Many strains of S. cerevisiae have a mating system
called homothallism in which some cells undergo a
conversion into the opposite mating type
• This allows matings between cells in what would
otherwise be a pure culture
57
Figure 9.31: Genetic basis of mating-type interconversion
58