Chapter 17 Gene Regulation in Eukaryotes

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Chapter 17 Gene Regulation in Eukaryotes

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• 200431060045

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This chapter can be studied with comparison to
chapter 16
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F O R E W O R D

• The regulation in eukaryotes is very like
  that in prokaryote, but is more complex.
• The basic principles and steps of
  regulation are similar, but eukaryotic genes have
  additional steps and more regulatory binding
  sites and are controlled by more regulatory
  proteins. Besides, nucleosomes and their
  modifiers influence access to genes.

4
The increasing complexity of regulatory
sequences from a simple bacterial gene controlled
by a repressor to a human gene controlled by
multiple activators and repressors.
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O U T L I N E

• Conserved Mechanisms of Transcriptional
  Regulation from Yeast to Mammals
• Recruitment of Protein Complexes to Genes by
  Eukaryotic Activators
• Signal Integration and Combinatorial Control
• Transcriptional Repressors
• Signal Transduction and the Control of
  Transcriptional Regulators
• Gene Silencing by Modification of Histones and
  DNA
• Eukaryotic Gene Regulation at Steps after
  Transcription Initiation
• RNAs in Gene Regulation

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1 CONSERVED MECHANISMS OF TRANSCRIPTIONAL
REGULATION

• FROM YEAST TO MAMMALS

7
Not all details of gene regulation are the same
in all eukaryotes, though they have much in
common. A typical yeast gene has less extensive
regulatory sequences than its multicellular
counterpart. Repressors work in a variety of
ways.
8
?Activators have separate DNA binding and
activating functions. The yeast activator Gal4
binds as a dimer to a 17bp site on DNA. The
activation domain and the binding domain are
separated.
9
Gal4 binds to four sites located 275bp upstream
of Gal1, and activates transcription Of Gal1
gene 1000-fold in the presence of galactose.
10

• The DNA-binding
• domain of Gal4, without that
• proteins activation domain,
• can still bind DNA, but cannot
• activate transcription.

(b) Attaching the activation Domain of GA4 to the
DNA- binding domain of the bacterial protein
LexA, creates a hybrid protein that activates
transcription of a gene in yeast as long as
that gene bears a binding site for LexA.
Domain swap experiment
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The Two Hybrid Assay This assay is used
to identify proteins that interact with each
other.
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? Eukaryotic Regulators Use a Range of
DNA-Binding Domains, but DNA Recognition
Involves the Same Principles as Found in Bacteria

• Several of the regulatory proteins in eukaryotes
  bind DNA as heterodimers, and in some cases even
  as monomers. Heterodimers extend the range of
  DNA-binding specificities available.
• Homeodomain Proteins. The homeodomain is a class
  of helix-turn-helix DNA-binding domain and
  recognizes DNA in essentially the same way as
  those bacterial proteins.
• Zinc Containing DNA-Binding Domains. There are
  various different forms of DNA-binding domain
  that incorporate a zinc atoms zinc finger and
  zinc cluster.
• Leucine Zipper Motif. This motif combines
  dimerization and DNA- binding surfaces within a
  single structural unit.
• Helix-Loop-Helix Proteins. AS in the example of
  the leucine zipper, an extended ? helical region
  from each of two monomers inserts into the major
  groove of the DNA.

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DNA recognition by a homeodomain.
The homeodomain consists of three ? helices, of
which two form the structure resembling the
helix- turn-helix motif. Thus, helix 3 is
the recognition helix and is inserted into the
major groove of DNA.
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Zinc finger domain
The ? helix on the left of the structure is the
recognition helix, and is presented to the DNA
by the ß sheet on the right. The zinc is
coordinated by the two His residues in the ?
helix and two Cys residues in the ß sheets as
shown. This arrangement stabilizes the structure
and is essential for DNA binding.
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Two large ? helices, one from each monomer, form
both the dimerization and DNA-binding domain at
different sections along their length. Thus, as
shown, toward the top the two helices interact
to form a coiled-coil that holds the monomers
together further down, the helices separate
enough to embrace the DNA, inserting into the
major groove on opposite sides of the DNA-helix.
Once again, specificity is provided by contacts
made between amino acid side chains on the ?
helices and the edge of base pairs in the major
groove.
zipper bound to DNA.
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Helix-loop-helix motif. A long ? helix involved
in both DNA recognition and , in combination
with a second shorter, ? helix dimerization.
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? Activating Regions Are Not Well-Defined
Structures

• The activating regions are grouped
• on the basis of amino acids content
• Acidic activation domains
• Glutamine-rich domains
• Proline-rich domains

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2 RECRUITMENT OF PROTEIN COMPLEXES TO GENES

• BY EUKARYOTIC ACTIVATORS

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?Activators Recruit the Transcriptional
Machinery to the Gene

• The activator recruits polymerase indirectly in
  two ways

• 1 . The activator can interact with parts of the
• transcription machinery other than
• polymerase, and , by recruiting them,
  recruit
• polymerase as well .
• 2 . Activators can recruit nucleosome modifiers
• that alter chromatin in the vicinity of a
  gene
• and thereby help polymerase bind.
• In many cases, a given activator can work in
• both ways.

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Activation of transcription initiation in
eukaryotes by recruitment of the
transcription machinery.
A single activator is shown recruiting two
possible target complexes the Mediator and,
through that, RNA polymerase ? and also the
general transcription factor TFIID. Other
general transcription factors are recruited as
part of the Mediator\ Pol ? complex separately,
or bind spontaneously in the presence of the
recruited components.
21
Activation of transcription through direct
tethering of mediator to DNA
The GAL1 gene is activated, in the absence of
its usual activator Gal4, by the fusion of the
DNA- binding domain of LexA to a component of
the Mediator Complex. Activation depends on
LexA DNA-binding sites being inserted upstream
of the gene. Other components required for
transcription initiation presumably bind
together with Mediator and Pol ?.
22
At most genes, the transcription machinery is not
prebound, and appear at the promoter only
upon activation. Thus, no allosteric activation
of the prebound polymerase has been evident in
eukaryotic regulation.
23
?Activators also Recruit Nucleosome Modifiers
that Help the Transcription Machinery Bind at
the Promoter

• Nucleosome modifiers come in two types
• Those that add chemical groups to the tails of
• histone such as histone acetyl transferases
• (HATs) , which add acetyl groups
• Those that remodel the nucleosomes, such as
• the ATP-dependent activity of SWI\ SNF.

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How do these modifications help activate a gene?

• Two basic models to explain how changes in
• nucleosomes can help the transcriptional
  machinery
• bind at the promoter
• Remodeling, and certain modifications,
• can uncover DNA-binding sites that
• would otherwise remain inaccessible
• within the nucleosome.
• Creating specific binding sites on
• nucleosomes for proteins bearing
• so-called bromodomains.

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Local alterations in chromatin structure
directed by activators
Activators, capable of binding to their sites
on DNA within a nucleosome are shown bound
upstream of a promoter that is
inaccessible within chromain.

• The activator is shown recruiting
• a histone acetylase. That enzyme
• adds acetyl groups to residues within
• the histone tails. This alters the
• packing of the nucleosomes
• somewhat, and also creates binding
• sites for proteins carrying the
• appropriate recognition domains.

(b)The activator recruits a nucleosome
remodeller, which alters the structure of
nucleosomes around the promoter, rendering it
accessible and capable of binding the
transcription machinery.
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? Action at a Distance Loops and Insulators
Many eukaryotic activators---particularly in
higher eukaryotes---work from a distance.
Various models have been proposed to explain how
proteins binding in between enhancers and
promoters might help activation in the cells of
higher eukaryotes.

• In Drosophila, the cut gene is activated from an
  enhancer
• some 100 kb away. A protein called Chip aids
  communication
• between enhancer and gene.
• In eukaryotes, the DNA is wrapped in nucleosomes,
  and
• the histones within those nucleosomes are
  subject to
• various modifications that affect their
  disposition and
• compactness.

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Specific elements called Insulators control the
actions of activators, preventing the
activating of the non-specific genes.
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a) A promoter activated by activators bound
to an enhancer. b) An insulator is
placed between the enhancer and the
promoter. When bound by appropriate
insulator- binding proteins, activation
of the promoter by the enhancer is blocked,
despite activators binding to the
enhancer. c) The activator can activate
another promoter nearby. s) The original
promoter can be activated by another
enhancer placed downstream.
Insulators block activation by enhancers.
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Transcriptional silencing
Silencing is a specialized form of repression
that can spread along chromatin, switching off
multiple genes without the need for each to bear
binding sites for specific repressors. Insulator
elements can block this spreading, so insulators
protect genes from both indiscriminate
activation and repression.
Agene inserted at random into the mammalian
genome is often silenced because incorporated
into a particularly dense form of chromatin
called heterochromatin. But if insulators are
placed up- and downstream of that gene they
protect it from silencing.
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? Appropriate Regulation of Some Groups of Genes
Requires Locus Control Regions
Only in adult bone marrow are the correct
regulators all active and present in appropriate
concentrations to bind these enhancers. But more
than this is required to switch on these genes
in the correct order.
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A group of regulatory elements collectively
called the locus control region, or LCR, is
found 30-50 kb upstream of the whole cluster of
globin genes. It binds regulatory proteins that
cause the chromatin structure around the whole
globin gene cluster to OPEN UP, allowing
access to the array of regulators that control
expression of the individual genes in a defined
order.
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• The human
• globin genes and the
• LCR that ensures
• their ordered
• expression.
• (b) The globin genes
• from mice, which are
• also regulated by an
• LCR.
• (C) The HoxD gene
• cluster from the
• mouse controlled by
• an element called
• the GCR which like
• the LCRs appears to
• impose ordered
• expression on the
• gene cluster.

Regulation by LCRs
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3 SIGNAL INTERATION AND

• COMBINATORIAL CONTROL

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? Activators Work Together Synergistically to
Integrate Signals

• In multicellular organisms signal integration is
  used
• extensively. In some cases numerous signals are
  required
• to switch a gene on. So at many genes multiple
  activators
• must work together to switch the gene on.
• When multiple activators work together, they do
  so
• synergistically. That is , the effect of two
  activators
• working together is greater than the sum of each
  of them
• working alone.
• Three strategies of synergy
• Two activators recruit a single complex
• Activators help each other binding cooperativity

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• Cooperative
• binding through
• direct interaction
• between the two proteins.
• (b) A similar effect is
• achieved by both
• proteins interacting with
• a common third protein.
• (c) The first protein recruits
• a nucleosome remodeller
• whose action reveals a
• binding site for a second
• protein.
• (d) Binding a protein
• unwinds the DNA from
• nucleosome a little, r
• revealing the binding site
• for another protein.

Cooperative binding of activators
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? Signal Integration the HO Gene Is Controlled
by Two Regulators One Recruits Nucleosome
Modifiers and the Other Recruits Mediator. The
HO gene is involved in the budding of yeast. The
HO gene is expressed only in mother cells and
only at a certain point in the cell cycle. These
two conditions are communicated to the gene
through two activators SWI5 and SBF.
37

• Why does expression of the gene depend
• on both activators?
• SBF( which is active only at the correct stage
• of the cell cycle) cannot bind its sites
  unaided
• their disposotion within chromatin prohibits it.
  
• SWI5( Which acts only in the mother cell) can
• bind to its sites unaided but cannot, from that
• distance, activate the HO gene.
• SWI5 can recruit nucleosome modifiers.
• These acts on nucleosomes over the SBBF sites.
• Thus, if both activators are present and active,
• the action of SWI5 enables SBF to bind, and
• that activator, in turn, recruits the
  transcriptional
• machinery and activates expression of the gene.

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SWI5 can bind its sites within chromatin
unaided, but SBF cannot. Remodellers and
histone acetylases recruited by SWI5 alter
nucleosomes over the SBF sites, allowing that
activator to bind near the promoter and
activate the gene.
Control of the HO gene
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? Signal Integration Cooperative Binding of
Activators at the Human ß-Interferon Gene The
human ß-interferon gene is activated in cells
upon viral infection. Infection triggers three
activators NF?B, IRF, and Jun\ ATF. They
bind cooperatively to sites within an enhancer,
form a structure called enhanceosome.
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The human ß-interferon enhanceosome
Cooperative binding of the three activators,
to gether with the architectural protein
HMG-1, activates the ß-interferon gene.
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? Combinatorial Control Lies at the Heart of the
Complesity and Diversity of Eukaryotes There
is extensive combinatorial control in
eukaryotes. In complex multicellular organisms,
combinatorial control involves many more
regulators and genes, and repressors as well as
activators can be involved.
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Combinatorial control
Each of the two gene controlled by multiple
signals- four in the case of gene A three in
the case of gene B. Each signal is communicated
to a gene by one regulatory protein. Regulatory
protein 3 acts at both genes, in combination with
different additional Regulators in the two cases.
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? Combinatorial Control of the Mating-Type Genes
from Saccharomyces cerevisiae
The yeast S. cerevisiae exists in three forms
Two haploed cells of different mating types-a
and ?-and the diploid formed when and an a and
an ? cell mate and fuse. Cells of the two
mating types differ because they express
different sets of gene a specific genes and ?
specific genes.
44

• The a cell and the ? cell each
• encode cell type specific regulators
• a cells make the regulatory protein a1
• ? cells make the proteins ?1 and ?2.
• A fourth regulatory protein called Mcm1,
• is also involved in regulating the
• mating-type specific genes and is
• present in both cell types.

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The three cell types of the yeast S. cerevisiae
are defined by the sets of genes they express.
One ubiquitous regulator( Mcm1) and three
cell-type specific regulators( a1, ?1 and ?2)
together regulate three classes of target genes.
The MAT locus is the region of the genome which
encode the mating type regulator.
Control of cell-type specific genes in yeast
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4 TRANSCRIPTIONAL

• REPRESSORS

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Repressors dont work by binding to sites that
overlap the promoter and thus block binding of
polymerase. Another form of repression different
from bacteria, which is the most common in
eukaryotes. It works as follows As with
activators, repressors can recruit nucleosome
modifiers, but in this case the enzymes have the
opposite effects to those recruited by
activators---they compact the chromatin or
remove groups recognized by the transcriptional
machinery.
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a) By binding to a site on DNA that overlaps
the binding site of an activator, a repressor
can inhibit binding of the activator to a gene,
and thus block activation of that gene. b) A
repressor binds to a site on DNA beside an
activator and interacts with that activator,
occluding its activating region. c) A repressor
binds to a site upstream of a gene and, by
interacting with the transcriptional machinery
at the promoter in some specific way, inhibits
transcription initiation. d) repression by
recruiting histone modifiers that alter
nucleosomes in ways that inhibit transcription.
Ways in which eukaryotic repressors work
49
In the presence of glucose, Mig1 binds a site
between the UASG and the GAL1 promoter. By
recruiting the Tup1 repressing complex, Mig1
repress expression of GAL1. Repression is a
result of deacetylation of local nucleosomes,
and also probably by directly contacting and
inhibiting the transcription machinery.
Repression of the GAL1 gene in yeast
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5 SIGNAL TRANSDUCTION AND THE CONTROL OF

• TRANSCRIPTIONAL REGULATORS

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? Signals Are Often Communicated to
Transcriptional Regulators through Signal
Transduction Pathways
The term signal refers to the initiating ligand
itself---that is , the sugar or protein or
others. It can also refer to the information
as it passes from detection of that ligand to
the regulators that directly control the
genes---that is, as it passes along a signal
transduction pathway.
52
In a signal transduction pathway, the initiating
ligand is typically Detected by a specific cell
surface Receptor. From there the signal is
relayed to the relevant transcriptional
regulator, often through a cascade of
kinases. Through an allosteric change in the
receptor ,the binding of ligand ot the
extracellular domain is communicated to the
intracellular domain.
53

• Two signal transduction pathways from mammalian
  cells
• The STAT pathway (b) The MAP
  kinase pathway

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? Signals Control the Activities of Eukaryotic
Transcriptional Regulators in a Variety of Ways
Once a signal has been communicated, directly or
indirectly, to a transcriptional regulator, how
does it control the activity of that regulator?
55

• In eukaryotes, transcriptional
• regulators are not typically
• controlled at the level of DNA binding.
• Regulators are instead usually controlled
• in one of two basic ways
• Unmasking an Activating Region.
• Transport Into and Out of the Nucleus.

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Activator Gal4 is regulated by masking
protein
The yeast activator Gal4 is regulated by the
Gal80 protein
57
? Activators and Repressors Sometimes Come in
Pieces For example, the DNA binding domain and
activating region can be on different
polypeptides, which come together on DNA to
form the activator. In addition, the mature of
the complex can determine whether the
DNA-binding protein activates or represses
nearby genes.
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6 GENE SILENCING BY MODIFICATION OF HISTONES

• AND DNA

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Silencing is a position effect---a gene is
silenced because of where it is located, not in
response to a specific environmental signal.
Also, silencing can spread over large
stretches of DNA ,switching off multiple genes,
even ones quite distant from the initiating
event. The most common form of silencing is
associated with a dense form of chromatin called
heterochromatin. Both activation and repression
of transcription often involve modification of
nucleosomes to alter the accessibility of a gene
to the transcriptional machinery and other
regulatory proteins. Transcription can also be
silence by methylation of DNA by enzymes called
DNA methylases.
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? Silencing in Yeast Is Mediated by
Deacetylation and Methlation of Histones
The telomeres, the silent mating-type locus, and
the rDNA genes are all silent regions in S.
cerevisiae. Methylation of DNA sequence can
inhibit binding of proteins, including the
transcriptional machinery, and thereby block
gene expression.
61

• Consider the telomere as an example.
• The final 1-5 kb of each chromosome is found in a
  
• folded , dense structure. Genes taken from
  other
• chromosomal locations and moved to this region
  are
• often silenced, particularly if they are only
  weakly
• expresse din their usual location.
• Mutations have been isolated in which silencing
  is relieved
• ---that is, in which a gene placed at the
  telomere is
• expressed at higher levels.
• These studies implicate three genes encoding
  regulators
• of silencing SIR2,3 and 4.
• The three proteins encoded by these genes form
  a complex
• that associates with silent chromatin, and Sir2
  is a histone
• deacetylase.
• The silencing complex is recruited to the
  telomere by a
• DNA-binding protein that recognizes the
  telomeres repeated
• sequences.
• Histone methyl transferases attach methyl groups
  to histone
• tails.

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Silencing at the yeast telomere
Rap1 recruits SIR complex to the telomere. SIR2,
a component of that ocmplex, deacetylates nearby
nucleosomes, The unacetylated tails themselves
then bind Sir3 and SIR4, recruiting more SIR
complex, allowing the SIR2 within it to act on
nucleosomes further away, and so on. This
explains the spreading of the silencing effect
produced by deacetylation.
63
?Histone Modifications and the Histone Code
Hypothesis
According to the histone code edea, different
patterns of modifications on histone tails can
be read to mean different things. The meaning
would, in part, be the result of the direct
effects of these modifications on chromatin
density and form. But in addition, the particular
pattern of modifications at any given location
would recruit specific proteins, the particular
set depending on the number, type, and
disposition of recognition domains those
proteins carry. There are also proteins that
phosphorylate serine residues in H3 and H4 tails
and proteins that bind thosse modifications. Add
to this the observation that many of the proteins
that carry modification recognizing domains are
themselves enzymes that modify histones
further.
64
Switching a gene off through DNA methylation and
histone midification
65
? DNA Methylation Is Associated with Silenced
Genes in Mammalian Cells
Some mammalian genes are kept silent by
methylation of nearby DNA sequences.
Methylation of DNA can mark sites where
heterochromatin subsequently forms. DNA
methylation lies at the heart of a phenomenon
called imprinting. Two regulatory sequences are
critical for the differential expression of the
human H19 and Igf2 genes An enhancer and an
insulator.
66
Imprinting
Two examples of genes controlled by imprinting-
the mammalian Igf2 and H19 genes. The H19 genes
is expressed from only the matermal chromosome,
Igf2 from the paternal chromosome. The
methylation state of the insulator element
determines whether or not the insulator binding
protein( CTCF) can bind and block activation of
the H19 gene from the downstream enhancer.
67
? Some States of Gene Expression Are Inherited
through Cell Division even when the Initiating
Signal Is No Longer Present
Patterns of DNA methylation can be maintained
through cell division
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7 EUKARYOTIC GENE REGULATION AT STEPS

• AFTER TRANSCRIPTION INITIATION

69
? Some Activators Control Transcriptional
Elongation rather than Initiation
At some genes there are sequences downstream of
the promoter that cause pausing or stalling of
the polymerase soon after initiation. At those
genes, the presence or absence of certain
elongation factors greatly influences the level
at which the gene is expressed. One example is
the HSP70 gene from Drosophila. The HIV virus ,
which causes AIDS, transcribes its genes from a
promoter controlled by P-TEF, which is brought
to the stalled polymerase by TAT. Tat
recognizes a specific sequence near the start of
the HIV RNA and present in the transcript made by
the stalled polymerase. Another domain of TAT
interacts with P-TEF and recruits it to the
stalled polymerase.
70
? The Regulation of Alternative mRNA Splicing
Can Produce Different Protein Products in
Different Cell Types
In some cases a given precursor mRNA can be
spliced in alternative ways to produce different
mRNAs that encode different protein products.
The choice of splicing variant produced at a
given time or in a given cell type can be
regulated. The regulation of alternative splicing
works in a manner reminiscent of transcriptional
regulation. In other cases, sequences called
splicing enhancers are found near splice sites.
71
The sex of a fly is determined by the ratio of X
chromosomes to autosomes. This ratio is
initially measured at the level of transcription
using two activators SisA and SisB. The genes
encoding these regulators are both on the X
chromosome, and so , in the early embryo, the
prospective female makes twice as much of their
products as does the male. These activators bind
to sites in the regulatory sequence upstream of
the gene Sex-lethal( Sxl). Another regulator
that binds to and controls the Sxl gene is a
repressor called Dpn( Deadpan) this is encoded
by a gene found on one of the autosomes(
chromosome2). Thus the ratio of activators to
repressor differs in the two sexes, and this
makes the difference between the Sxl gene being
activated( in females) and repressed( in males).
72
Early transcriptional regulation of Sxl in male
and female flies.
73
A cascade of alternative splicing events
determines the sex of a fly
74
? Expression of the Yeast Transcriptional
Activator Gcn4 Is Controlled at the Level of
Translation
Gcn 4 is a yeast transcriptional activator that
regulates the expression of genes encoding
enzymes that direct amino acid biosynthesis.
Although it is a transcriptional activator, Gcn4
is itself regulated at the level of translation.
The mRNA encoding the Gcn4 protein contains four
small open reading frames called uORFs upstream
of the coding sequence for Gcn4.The most
upstream of these short open-reading frames(
Uorf1) is efficiently recognized by ribosomes
that scan along the message from the 5 end.
Before initiating translation of any downstream
open-reading frame scanning 40s ribisome
subunits must bind the translation factor Eif2
complexed with the initiating tRNAs molecule
Met-tRNAiMet.
75
The open-reading frame encoding the yeast
activator Gcn4 is preceeded by four other ORFs.
The first of these upstream ORFs is translated
initially. When amino acids are scarce(
starvation conditions), it takes longer for the
translational machinery to re-initiate
translation, and so it tends to reach the
Gcn4- encoding open-reading frame before
re-initiating and translates that to give Gcn4
protein, When amino acids are plentiful(
nonstarvation conditions) re-initiation takes
place at intervening open- reading frames, and
the translation machinry then dissociates from
the RNA template and Gcn4 is never translated.
High levels of amino acids the Gcn4 mRNA is
not translated
Low levels of amino acids the Gcn4 mRNA is
translated
Translational control of Gcn4 in response to
amino acid starvation
76
8 RNAs IN GENE

• REGULATION

77
RNAs have a more general and mechanistically
distinct role in gene regulation. Short RNAs,
generated by the action of enzymes can direct
repression of genes with homology to those short
RNAs. This repression, called RNA interference(
RNAi), can manifest as translational inhibition
of the mRNA, destruction of the mRNA or
transcriptional silencing of the promoter that
directs expression of that mRNA. The
role of these RNAs ranges from developmental
regulation to the protection against infection
by certain viruses. RNAi has also been adapted
for use as a powerful experimental technique
allowing specific genes to be specific genes to
be switched off in any of many organisms.
78
? Double-Stranded RNA Inhibits Expression of
Genes Homologous to that RNA
The discovery that simply introducing
double-stranded RNA( dsRNA) into a cell can
repress genes containing sequences identical to(
or very similar to) that dsRNA was remarkable in
1998 when it was reported. In that case, the
experiment was done in the worm C. elegans. A
similar effect is seen in many other organisms
in which it has subsequently been tried. Earlier
than this report, however, it had been known
that in plants genes could be silenced by copies
of homologous genes in the same cell. Those
additional transgenes were often found in
multiple copies, some Integrated in direct
repeat orientation. Also, in plants, it was
known that infection by viruses was combated by
a mechanism that involved destruction of viral
RNA. These two cases were brought together in
the following observation infection of a plant
with an RNA virus that carried a copy of an
endogenous plant gene led to silencing of that
endogenous gene. All these phenomena are now
known to be mechanistically linked.
79
? Short Interfering RNAs( siRNA) Are Produced
from dsRNA and Direct Machinery that Switches
Off Genes in Various Ways
Dicer is an RNAse?-like enzyme that recognizes
and digests long dsRNA. The products of this are
short double-stranded fragments about 23
nucleotides long. These short RNAs( often called
short interfering RNAs, or siRNAs) inhibit
expression of a homologous gene in three
ways they trigger destruction of its mRNA they
inhibit translation of its mRNA or they induce
chromatin modifications within the promoter that
silence the gene.

80
That machinery includes a complex called RISC (
RNA- induced silencing complex). A RISC complex
contains, in addition to the siRNAs themselves,
various proteins including members of the
Argonaut family, which are believed to interact
with the RNA component. There is another feature
of RNAi silencing worth noting- its extreme
efficiency. Very small amounts of dsRNA are
enough to induce complete shutdown of target
genes. Why the effect is so strong? It might
involve an RNA-dependent RNA polymerase which is
required in many cases of RNAi. The involvement
of this enzyme suggests some aspect of the
inhibitory signal might be amplified as part
of the process.
81
? MicroRNAs Control the Expression of some Genes
during Development
There is another class of maturally occurring
RNAs, called microRNAs( miRNAs), that direct
repression of genes in plants ans worms. Often
these miRNAs are expressed in developmentally
regulated patterns. The miRNAs, typically 21 or
22 nts long, arise from larger precursors( about
70-90 nts long) transcribed from non-protein
encoding genes. These transcripts contain
sequences that form stem loop strutures, which
are processed by Dicer. The miRNAs they produce
lead to the destruction ( typically the case in
plants) or translational repression( in worms)
of target mRNAs with homology to the miRNA.
82
Summary

• There are several complexities in the
• organization and transcription of
• eukaryotic genes not found in bacteria
• Nucleosomes and their modification.
• Many regulators and larger distances.
• The elaborate transcriptional machinery.
• 2.In eukaryotes, activators predominantly
• work by recruitment, but do not recruit
• polymerase directly or alone. They recruit
• the other protein complexes required to
• initiate transcription of a given gene.

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3.Some activators work from sites far from the
gene, requiring that the DNA between their
binding sites and the promoter loops
out. 4.Most commonly, eukaryotic repressors
work by recruiting histone modifiers that
reduce transcription. 5.Groups of genes can be
kept in a silent state without the need for
specific repressors bund at each individual
gene. In some eukaryotic organisms, such as
mammals, silent genes are also associated with
methylated DNA.
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6.There are various steps in gene regulation
after transcription initiation can be regulated.
These include transcriptional elongation and
translation, but most striking is the regulation
of splicing. 7.Another form of gene regulation
involves small RNA molecules that inhibit
expression of homologous genes. 8.The
mechanisms by which these RNAs inhibit
expression of genes can involve destruction of
mRNA, inhibition of translation, and
RNA-directed modification of nucleosomes in the
promoters of genes.
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• Important conceptions
• Promoter
• Regulator binding site
• Enhancer
• Insulator
• Reporter gene
• Gene silencing

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Thank you!