1. Chapter 17 Gene Regulation in Eukaryotes
04级生物学基地班
姜冠男

2. This chapter can be
studied with comparison
to chapter 16

3. 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.

5. 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

6. 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. ⅠActivators 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. Domain swap experiment
The DNA-binding
domain of Gal4, without that
protein’s 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

11. The Two Hybrid Assay
This assay is used to identify proteins that interact
with each other.

12. Ⅱ 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 ɑ helical region from each of two monomers inserts into the major groove of the DNA.

13. DNA recognition by a homeodomain.
The homeodomain
consists of three ɑ
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.

14. Zinc finger domain The ɑ helix on the left of the
structure is the recognition helix,
and is presented to the DNA by
the β sheet on the right. The
zinc is coordinated by the two
His residues in the ɑ helix and
two Cys residues in the β
sheets as shown. This
arrangement stabilizes the
structure and is essential for
DNA binding.

15. zipper bound to DNA. Two large ɑ 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 ɑ helices and the edge of base pairs
in the major groove.
zipper
bound to
DNA.

16. Helix-loop-helix motif. A long ɑ helix
involved in both DNA recognition and , in
combination with a second shorter, ɑ helix
dimerization.

17. Ⅲ 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

18. 2 RECRUITMENT OF PROTEIN COMPLEXES TO GENES
BY EUKARYOTIC ACTIVATORS

19. ⅠActivators 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.

20. Activation of
transcription
initiation in
eukaryotes by
recruitment
of the
machinery.
A single activator is shown recruiting two possible target complexes:
the Mediator; and, through that, RNA polymerase Ⅱ; and also the
general transcription factor TFIID. Other general transcription factors
are recruited as part of the Mediator\ Pol Ⅱ 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 Ⅱ.

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. ⅡActivators 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.

24. 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.

25. Local alterations in chromatin structure directed by 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.

26. Ⅲ 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.

27. Specific elements called
Insulators control the
actions of activators,
preventing the activating
of the non-specific genes.

28. Insulators block activation by enhancers.
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.

29. 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.

30. Ⅳ 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.

31. A group of regulatory elements
collectively called the locus control
region, or LCR, is found 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.

32. Regulation by LCRs 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

33. COMBINATORIAL CONTROL
3 SIGNAL INTERATION AND
COMBINATORIAL CONTROL

34. Ⅰ 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

35. 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

36. Ⅱ 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.

38. Control of the HO gene 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

39. The human β-interferon gene is activated in cells
Ⅲ Signal Integration: Cooperative
Binding of Activators at the Human
β-Interferon Gene
The human β-interferon gene is activated in cells
upon viral infection. Infection triggers three
activators: NFĸB, IRF, and Jun\ ATF. They bind
cooperatively to sites within an enhancer, form a
structure called enhanceosome.

40. The human β-interferon enhanceosome
Cooperative
binding of the
three activators,
to gether with the
architectural
protein HMG-1,
activates the
β-interferon
gene.

41. Ⅳ 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.

42. 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.

43. Ⅴ 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 ɑ-and the diploid formed when and an a and
an ɑ cell mate and fuse.
Cells of the two mating types differ because they
express different sets of gene: a specific genes
and ɑ specific genes.

44. The a cell and the ɑ cell each
encode cell type specific regulators:
a cells make the regulatory protein a1;
ɑ cells make the proteins ɑ1 and ɑ2.
A fourth regulatory protein called Mcm1,
is also involved in regulating the
mating-type specific genes and is
present in both cell types.

45. Control of cell-type specific genes in yeast
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, ɑ1 and ɑ2) 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

46. 4 TRANSCRIPTIONAL
REPRESSORS

47. As with activators, repressors can recruit
Repressors don’t 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.

48. Ways in which eukaryotic repressors work
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. Repression of the GAL1 gene in yeast
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

50. 5 SIGNAL TRANSDUCTION AND THE CONTROL OF
TRANSCRIPTIONAL REGULATORS

51. The term “signal” refers to the initiating ligand
Ⅰ 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

54. Once a signal has been communicated,
Ⅱ 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.

56. Activator
Gal4 is
regulated
by masking
protein
The yeast activator Gal4 is regulated
by the Gal80 protein

57. Ⅲ 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.

58. 6 GENE “SILENCING” BY MODIFICATION OF HISTONES
AND DNA

59. 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.

60. Ⅰ 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 telomere’s repeated
sequences.
Histone methyl transferases attach methyl groups to histone
tails.
Just as acetylated residues within histones are recognized
by proteins bearing bromodomains, methylated residues
bind proteins with chromodomains.

62. 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. ⅡHistone 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. Ⅲ 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. Patterns of DNA methylation can be maintained through cell division
Ⅳ 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

68. 7 EUKARYOTIC GENE REGULATION AT STEPS
AFTER TRANSCRIPTION INITIATION

69. Ⅰ 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. Ⅱ 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. Ⅲ 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. Translational control of Gcn4 in response to amino acid starvation
High levels of
amino acids:
the Gcn4 mRNA
is not translated
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.
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. Ⅰ 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. Ⅱ Short Interfering RNAs( siRNA) Are Produced from dsRNA and Direct
Machinery that Switches Off Genes in
Various Ways
Dicer is an RNAseⅢ-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. Ⅲ 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 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.

83. 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.

84. 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.

85. Important conceptions:
Promoter
Regulator binding site
Enhancer
Insulator
Reporter gene
Gene silencing

86. Thank you!