wh

Document Sample
wh Powered By Docstoc
					Accomplish the gene
regulation of prokaryotes,
we comeback to the
eukaryotes.
You well exclaim with it’s
complication and accuration.
   Chapter 17
Gene Regulation in
   Eukaryotes
   Similarity And
   Difference of
regulation between
  eukaryotes and
    prokaryote
Similarity :                    Difference:

•   Principles are the same:    •    Pre-mRNA splicing adds an
    signals, activators and          important step for
    repressors, recruitment          regulation.
    and allostery,              •    The eukaryotic
    cooperative binding              transcriptional machinery
•   Expression of a gene can         is more elaborate than
    be regulated at the              its bacterial counterpart.
    similar steps, and the      •    Nucleosomes and their
    initiation of                    modifiers influence
    transcription is the most        access to genes.
    pervasively regulated       •    Many eukaryotic genes
    step.                            have more regulatory
                                     binding sites and are
                                     controlled by more
                                     regulatory proteins than
                                     are bacterial genes.
         Topic 1
Conserved Mechanisms of
Transcriptional Regulation
       from Yeast
      to Mammals
The basic features of gene
regulation are the same in all
eukaryotes, because of the
similarity in their
transcription and nucleosome
structure.
The typical eukaryotic
activators works in a manner
similar to the simplest
bacterial case.
Repressors work in a variety
Domain swap
experiment
Moving domains
among proteins,
proving that
domains can be
dissected into
separate parts of
the proteins.


Many similar
experiments
shows that DNA
binding domains
and activating
regions are
DNA-binding domains and
   activating regions are
   separable:
1. Activator produces a protein
   bound to the DNA normally but
   did not activate transcription.
2. Fusion of the C-terminal
   region of the activator to the
   DNA binding domain of a
   bacterial repressor, LexA
   activates the transcription of
   the reporter gene. Domain swap
   1-2 Eukaryotic regulators use
  a range of DNA binding
  domains, but DNA recognition
  involves the same principles as
  found in bacteria
 Homeodomain proteins

 Zinc containing DNA-binding
  domain
 Leucine zipper motif

 Helix-Loop-Helix proteins
Both of these proteins
 use hydrophobic amino
 acid residues for
 dimerization.
Bactrial regulatory proteins
• Most use the helix-turn-helix
  motif to bind DNA target
• Most bind as dimers to DNA
  sequence: each monomer inserts an
  a helix into the major groove.
Eukaryotic regulatory proteins
1. Recognize the DNA using the
   similar principles, with some
   variations in detail.
2. Some form heterodimers to
   recognize DNA, extending the
   range of DNA-binding specificity.
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




     Figure 17-5
Zinc containing DNA-binding
 domains finger domain: Zinc
  finger proteins (TFIIIA) and Zinc
  cluster domain (Gal4)




Figure 17-6
Leucine Zipper Motif: The Motif
combines dimerization and DNA-
binding surfaces within a single
structural unit.

  Figure 17-7
  Helix-Loop-Helix motif: Because the
                          region of the
                          a-helix that
                          binds DNA
                          contains baisc
                          amino acids
                          residues,
                          Leucine zipper
                          and HLH
                          proteins are
                          often called
                          basic zipper
                          and basic HLH
                          proteins.
Figure 17-8
 1-3 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
  Ⅱ Recruitment of
 Protein Complexes
      to Genes
         by
Eukaryotic Activation
2-1 Interacting with parts of the
  transcription machinery.

   Some activators not only recruit
    parts of the transcriptional
    machinery, they also induce
    allosteric changes in them
The eukaryotic transcriptional machinery
contains polymerase and numerous proteins
being organized to several complexes,
such as the Mediator and the TFⅡD
complex. Activators interact with one or
more of these complexes and recruit them
to the gene.




Figure 17-9
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
2-2 Activators also recruit
nucleosome modifiers that help
the transcription machinery bind
at the promoter

 1. Modifiers direct recruitment
    of the transcriptional
    machinery
 2. Modifiers help activate a
    gene inaccessibly packed
    within chromatin
Two types of Nucleosome
 modifiers :
 Those add chemical groups to
 the tails of histones, such
 as histone acetyl
 transferases (HATs)
 Those remodel the
 nucleosomes, such as the
 ATP-dependent activity of
 SWI/SNF
Two basic models for how these
modification help activate a gene :
  Remodeling and certain
  modification can uncover DNA-
  binding sites that would otherwise
  remain inaccessible within the
  nucleosome.
  By adding acetyl groups, it
  creates specific binding sites on
  nucleosomes for proteins bearing
  so-called bromodomains.
Fig 17-11 Local alterations in chromatin
2-3 Action at a distance: loops
and insulators

 Many enkaryotic activators-
   particularly
 in higher eukaryotes-work from a
   distance.
1. Some proteins help, for example Chip
   protein in Drosophila.
2. The compacted chromosome structure
   help. DNA is wrapped in nucleosomes
   in eukaryotes.So sites separated by
   many base pairs may not be as far
   apart in the cell as thought.
Specific elements called
insulators control the
actions of activators,
preventing the activating
the non-specific genes
    Insulators
      block
    activation
        by
    enhancers

Figure 17-12
       Transcriptional Silencing
Silencing is a specializes form of
  repression that can spread along
  chromatin, switching off multiple
  genes without the need for each to
  bear binding sites for specific
  repressor.
Insulator elements can block this
  spreading, so insulators protect genes
  from both indiscriminate activation
  and repression.
E.P: A gene inserted at random
into the mammalian genome is
often “silenced” because it
becomes 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.
2-4 Appropriate regulation of some
 groups of genes requires locus control
 region (LCR).



  Figure 17-13
A group of regulatory elements
collectively called the locus control
region (LCR), is found 30-50 kb upstream
of the cluster of globin genes. It’s made
up of multiple-sequence elements :
something like enhancers, insulators or
promoters.
It binds regulatory proteins that cause
the chromatin structure to “open up”,
allowing access to the array of regulators
Another group of mouse genes
whose expression is regulated in
a temporarily and spatially
ordered sequence are called HoxD
genes. They are controlled by an
element called the GCR (global
control region) in a manner very
like that of LCR.
Ⅲ Signal Integration
       and
  Combinatorial
     Control
3-1 Activators work together
synergistically to integrate
signals
In eukaryotic cells, numerous signals are
  often required to switch a gene on. So at
  many genes multiple activators must work
  together.
They do these by working synergistically:
  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
  One activator recruit something that helps
  the second activator bind
                                   a.“Classica
                                           l”
                                   cooperative
                                      binding
                                      b. Both
                                      proteins
                                    interacting
                                   with a third
                                      protein

                                    d. Binding a
                                        protein
                                     unwinds the
                                       DNA from
c. A protein recruits               nucleosome a
a remodeller to                         little,
reveal a binding site              revealing the
for another protein Figure 17-14    binding site
3-2 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.
It has two activators : SWI5 and SBF.


                                 alter the
                                nucleosome




                          Figure 17-15
SBF cannot bind its sites
unaided; their disposition
within chromatin prohibits it.
SWI5 can bind to its sites
unaided but cannot, from that
distance, activate the HO gene.
SWI5 can, however, recruit
nucleosome modifiers. These act
on nucleosomes over the SBF
sites
3-3 Signal integration:
Cooperative binding of
activators at the human b-
interferon gene.
The human β-interferon gene is activated
in cells upon viral infection. Infection
triggers three activators :
 NFκB, IRF,
 and Jun/ATF.
 They bind
 cooperatively
 to sites within
 an enhancer,
 form a                     Figure 17-16
 structure
 called
 enhanceosome.
3-4 Combinatory control lies at
the hear of the complexity and
diversity of eukaryotes
There is extensive combinatorial
 control in eukaryotes.
   Four
  signals


                             Figure 17-17
  Three
  signals



 In complex multicellular organisms,
 combinatorial control involves many
 more regulators and genes than shown
 above, and repressors as well as
3-5 Combinatory control of the
mating-type genes from S.
cerevisiae
The yeast S.cerevisiae exists in
 three forms: two haploid cells
 of different mating types- a
 and a -and the diploid formed
 when an a and an a cell mate and
 fuse.Cells of the two mating
 types differ because they
 express different sets of genes :
 a specific genes and a specific
genes.
a cell make the regulatory
 protein a1,a cell make the
 protein a1 and a2.
A fourth regulator protein Mcm1
 is also involved in regulatory
 the mating-type specific genes
 and is present in both cell
 types.
Control of cell-type specific genes in yeast




   Figure 17-18
      Ⅳ
Transcriptional
  Repressors
In eukaryotes,
repressors don’t work by
binding to sites that
overlap the promoter and
thus block binding of
polymerase, but most
common work by recruiting
nucleosome modifiers.
                 Ways in
                   which
               eukaryotic
                repressor
                   Work
                 a and b




Figure 17-19
 Ways in
   which
eukaryotic
repressor
   Work      Silencing
 c and d
A specific example:
 Repression of the GAL1
 gene in yeast




In the presence of glucose, Mig1
binds a site between the USAG and
the GAL1 promoter. By recruiting
the Tup1 repressing complex, Mig1
represses expression of GAL1.
  Ⅴ Signal Transduction
            and
      the Control of
Transcriptional Regulators
5-1 Signals are often
communicated to
transcriptional regulators
through signal transduction
pathway
For example, histone
deacetylases repress
transcription by removing
actetyl groups from the
tails of histone.
Other enzymes add methyl
groups to histone tails, and
this frequently represses
transcription.
In a signal transduction pathway:
initiating ligand binds to an
extracellular domain of a
 specific cell surface receptor
 this binding bring an
 allosteric change in the
intracellular domain of receptor
the signal is relayed to the
 relevant transcriptional
 regulator often through a
 cascade of kinases.
5-2 Signals control the
activities of eukaryotic
transcriptional regulators in a
variety of ways
 a. The STAT pathway




b. The MAP kinase pathway
Once a signal has been
 communicated, directly or
 indirectly, to a transcriptional
regulator,In eukaryotes,
 transcriptional regulators
are not typically controlled at
 the level of DNA binding. They
 are usually controlled in one of
 two basic ways :
  Unmasking an activating region
  Transport in or out of the
 nucleus
Activator Gal4 is regulated by masking protein Gal80
The signalling ligand causes activators (or
repressors) to move to the nucleus where
they act from cytoplasm.
5-3 Activators and repressors
 sometimes come in pieces.
For example, the DNA binding domain
 and activating region can be on
 different polypeptides. same of an
 activator
In addition, the nature of the
 protein complexes forming on DNA
 determines whether the DNA-binding
 protein activates or represses
 nearby genes. For example, the
 glucocorticoid receptor (GR).
Ⅵ Gene “Silencing”
         by
  Modification of
 Histones and DNA
Gene “silencing” is a position
 effect-a gene is silenced
 because of where it is located,
 not in response to a specific
 environmental signal.
The most common form of
 silencing is associated with a
 dense form of chromatin called
 heterochromatin. It is
 frequently associated with
 particular regions of the
 chromosome, notably the
 telomeres, and the centromeres.
6-1 Silencing in yeast is
mediated by deacetylation ane
methylation of the histones
The telomeres, the silent mating-type
  locus, and the rDNA genes are all
  “silent” regions in S.cerevisiae.
Three genes encoding regulators of
  silencing, SIR2, 3, and 4 have been
  found (SIR stand for silent
  information regulator).




     Silencing at the yeast telomere
Transcription can also be
 silenced by methylation of
 DNA by enzymes called DNA
 methylases.
This kind of silencing is not
 found in yeast but is common
 in mammalian cells.
Methylation of DNA sequence
 can inhibit binding of
 proteins, including the
 transcriptional machinery,
 and thereby block gene
Switching a gene off :

A mammalian gene marked by
 methylation of nearby DNA
 sequence     recognized by DNA-
 binding proteins
recruit histone decetylases and
 histone methylases
modify nearby chromatin
This gene is completely off.
Figure 17-24




               Switching a gene off
DNA methylation lies at the heart of a
phenomenon called imprinting. Two
  examples :Human H19 and Igf2 genes.
Here an enhancer and an insulator are
  critical.
Nucleosome and DNA odifications
 can provide the basis for
 epigenetic inheritance. DNA
 methylation is even more
 reliably inherited, but far
 more efficiently is the so-
 called maintenance methylases
 modify hemimethylated DNA-the
 very substrate provided by
 replication of fully ethylated
 DNA.
Patterns of DNA methylation can be
   maintained through cell division
    Ⅶ Eukaryotic
   Gene Regulation
       at Steps
         after
Transcription Initiation
 At some genes there are sequence
 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.
Two examples :
Early transcriptional regulation of Sxl in
         male and female flies
A cascade
     of
alternative
  splicing
   events
determines
  the sex
  of a fly
Gcn4 is a yeast transcriptional
  activator that regulates the
  expression of genes encoding
  enzymes that direct amino acid
biosynthesis.The mRNA encoding the
  Gcn4 protein contains four small
  open reading frames upstream of
  the coding sequence for Gcn4.
Although it is a activator, Gcn4
  is itself
regulated at the level of
  translation.
   In the presence of low levels of
  amino acids, the Gcn4 mRNA is
  translated (and so the
  biosynthetic are expressed).
   In the presence of high levels,
  it is not translated.
High
  levels
of amino
acids :
the Gcn4
mRNA is
  not
translated
Low levels
of amino
acids :
the Gcn4
mRNA is
translated
   Ⅷ RNAs
      in
Gene Regulation
Short RNAs 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.
  RNA
silencing
RNAi silencing is extreme
 efficiency. Very small
 amounts of dsRNA are enough
 to induce complete shutdown
 of target genes.
There is another class of
naturally occurring RNAs,
called microRNAs (miRNAs),
that direct repression of
genes in plants and worms.
The mechanism of RNAi may have
evolved originally to protect
  cells from any infectious, or
  otherwise disruptive, element
  that employs a dsRNA
  intermediate in its replicative
  cycle.

				
DOCUMENT INFO
Shared By:
Categories:
Tags:
Stats:
views:0
posted:4/5/2013
language:Unknown
pages:86