Cell Nucleus

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Cell Nucleus

• Gene Structure
• Control of Transcription

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Agenda

• Gene control
• core, proximal and distal promoters
• enhancers
• transcription factors (proteins)
• response elements (DNA code)
• Example glucocorticoid receptor
• Co-activators
• Mediators of the effect of transcription factors.

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- Entire genome in each cell.
How does cell accomplish protein production? How
does it make sure the correct proteins are made,
and in the right amount?
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Control of Gene Expression in Eukaryotes

• Transcription level
• if and how often a gene is transcribed.
• Processing level
• different messenger RNAs made from a given gene
  (alternative splicing)
• Translational level
• How much of the mRNA is made into protein.(and
  mRNA lifetime)
• Post-translation
• Protein lifetime

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Review

• Last class chromatin structure
• DNA associated protein
• Histones help make the fundamental unit of
  chromatin the nucleosome
• Structure of DNA is related to its
  transcriptional activity
• Constitutive always heterochromatin
• Facultative sometimes heterochromatin
• Chromatin packing
• making it more or less accessible for
  transcription
• regulated by the cell.

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Transcriptional Control

• RNA polymerase II transcribes some genes much
  more frequently than others
• This depends on regulatory sites on the DNA and
  the presence of transcription factors.

hnRNA
factors
3
RNA pol II
5
Regulatory sites
gene
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Components of the Promoter

• A) Core promoter. DNA sequence, -1 to -40 bases
  from the start of the coding DNA. On/Off
  regulation of the gene
• It is recognized by a series of DNA-binding
  proteins general transcription factors,
  comprising the pre-initiation complex, including.
• TBP (tata binding protein) recognizes the
  nucleotide sequence TATA, about 30 bases back
  from the start of the gene.
• TAFs (TBP-associated Factors) part of a group of
  General Transcription factors accessory proteins
  necessary for RNA polymerase II
• RNA polymerase II, which produces hnRNA,
  (transcription)

regulatory sequence
gene
TAF
RNA polymerase
TBP
TATA
RNA start
core promoter
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Components of the PromoterB. Proximal promoter
(approx. -40 to -150 bases away)
gene
regulatory sequence
TAF
NF1
TBP
RNA polymerase
TATA
CAAT
GC
RNA start
Proximal promoter

• CAAT and GC boxes are bound to transcription
  factors such as NF1
• NF1 recruits a co-activator needed for RNA
  polymerase to work
• Whereas the core promoter determines whether or
  not transcription can take place, the proximal
  elements regulate frequency of transcription.
• When methylated by the cell, GC regions
  inactivate the gene
• (note, the cell has control, because nothing
  happens at the core or proximal promoter unless
  all the transcription factors are present)

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Experiment using recombinant DNA method, change
or remove DNA sequence, then measure the
appearance of the mRNA in the cytoplasm.
Note to students to reduce the file size I left
this figure off the web-notes. You can find the
figure in your textbook. Fig. 12.32 third
edition, and Figure 12.41 fourth edition.
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Components of the PromoterC) Distal promoter.
Contains response elements. -500 to -1000 bases.

• Response elements. Special DNA sequence which
  bind to proteins called specific transcription
  factors
• Specific proteins called transcription factors
  may activate or repress transcription activity.
  Specific to one gene (or a few genes)
• The cell controls gene activity by regulating the
  presence or absence of the specific transcription
  factor.
• Eg. Cyclic AMP activates CREB, which binds to its
  own response element and then turns on a gene
• Transcription factors are often activated by
  dimerization
• Every gene has its own set of response elements.
  an integrating function.

GR
Transcription factor
GRE
500 to 1000 bases
Response element
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Components of the PromoterD) Enhancers

• Specific DNA sequences
• Which bind specific transcription factors, and
  activate gene expression
• Tens of thousands of bases away, but strong
• One enhancer, when activated, activates a number
  of genes. Coordinating function.
• All the genes are usually in one loop, enhancers
  are separated from other loops by insulator
  proteins.

Preinitiation complex (one per gene)
DNA loop, 10,000 bases
Insulator proteins
Enhancer Transcription factor
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Example of Transcriptional Regulation

• 1. Glucocorticoids released from adrenal gland
  when multicellular animal is injured or ill
• 2. enter blood stream
• 3. hormone is noticed by responsive cell.
  Receptor/hormone complex is formed in cytoplasm
• 4. certain genes are turned on (need a response
  element)
• 5. new proteins (PEPCK) are made to do the
  function
• (gluconeogenesis) (provides glucose to cells to
  help them survive the trauma)

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The Glucocorticoid Recepter Glucocorticoid
receptor
protein PEPCK
translation
mRNA
glucocorticoid receptor
transcription
Binds, forms dimer, activates NLS
activates gene requires correct response element
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Glucocorticoid receptor example of a specific
transcription factor.

• Structure
• DNA binding domain, recognizes specific DNA
  sequence
• Since it is a dimer it recognizes a palindromic
  sequence
• Activation domain, alters transcription, usually
  through a co-repressor or co-activator
• Function how does it turn on the gene?
• Brings in Coactivators which
• Supply general transcription factors for RNA
  polymerase II, TAFs
• Alter chromatin structure

5 nnnnnnnnnnnnAGAACAnnnTGTTCTnnnnnn3 3
nnnnnnnnnnnnTCTTGTnnnACAAGAnnnnnn5
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Response Elements on the PEPCK Gene
CREB/ CREM
HNF-3
Fos/Jun
DBP C/EBP
T3 Receptor
Insulin
C/EBP
PPAR?/RXR
HNF-1
GR
PolII
Fos/Jun
RAR
NF1
TBP
GRE
PPARRE
AF1
P4
P3I
P1
TATA
CRE-1
TRE
P3II
IRE
P2

• PEPCK regulates glucose metabolism
• Regulated by many different hormones
• A site of integration

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Question How do transcription factors affect
gene transcription?

• Answer
• By altering histone-binding and making gene
  accessible to polymerase activity

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How Transcription Factors Work

• Binds to the response elements in the Distal
  promoter region (recognizes the nucleotide base
  sequence)
• Recruits proteins which help the pre-initiation
  complex work. These are called Coactivators.
• Enhances the RNA polymerase activity

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More on Coactivators

• Coactivators are proteins which link
  transcription factors, including the
  glucocorticoid receptor to
• general transcription factors needed for
  transcription
• Chromatin re-modeling enzymes
• For the glucocorticoid receptor the coactivator
  protein is called CBP coactivator, a type of
  Histone Acetyltransferase (HAT)

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Histone Acetyltransferase

• acetylates the lysine residues of the histones
• this has two effects
• a) reduces the strength (destabilizes) of the
  histone-DNA interaction and
• b) reduces interactions between the histone
  proteins

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Histone AcetyltransferaseStep I
TATA
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Histone AcetyltransferaseStep II
Acetylates histones
RNA polymerase
TAFII250
TATA
Acetylates histones

• Preinitiation complex has its own histone
  acetyltransferase activity
• keeps acetylating the histones as it transcribes
  subunit TAFII250

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Transcriptional Repression

• Histone Deacetyltransferases
• DNA Methyltransferases

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Histone Deacetyltransferases

• Histone Deacetylases (HDACs) return histones to
  normal state
• HDAC activity is a property of co-repressors

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DNA Methyltransferases

• Add methyl groups to DNA
• Always at carbon 5 of cytosine
• This essentially tags regions of DNA so that
  they are utilized (transcribed) differently
• This is a reversible process, but DNA methylation
  is passed-on ..

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Genomic Imprinting

• The state of methylation is passed on to daughter
  cells
• Beta-globin genes are less methylated in the
  fetal liver
• One of the two X-chromosomes is methylated
• Imprinted genes, which are inherited from one
  parent only, are turned off when gametes are
  made, stay off in the adult organism.
• In embryonic development there is a wave of
  de-methylation in first few cell divisions,then
  re-methylation as cell lineages are
  established.(fig. 12.51, fourth ed.) As the
  organism grows, the cells turn off the genes they
  and their progeny- wont need in the future.

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• Methylated sites promote gene inactivation by
  de-acetylating histones
• 1. Methylated GC islands (in the proximal
  promoter) recruit the binding of the protein
  MeCP2
• 2. MeCP2 in-turn recruits 3. Sin3 co-repressor is
  a histone de-acetylase (HDAC)
• 4. which acts by maintaining chromatin
  condensation, inaccessible to RNA polymerase.
• Note, the big question here is how the cell
  accomplishes the methylation in the first place
  and how it recognizes which genes are to be
  inactivated. Largely still an open question.
• Also note, in this case the DNA itself is
  methylated. The histones can be acetylated, as
  discussed, and they can be methylated too, which
  we didnt discuss much.
• Also note. A co-repressor is much like a
  co-activator it passes along an inactivating
  rather than an activating signal.

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methylated GC island
stops
RNA polymerase
TATA
MeCP2
Sin3
H1
H1
H1
de-acetylates
recruitment
MeCP2
Inactivation of DNA regions by MeCP2/Sin3
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Main levels of gene expression

• Genome. (nucleus) Makes gene available for
  expression
• Chromosome de-condensation
• DNA methylation
• Histone acetylation
• Changes in HMG proteins, nuclear matrix
• Transcription. Makes primary RNA transcript hnRNA
• Control by transcription factors
• RNA processing, and nuclear export
• RNA splicing, other processing events
• Movement into the cytoplasm, where translation
  happens
• Translation (cytoplasm)
• mRNA degradation and turnover
• Translation control by initiation factors,
  repressors, microRNAs
• Post-translation
• Protein folding
• Polypeptide cleavage
• Modifications
• Destination to correct location in the cell, or
  for secretion
• Resulting Functional protein.