Regulation of Transcription Chapter 11

1
Regulation of Transcription Chapter 11

• Attenuation, phage l, eukaryotic txn factors,
  enhancers, promoters, and epigenetics

2
Activator and repressor factors

• May contain at minimum 2 domains in the folded
  structure of the protein
• DNA binding domain drives the factor to a
  specific site in genome
• Allosteric site makes DNA binding domain
  functional or not by interaction with small
  molecules (allosteric effectors).
• Allosteric effectors, upon association or
  dissociation with the target domain, influence
  the structure of the ptn around the DNA binding
  site.

3
Effects of allostery on DNA binding
Weak affinity for DNA
4
ara operon Dual controls
Arabinose present araC ptn binds to Initiator
region. In combination with CAP/cAMP, Rpol is
attracted to enter and txn proceeds
No Arabinose araC ptn binds to both Initiator
and an operator (araO) region. DNA forms a loop
structure which blocks TXN
5
Linear array of genes in the genome corresponds
to metabolic pathways
trp operon in E. coli. Genes having connected
roles in metabolism are frequently clustered in
prokaryotic systems in the same sequence as the
reactions they control. This enhances gene group
specific control and facilitates operon
regulation. This is logical and intuitive!
6
Other controls are in place to regulate activity
of genes
Feedback inhibition at level of enzyme
activity, tryptophan inhibits early pathway
activities
7
In trp operon repressor is tryptophan (final
product)

• In presence of final product, txn of operon down
  by 10 foldmakes sense

Tryptophan
trpR
Cannot bind to DNA w/o trypo.
OFF
8

• Attenuation Yanofsky experiments with trp
  operon.
• WT cells tryptophan interacts with trpR
  represses TXNthis makes sense Why make enzymes
  for this aa when the aa is present?? So.cells
  shut it downeconomy of life and all that.
• Mutate trpR Operon is constitutive or always
  on
• BUT something odd happens when you REMOVE the
  trypto. Effector in the trpR mutant TXN goes up
  another 10 fold. This is oddwhat is the deal??
• Good ol Chuck found a leader sequence of 160 bp
  5 of the operon!

9
Here is the leader sequence (Fig. 11-18)

• Leader 160 bp just BEFORE the 1st triplet in
  trpE gene.
• Deleting about 30 bp (attenuator region) gives
  FULL txn of operon in the presence of
  tryptophan.so what does the other 130 bp do?
• The First 140 bases of the leader were
  transcribed at high rates even though the
  remaining operon mRNA was off.
• Thuseven when tryptophan is present.leader mRNA
  is being made at high levels however, operon
  mRNA is notits attenuated.

2 tryptophan codons
10
In absence of amino acid tryptophan, attenuation
stopshere is how

• Fig 11-20 proposes the intuitive model to
  describe attenuation.

Note the extensive folding of the leader RNA. It
forms 3 stem loop structures
11
In this case segment 1 is translated
through When ribosome passes over segment 2
segments 3,4 base pair. This forms a stem/loop
that terminates txn.recall txn termination in
Ch. 10
12

• In absence of tryp cell needs to upregulate
  operon.
• Ribosome stalls at segment 1 allowing 2,3
  interactions
• No termination since the termination structure
  cannot form
• The 2,3 stem loop structure does not terminate

What causes the ribosome to stall when tryptophan
is scarce?
13

• Recall Txn and Tln are coupled in E. coli. For
  this reason, amino acids can directly influence
  mRNA productionhere is how it may work in the
  trp operon.
• The leader encodes a 14 aa peptide that contains
  two trp aa see 11-21
• When tryptophan is present there is enough of
  it to allow complete synthesis of the short
  peptide-During TLN, as ribosome moves, the
  2ndary structure of the mRNA favors a stem loop
  structure (regions 3-4) that releases the mRNA
  and terminates txn.
• When tryptophan absent -There is insufficient
  aa to complete tln, thus, the ribosome stalls
  out-The 2ndary structure forms in mRNA that
  facilitates read through and txn does not
  terminate.NEAT!

14
Phage l
Please read about how lysogeny is regulated (vs.
lytic activity) Pay attention to Fig. 11-23 and
11-24 Conceptually the same as lac and trpjust
more complex.
15
Eukaryotic Gene Regulation.

• Relevant points
• Signals are interpreted from the environment but
  also as developmental cues (not seen with
  unicellular low lifes!)
• Txn control is prominent and shares some
  features with E.coli
• Trans-acting factors are significant players as
  are cis active targets of their action.
• There are some major differences due complexity
  and high genetic capacity.

16
Cis-acting sites

• Relevant points
• Three R.pol (I,II,III with pol II mRNA driver)
• Pol II requires LOTS of cooperation from other
  ptn factors
• There are classes of cis active sites that
  differ by location
• Core promoter elements
• Promoter-proximal elements
• Distance-independent elements

17
Cis-acting sites
Core promoters and promoter proximal elements.
Fig. 11-25, 26
Core
Proximal elements

• Note
• Not all promoters have all of these elements
• Some lack TATA and are called TATA-less
• Others lack proximal elements

18
Distance Independent, Cis-acting sites
Enhancers and Silencers. Fig. 11-28. Enhancers
cis acting sites that work at a great distance
and in general are distance INDEPENDENT. They
stimulate txn at a distance. Silencers also cis
active. Reduce txn by interactions with
repressors MAY ACT MANY KB AWAY FROM THE LINKED
GENE IN CIS.
50-200 kilobases away
ENH
Linked, Target Gene
19
Trans acting factors in eukaryotes

• Large of these
• Bind DNA (although some do not)
• Interact with other factors
• Many probably have allosteric effectors
• Many are post-translationally modified
  (acetylation, phosphorylation, methylation)
• Some have multiple domain structures (interact
  DNA and ptn or effectors)

20
General Txn Factors

• Assist in recruiting (binding or entry) of the
  R.Pol machinery
• Many interact w/ R.pol
• TFIID Complex with 8 factors, interacts R.pol
  (these are basal or general Txn factors or TAFs)
• TAF minimal factors required for txn

21
Core promoter assembly -TAFs TATA associated
Factors -Names TFIID, TFIIA etc. The II
corresponds to RNA Pol II. -Assembly process is
thought to be sequential in the order
shown. Inhibitors interrupt access to TATA
DISTANCE ACTION FACTORS have also been
identified GCN4 in yeast binds to enhancers
call UAS (upstream activation Seq.) These bind
to ATGACTCATTACTGAGTA NOTE symmetrical
22
Note how TATA binding ptn (blue) induces a DNA
bendrecall this from prokaryotes (CAP) can help
bind TAFs and/or R.Pol.
23
Tissue Specific Enhancers

• Multiple enhancers drive/control expression of
  tissue specific genes
• Distances are large and cis site may be either 5
  or 3 of gene (even in introns)
• One enhancer may control expression in one tissue
  and another may control expression is some other
  tissue
• Sounds complicatedhow to dissect??

24
Reporter genes how to find tissue specific
enhancers

• Chunks of a reporter gene fused to a given cis
  element to test for expression in cells.

P element transformation places construct in
germ line of Drosophila. Weak promoter driving
lacZ (b-gal) does not give high
expression Unless some testor DNA linked up
that stimulates expression. To find the tissue
histochemical staining for b-gal expression
P- element encodes a transposase that allows
insertion into the genome in flies (only germ
line insertion, requires 31 bp repeats).
25
Mouse embryo this approach did NOT use
P-element but instead used the transgenic mouse
model. Clone 258 bp test DNA to lacZ
reportershows muscle precursor cells (muscle
specific expression patterns).
Once a Tissue enhancer is identifiednext
identify the ptns that bind and regulate its
activity.
26
Gene Fusions in eukaryotes

• Gene rearrangements (known to occur) can bring
  distance acting segments to bear on novel
  promoters (Fig. 22-22)

This can activate a gene that promotes cell
proliferation cancer ALSO a good mechanism
for evolution!
27
Regulation of Txn Factors

• Some Txn Factors are tissue specific
• Tissue specific enhancers have to communicate
  within the whole organism
• Good example Steroid hormones that target the
  genome.
• Hormones produced in one tissue and target
  another through circulation (estrogen,
  testosterone, prolactin)
• Small, generally hydrophobic character
• Pass effectively through membranes
• Bind/regulate Txn factors (must target nucleus)
• Example steroid hormone/receptor targeting the
  HRE (hormone response element).

28
Hormone Receptor
hormone
Cell
N
29
Structures of DBP
Protruding a-helices engage major groove of DNA
DNA binding ptns
30
21 bp operator DNA
2 operators, 2 monomers its very
symmetrical. The other two monomers are available
for binding They allow recognition of operator
when sliding 3 or 5 (ie either direction) into
the operator.
Lac repressor/DNA complex tetramer bound to
operator
31
DNA binding motifs that are commonly seen
Zinc Fingers coordinated complex with cysteine
and histidine. Sequence specific binding employs
several fingers in one molecule.
Lac repressor motif
32
Epigenetic Inheritance also called gene silencing

• Methylation of Cytosine residues by DNA
  methyltransferases (DNMT) can suppress expression
  of linked genes.
• Methylation states tend to be inherited

Methyl C
Promoter
DNMT
Gene is on unmethylated prommoter
Gene is off
Inherited Methylation pattern passed on with
DNA replication
33
Epigenetics and imprinting

• Allele specific gene inactivation maternal or
  paternal genes differentially active.
• If maternally imprinted gene from mother is off
  for example.
• The imprinting is based on DNA methylation which
  affects enhancer action
• Thus even though diploid (2 alleles), maternal
  and paternally derived alleles may be differently
  regulated at txn level.
• The cells behave as hemizygous despite having
  two copies in autosomal cells.