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• The cap at the 5 end of the mRNA is synthesized
  once the poly(A) tail is completed
• The poly(A) tail is required for proper
  translation of the mRNA
• A nucleosome consists of DNA wrapped around a
  histone complex
• The acronym MAR stands for matrix attachment
  region
• Only protein encoding genes are transcribed by
  RNA polymerase II
• snRNPs play a role in the transport of the mRNA
  out of the nucleus
• Euchromatin is DNA that is relatively loosely
  packaged and transcriptionally active.
• The TATA box is a sequence in the promoter that
  is required for transcription.

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• Overview
• Global mechanisms controlling gene expression
• - Histone acetylation
• DNA methylation
• Activation of gene expression by transcription
  factors
• Promoter structure
• Promoter analysis
• Signal transduction pathways
• MAPkinases
• Review of translation
• tRNA
• Ribosomes
• Post-translational processing
• Protein degradation

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Control of gene expression histone acetylation
and DNA methylation
The structure of the chromatin determines the
accessibility of the DNA and therefore
transcription. Chromatin accessibility is
controlled in part by HATs (histone acetyl
transferases). This is a class of proteins that
can acetylate the histones. Acetylation reduces
the histones affinity for the DNA, and may also
reduce the interaction between individual
nucleosomes. The result is a more open chromatin
structure, so that transcription can occur.
Histone remodeling proteins are also involved
in controlling chromatin structure. They can
slide histones, remove them or change their
conformation to improve the accessibility of the
DNA. Histone deacetylases are proteins that
remove acetyl groups from the histones to repress
gene activity. DNA methylation is used in
conjuncton with histone acetylation to control
gene expression. Expressed genes are not
methylated. Methylation-sensitive restriction
enzymes can be used to get an idea of the extent
of DNA methylation. The DNA methylases that are
involved in controlling gene expression are
different from the ones involved in maintenance
methylation. Methyl CpG binding proteins are
thought to bind to CpG islands upstream of the
gene, methylate it, and render the adjacent gene
inactive. These proteins are sometimes components
of histone deacetylases, so that there is synergy
between the two mechanisms.
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Control of gene expression transcription factors
Transcription factors are DNA binding proteins
that bind to cis-acting elements in the promoter.
These cis-acting elements are oftentimes (but not
always) short (6-10 bp) palindromic
sequences. The binding of a transcription factor
facilitates the recruitment of the TATA-binding
protein and the other components of the
transcription initiation complex. The
transcription factors themselves are controlled
by internal or external cues. They may be
present in the cystosol in an inactive state, and
after becoming activated get transferred to the
nucleus. There are several classes of
transcription factors, including Helix-turn-helix
transcription factors two helices, one
bindingthe minor groove of the DNA, the other
lying across it at an angle. Leucine zippers
has a stretch of aa with leucine residues at
every 7th position the zipper in one protein may
interact with that of another to form a diner.
The DNA is bound by positively charged amino
acids adjacent to the zippers. Zinc fingers
these transcription factors contain a
loop-forming stretch of aa that binds Zn2
Cys2/His2 fingers Cys-X2-4-Cys-Phe-X5-Leu-X2-H
is-X3-His Cys2/Cys2 fingers Cys-X2-Cys-X13-Cys-
X2-Cys
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Leucine zipper
Zinc fingers
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Methods by which a transcription factor can
become activated
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Promoter bashing see article by Yamamoto et
al. 1991
In order to identify cis-acting elements in
promoters, you can construct a series of
truncated promoter fragments, fuse them with a
reporter gene (via cloning) and evaluate the
expression in transgenic plants. This is referred
to as promoter bashing. The major criticism of
this method is that you eliminate the possibility
of interactions between different transcription
factors. A more informative
alterative is a linker scanning. In this case a
window of 10 bases is mutated along a stretch of
the promoter and the effect on reporter gene
expression is evaluated.
Expression
100
100
80
60
10
0
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Article Yamamoto et al. (1991) Plant Cell 3
371-382 This article describes the isolation and
characterization of a gene, TobRB7, from tobacco.
A TobRB7 cDNA was isolated from a root-specific
library. The corresponding gene was isolated
from a genomic library. The promoter thus
isolated was fused to the GUS reporter gene and
introduced in tobacco via transformation. To
identify cis-acting elements, a series of
deletions of the promoter were generated and also
fused to the GUS gene (Figure 4). The effect of
these deletions was examined (Figure 7). This
shows that in general the longer promoter
fragments result in higher expression levels. A
major reduction in expression was observed when
the region between 600 and 300 was eliminated.
There also appeared to be a negative cis-element
between position 800 and 600. The deletion of
this element increases GUS activity.
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Spatial and temporal control of gene expression
Model internal or external signals set in motion
a signal transduction cascade that results in
the activation of a transcription factor. The
transcription factor binds to the promoter,
recruits the transcription machinery, and the
gene gets transcribed. External or internal
stimuli can be perceived by receptor proteins,
which sometimes have kinase activity. Phosphoryla
tion of a target protein (such as a transcription
factor) results in a change in conformation so
that the target becomes active. Article Jonak
et al. (1994) Plant Mol. Biol. 24 407-416 This
is a review article describing MAP (mitogen
activated protein) kinases. The best described
MAP kinase-based signal transduction pathway is
the one involved in mating of haploid yeast. The
binding of a mating factor results in arrest of
DNA replication, and the activation of a
machinery that allows the mating to occur (p.
409-410). A nice comparison of similar pathways
in other organisms is shown on p. 408. Even
though not all components have been identified,
the mechanisms are very similar. The ethylene
response pathway may rely on MAPK-based
signaling, as described on p. 411 and further.
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Translation This is the process during which the
mRNA is read and translated into a sequence of
amino acids. Two important players are tRNAs and
ribosomes. After nuclear export, the mRNA is
targeted to specific locations in the cytosol,
based on an mRNA ZIP code. This is a sequence
found in the 3 UTR of the mRNA. In the current
models, an mRNA binding protein recognizes the
specific sequence in the 3UTR and through
interaction with the cytoskeleton directs the
mRNA to its destination tRNA tRNAs are small
RNA molecules (74-90 nt) involved in translation.
They form the link between the mRNA and the amino
acids. tRNA molecules can be displayed as a
clover leaf, although the 3D structure is
L-shaped. Each tRNA has a number of
features The codon is recognized by the tRNAs
anticodon. The anticodon is the end of the
anti-codon arm. The acceptor arm links up with
the amino acid. The 3 end is always 5-CCA-3.
The acceptor arm contains a stretch of 7
basepairs. There are two arms with unusual bases,
pseudo-uridine in the T?C arm, and
dihydro-uridine in the D-arm. The V-arm contains
a variable number of nucleotides. Aminoacyl-tRNA
synthetases are enzymes that attach amino acids
to tRNA molecules. Amino acids are activated
through a reaction with ATP, and are subsequently
linked to the 3 end of the tRNA. Class I
aminoacyl-tRNA synthetases place the amino acid
on the 2OH group of the A-residue, whereas Class
II aminoacyl-tRNA synthetases use the 3 OH
group. There are also some differences in the
structure of the V-arm.
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Ribosomes The ribosomes mediate the translation.
There are two parts to the ribosomes, a large
(60S) and a small (40S) subunit. The 60S unit is
made of 28S, 5.8S and 5S rRNA and approx. 50
proteins, whereas the small unit is made of 18S
rRNA and approx. 33 proteins. The small
subunit scans the 5 end of the mRNA until it
recognizes the AUG start codon. A number of
initiation factors play a role in this process,
which may involve interactions with the 5 cap
and the 3 poly(A) tail. The Kozak sequence is a
conserved sequence surrounding the start codon
5-ACCAUGG-3. Once the start codon has been
identified, the large subunit of the ribosome
attaches. Protein synthesis can now start. The
ribosome contains two sites at which amino-acyl
tRNAs can bind the P- or peptidyl site and the
A- or aminoacyl site. The P-site is occupied by
the initiator tRNAMet, charged with methionine.
The A-site covers the second codon in the ORF.
The A-site becomes filled with the appropriate
amino-acyl tRNA, and a peptide bond between the
two amino acids is formed through the action of a
peptidyl transferase. This enzyme releases the
amino acid from the tRNA and forms the peptide
bond. The A-site is now occupied by the two amino
acids, linked to the tRNA. Translocation is
the process by which the ribosome moves over 3
nucleotides, the dipeptide-tRNA moves to the
P-site, and empty tRNA from the P site is
ejected. This process requires GTP hydrolysis.
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Nonsense mediated mRNA degradation Once an mRNA
has been synthesized, it undergoes a pioneer
round of translation. This is to make sure that
there are no premature stop codons present in the
mRNA as a result of mutations or transcription
mistakes. If there were, the cell would be
wasting resources synthesizing dysfunctional
proteins. The ribosomes scan the mRNA. If a stop
codon is found within a window of 50 nt upstream
of the 3 exon-exon junction (which is likely to
be marked by proteins remaining there from the
splicing reaction), the mRNA is going to get
tagged for degradation. This model may not cover
all cases where reduced expression is observed as
a result of stop codons.
cDNA of interest
Control cDNA
TAA
(1,3,5)
wild type
TAA
TAG
(2)
bmr12
TAA
TGA
(4)
bmr18
TAA
TGA
(6)
bmr26
200 bp
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Post-translational processing

• The newly synthesized peptide is typically
  inactive and needs to undergo some modifications
• protein folding to achieve the correct tertiary
  structure. Folding is often mediated by
  chaperones, proteins that help the protein find
  its correct folding.
• proteolytic cleavage to remove parts of the
  protein. This is a mechanism to prevent a
  protein from being active too early or in the
  wrong spot.
• chemical modification of individual amino acids.
  The following modifications can occur
• SMALL CHEMICAL SUBSTITUTIONS
• Acetylation of lysine
• Methylation of lysine
• Phosphorylation of serine, threonine and tyrosine
• Hydroxylation of proline and lysine
• N-formylation of N-terminal glycine
• SUGARS
• O-linked glycosylation of serione and threonine
• N-linked glycosylation of asparagine
• LIPIDS
• Acylation of serine, threonine and cysteine
• N-myristoylation of N-terminal glycine

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Protein degradation Proteins are degraded after
they become conjugated to ubiquitin. Ubiquitin is
a 76 aa protein which is transferred to lysine
residues of the protein to be degraded. The
degradation signal is not quite understood, but
certain motifs seem to play a role in the binding
of the E3 protein. Three proteins are involved
in the conjugation with ubiquitin and subsequent
degradation E1 carries the ubiquitin protein.
Ubiquitin is then transferred to E2, while E3 is
linked to the substrate. E2 is then transferred
to a lysine in the substrate. Conjugation of
ubiquitin leads to the degradation of the protein
in proteasomes, large cylindrical structures with
a sedimentation coefficient of 26S (made up of a
20S cylinder and tow 19S caps). The protein first
needs to unfold to fit in the cylinder, and the
degradation results in the production of short
peptide of 4-10 aa long. These are subsequently
broken down into individual amino acids that are
recycled.
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