Gene expression Control in Eukaryotes

1. DNA recombination
2. Transcriptional Initiation
3. Transcript Processing and Modification
4. RNA Transport:
5. mRNA Stability:
6. Translational Initiation
7. Post-Translational Modification
8. Protein Transport
9. Control of Protein Stability

DNA RECOMBINATION

B cell maturation

Clonal Selection of B Cells Producing Polyclonal Antibodies

Out of 8 different naive B-cell clones, 3 are able to react with 3
different antigenic determinants on the infecting virus.

Clonal selection

Antibody production
Gen untuk rantai H tdpt di khromosom 12
Gen untuk rantai L tdpt di khromosom 16

B Cell Maturation

Heavy Chain Gene Structure

Located at chromosome 12
Consist of 300 V exons, 20 D exons, 4 J exons and 8 constant genes
Exons V, D, and J encode the variable domain of H chain

Heavy Chain Production

Waktu sel B masih muda, gen-gen daerah variabel sebesar 324 gen
Sewaktu sel B dewasa, gen-gen daerah variabel tinggal 3 gen

VDJ Recombination In Ig gene

Heavy Chain Class Switch

Ig Light Chain Genes

Located at chromosome ?
V and J exons encode variable domain of L chain

VJ Recombination of Ig Light chain gene

Chromatin remodelling
Chromatin Remodeling Is an Important Aspect of
Eukaryotic Gene Expression
Histone covalent modification : Histone
acetylation and deacetylation are best
understood. (lysine residues )

Regulation of Transcription on Eukaryote

Transcription controlled by :
cis elements = cis acting factors
on promoter region
on upstream region

trans acting factors

complex

single protein
peptide hormons
steroid hormone-receptor protein
vitamine- receptor protein complex
minerals or mineral protein complex
mixture of proteins and non-proteins

Control of Eukaryotic Transcription Initiation

Enhanceosome
Example

Formation and putative structure of the enhanceosome formed on

the human -interferon gene enhancer.

Formation and putative structure of the enhanceosome formed on

the human -interferon gene enhancer.
top is the distribution of the multiple cis-elements (HMG, PRDIV,
PRDI-III, PRDII, NRDI) composing the -interferon gene
enhancer.
The intact enhancer mediates transcriptional induction of the interferon gene (over 100-fold) upon virus infection of human
cells.
The cis-elements of this modular enhancer represent the binding
sites for the trans-factors HMG I(Y), cJun-ATF-2, IRF3-IRF7,
and NF-B, respectively.
The factors interact with these DNA elements in an obligatory,
ordered, and highly cooperative fashion as indicated by the arrow.

Formation and putative structure of the enhanceosome formed on

the human -interferon gene enhancer.
Initial binding of four HMG I(Y) proteins bends in the enhancer
the entire 7080 bp region to assume a high level of curvature.
This curvature is integral to the subsequent highly cooperative
binding of the other trans-factors. formation and stability of
the enhanceosome and generate recruitment of chromatinmodifying coregulators that carry
enzymatic activities (eg, Swi/Snf: ATPase, chromatin
remodeler and P/CAF: histone acetyltransferase)
general transcription machinery (RNA polymerase II and
GTFs).

Formation and putative structure of the enhanceosome formed on

the human -interferon gene enhancer.
Although four of the five cis-elements (PRDIV, PRDI-III, PRDII,
NRDI) independently can modestly stimulate (~10-fold)
transcription of a reporter gene in transfected cells, all five ciselements, in appropriate order, are required to form an enhancer
that can appropriately stimulate mRNA gene transcription (ie,
100-fold) in response to viral infection of a human cell.
This distinction indicates the strict requirement for appropriate
enhanceosome architecture for efficient trans-activation.
Similar enhanceosomes, involving distinct cis- and trans-factors
and coregulators, are proposed to form on many other mammalian
genes.

Transcriptional Initiation;
Example
Normally Myoblast fusion form the polynucleated
skeletal muscle.

Very few protein in myoblast. After fusing,

protein synthesis increase
A protein Myo D1, a single DNA-binding
protein (muscle specific ) is produced. Myo D1
is a transcription factor.
If preadipocyte or preepithelial cells culture
are inserted with Myo D1, these cells would
become myocytes.

Alternative
splicing

In Antibody class switching

Alternative
tailing

In antigen receptor of lymphocyte precursor

Alternative splicing & tailing

mRNA Stability

Proto oncogen Fos and myc.

Unstability is probably due to an A and
U-rich untranslated region.
Stability of mRNA are subject to
translational control of nutrient.

Translational Initiation
(Ferritin synthesis)
Translation can be blocked by specific proteins at specific
sites on the mRNA. (Synthesis of Ferritin by Iron).
Ferritin mRNA is not translated unless iron is bound to a
series of bases that comprise the iron response element
that is part of the massage.
When iron is present, the iron-response element is fold away,
make it available for use.
When iron is absent, the start site is covered up, make it
unavailable for use.

The eIF-2 cycle

The eIF-2 cycle

= regeneration of GTP-bound eIF-2 following the hydrolysis of
GTP during translational initiation

When the 40S preinitiation complex is engaged with

the 60S ribosome to form the 80S initiation complex,
the GTP bound to eIF-2 is hydrolyzed providing energy
for the process.
In order for additional rounds of translational initiation
to occur, the GDP bound to eIF-2 must be exchanged
for GTP.
This is the function of eIF-2B which is also called
guanine nucleotide exchange factor (GEF).

Heme controlled Inhibitor = HCI

Erythrocytes are enucleate and contain primarily globin mRNA.
When the level of heme is low it would be inefficient for
erythrocytes to synthesize globin protein.
As the level of heme falls the activity of HCI increases.
HCI is a kinase which phosphorylates eIF-2.
When phosphorylated, eIF-2 still hydrolyzes bound GTP to GDP
and still interacts with eIF-2B (GEF). However, the rate of eIF2B-mediated GTP exchange is greatly reduced.
This renders eIF-2 incapable of being used to form an new ternary
initiation complex and translational initiation is reduced.
When the level of heme again rises the activity of HCI is reduced
and translational initiation is once again active.

Regulation of translation by heme controlled inhibitor (HCI).

Post-Translational Modification;
Collagen
Collagen disintesis pada ribosom dalam bentuk
preprokolagen. Masih mempunyai rangkaian sinyal =
rangkaian pembimbing yang mengarahkan ke ruang
vesikuler RE.
Dalam RE terjadi hidolisis rangkaian sinyal,
hidroksilasi prolin dan lisin, dan glikosilasi
hidroksilisin.
Sesudah disekresi App. Golgi, hidrolisis enzimatik
menyebabkan terlepasnya prokolagen membentuk
triple heliks kolagen.
Sel penghasil kolagen juga penghasil fibronektin.
Keduanya membentuk matriks periseluler.

Signal sequence on amino terminus enters first and continues

to elongate