REGULATION

OF GENE
EXPRESSION
IN EUKARYOTE
Pertemuan ke 12
By. Puspa Julistia Puspita, S.Si, M.Sc
REGULASI EKSPRESI GEN pada EUKARIOT

Ekspresi gen eukariotik dapat dikontrol di enam tahap yang berbeda.

Contoh regulasi pada tiap tahap telah diketahui, tetapi hampir semua gen
memiliki situs kontrol/regulasi pada tahap 1, yaitu tahap transkripsi dari
sekuens DNA menjadi RNA.
REGULATION OF GENE EXPRESSION IN EUKARYOTE

- GENERAL EXPRESSION FACTOR

- OTHER EUKARYOTIC REGULATION OF
GENE EXPRESSION
 RNA Polymerase
 Transcription factors : a set proteins for initiate the transcription
 Basal Factors : bind to the promotor site
 Co-activators : the transcription factor for transmit signal from activator proteins to basal
factors
 Activators : the regulatory proteins that bind to enhancer to increase the transcription
 Enhancers : the binding site for activator
 Repressor : the regulatory proteins that bind to silencer to decrease the transcription
 Silencers : the binding site for repressor
 Promotor : the site for binding site of basal factors that contain the TATA box
sequences
Figure 1. Looping model of direct long-range enhancer action in control of transcriptional regulation. Enhancer sequences
located in cis to the gene coding region are bound by a number of enhancer binding transcription factors which make up an
activator complex. These then interact with a complex of basal transcription factors assembled at the promoter by a ‘looping
out’ of the intervening sequence. This interaction may occur directly or through a co-activator complex which transmits the
signal from one or more activator complexes to the basal transcription machinery to initiate transcription. Figure reproduced
from Donald Buckley.
 The promoter region of a gene attracts the enzyme RNA polymerase and correctly
orients the enzyme to begin its task of making an RNA copy of the gene.
 The promoters of both bacterial and eucaryotic genes include an initiation site,
where transcription actually begins, and a sequence of approximately 50
nucleotides that extends upstream from the initiation site (if one likens the
direction of transcription to the flow of a river). This region contains sites that are
required for the RNA polymerase to bind to the promoter. In addition to the
promoter, nearly all genes, whether bacterial or eucaryotic, have regulatory DNA
sequences that are used to switch the gene on or off.
 Some regulatory DNA sequences are as short as 10 nucleotide pairs
and act as simple gene switches that respond to a single signal. Such
simple switches predominate in bacteria.
 Other regulatory DNA sequences, especially those in eucaryotes, are
very long (sometimes more than 10,000 nucleotide pairs) and act as
molecular microprocessors, integrating information from a variety of
signals into a command that dictates how often transcription is
initiated.
 Regulatory DNA sequences do not work by themselves. To have any
effect, these sequences must be recognized by proteins called
transcription regulators, which bind to the DNA
 TRANSCRIPTIONAL REGULATION IN EUKARYOTE
● GENERAL TRANSCRIPTION FACTORS
TFIIF
Start of transcription TFIIE
RNA polymerase II
TFIIH
TFIID

Protein kinase (TFIIH) activity

TFIIB

Transcription begins
● OTHER/LONG DISTANCE TRANSCRIPTION FACTOR
Gene General transcription Gene
regulatory factors RNA regulatory
proteins polymerase II proteins
Gene x

TATA
Regulatory Spacer DNA Promotor
sequences
RNA transcript
The gene control region for gene x
 GENE REGULATION AT A DISTANCE: ENHANCER ELEMENT

DNA double helix

100 nucleotide pairs

500 nucleotide pairs

NtrC

Enhancer Promotor

Looped
activation
intermediate

Gene on
 GENE REGULATION AT A DISTANCE IN EUKARYOTE

● Trans acting element/protein

Strong activating
assembly Silent assembly of
regulatory proteins
Strongly
inhibiting
protein

Weakly RNA polymerase and general

activating transcription factors
protein
assembly

TATA
 LONG DISTANCE TRANSCRIPTION-REGULATION MECHANISMS

Activator Activation surface

Repressor
● Competitive
DNA binding
Binding site TATA
Binding site
for activator for repressor

● Masking the
activation surface TATA
Binding site Binding site
for activator Binding site
for repressor for activator

● Direct interaction
with the general
transcription factor
TATA
 GAL4-REGULATION

Acidic activation domain

GAL4 GAL4 DNA-
protein binding domain lacZ gene
DNA binding TATA Gene on
site for GAL4
RNA

GAL4 lexA-GAL4
chimeric protein
TFIID TFIIB

TATA Gene DNA-binding TATA Gene off

site for GAL4

DNA-binding TATA Gene on

site for lexA
RNA
Enhancer
• Enhancer memiliki panjang hingga 200 pasang basa dan
terdiri dari multiple binding sites untuk pengikatan
sequence-specific transcriptional activators.
• Enhancer sering dibedakan dari elemen promotor dengan
sifat yang unik, yaitu enhancer dapat dipindahkan dari satu
tempat ke tempat lainnya dalam sebuah molekul DNA atau
bahkan diubah (dirotasi hingga 180 derajat), tanpa
mempengaruhi kemampuan dari faktor transkripsi untuk
menstimulasi transkripsi.
• penghilangan enhancer dapat menurunkan level transkripsi
100 kali atau lebih.
Silencers bind repressor proteins that prevent the binding of
proteins to nearby enhancers, thereby down-regulating
expression, and like enhancers they can be located far from the
gene. It is also possible that silencers repress transcription directly
by binding transcriptional repressors that interact with the
promoter in the same way as enhancers (Gumucio et al. 1992) or
they may block access of enhancer activator complexes to the
promoter. Their proximity or even overlap with enhancers often
means the boundaries between these two types of elements can
be unclear, and a single functional module may act as both an
enhancer and a silencer depending on its cellular context (e.g.
Grice et al. 2005).
 The third type of regulatory region, an insulator, prevents
enhancers from inappropriately binding to and activating
the promoter of other genes in the same region of the
chromosome, as well as isolating independent
transcriptional regions from cross-reacting with
neighbouring regulatory elements (Brasset and Vauce,
2005).
 Transcriptional activator berikatan dengan sebuah enhancer mampu
untuk menstimulasi transkripsi pada daerah core promotor.
Transkripsi faktor ini dikenal sebagai co-activator.
 Co-activator dapat di bagi menjadi 2 fungsi; (1) co-activator yang
berinteraksi dengan basal transcription (faktor transkripsi dan RNA
polimerase), (2) Co-activator yang berinteraksi dengan chromatin,
mengubahnya dari keadaan yang inaccessible bagi transcription
machinery (faktor-faktor transkripsi) ke keadaan yang sangat
transcription ‘friendly” (dapat dilihat melalui video)
 (1) Co-activator yang berinteraksi dengan Basal
Transcription Factors
Co-activator yang berkomunikasi secara langsung antara
enhancer- transcription factor dan basal transcription disebut
mediator. Mediator merupakan multisubunit kompleks, besar
yang berinteraksi secara langsung dengan RNA polimerase II.
Mediator dibutuhkan oleh berbagai variasi transcriptional
activator dan juga sebagai sebuah element penting dalam
sebagian besar transkripsi, jika tidak sebagai gen penyandi
protein
 Transcription factors
 2. Co-activator yang mengganggu struktur
Kromatin
• DNA pada sel eukariot membungkus histon, yang kemudian
bersama sama membentuk nukleosom. Tiap inti nukleosom
terdiri atas suatu kompleks dari delapan protein histon yang
disebut juga histon oktamer, dengan DNA rantai ganda
dengan panjang 147 pasang nukleotida.
• Setiap molekul histone dari nukleosom mempunyai sebuah
ekor N-terminal flexible yang memanjang hingga keluar dari
pusat partikel histone melewati DNA helix.
• Modifikasi kovalen dari ekor ini berdampak pada struktur
kromatin dan fungsinya
• Modifikasi histon diantaranya yaitu metilasi, fosforilasi dan asetilasi
• Metilasi mengakibatkan terjadi pemadatan kromatin dan memadamkan
transkripsi. Sedangkan acetilasi atau penambahan gugus asetil ke
spesifik residu lisin pada “ core histone “ memiliki efek berkebalikan.
Acetilasi residu histon mencegah benang kromatin dari folding menjadi
struktur yang padat, sehingga daerah euchromatik menjadi tetap aktif.
• Acetilasi Histon meningkatkan akses dari daerah spesifik DNA cetakan
untuk menarik protein, sehingga mendorong aktivasi transkripsi
• Chromatin remodeling
 GENETIC EXPRESSION CONTROL
● RNA level (transcription) ● Protein level (translation)
● Initiation ● Autogenous control
● RNA control prokaryote
● Termination
● Codon usage
● mRNA-secondary structure eukaryote
● Phosphorylation
repressor/inducer gene structural sequence (x)
ds DNA of
Transcription-initiation control gene X
regulatory sequence (x)
negative control positive control

mRNA induser induser

active inactive inactive active
repressor repressor apoinducer apoinducer
Example: lactose operon Example: catabolite repression

korepresor
korepresor
inactive active active inactive
repressor repressor apoinducer apoinducer
Example: tryptophan operon Example:
 REGULATION BY CODON USAGE

● one aa coded by several codons  several type of tRNA for one aa

● different tRNA population of each codon
● high tRNA population of the use codon >>  translation >>  protein >>

REGULATION BY mRNA-SECONDARY STRUCTURE

AUG dengan loop  translasi <<

AUG tanpa loop  translasi >>

● loop (mRNA-secondary structure)  more difficult scanning by ribosome

● looped RNA more stable  stability >>  energy <<  translation <<
● most in eukaryote,  in prokaryote, translation begins directly before the end of
transcription  no mRNA-loop formation
 REGULATION BY PHOSPHORYLATION
Methionyl initiator tRNA mRNA 60S ribosomal subunit

Active
eIF-2
Protein
synthesis

40S ribosomal Inactive eIF-2

subunit Guanine nucleotide
releasing protein, eIF-2B

In the absence of
active eIF-2B, excess
eIF-2 remains in its eIF-2B
inactive, GDP-bound Protein kinase
form and protein phosphorylates eIF-2
synthesis slows
dramatically
Phosphorylated eIF-2 sequesters all eIF-2 as an inactive complex