Ekspresi Gen

1. Transkripsi
Drs. Sutarno, MSc., PhD.
 Pendahuluan

 Suatu organisme mengandung berbagai tipe sel

somatik, yang masing-masing berbeda bentuk
maupun fungsinya. Namun demikian semua sel
ini memiliki genom yang sama
 Gen-gen di dalam genom ini tidak akan memiliki
pengaruh apa-apa, kecuali setelah
di’ekspresi’kan.
 Tipe sel yang berbeda mengekspresikan gen-
gen yang berbeda, dengan demikian
mememperlihatkan bentuk dan fungsi yang
bervariasi pula.
Tahap-tahap utama dalam ekspresi
gen-gen pengkode protein.

The Central Dogma of Molecular Biology:

Garis besar tentang ekspresi gen

"Gene expression“/ ekspresi gen berarti

pembentukan protein atau RNA fungsional
oleh gen pengkodenya.
Tahapannya:
1. Transcription/ transkripsi: suatu untai DNA
digunakan sebagai pencetak untuk
mensintesis suatu untai RNA, yang disebut
transkrip primer/ primary transcript.
2. RNA processing/ pemrosesan
RNA: modifikasi primary transcript untuk
menghasilkan RNA yang dewasa /mature
mRNA (untuk gen pengkode protein) atau
tRNA maupun rRNA fungsional.
 Untuk gen pengkode RNA, (tRNA dan rRNA),ekspresi
gen selesai setelah terbentuknya rRNA atau tRNA
yang fungsional.
 Namun demikian, protein gen memelukan beberapa
tahap tambahan:
 Nuclear transport/ transportasi keluar inti: mRNA
harus ditransportasikan keluar dr inti ke sitoplasma
untuk proses sintesis protein.
 Protein synthesis/ sintesis protein: di dalam
sitoplasma, mRNA berikatan dengan ribosom, yang
dapat melakukan sintesis polipeptida berdasarkan
sekuen pada mRNA.
 Transkripsi

 Transcripsi: adalah proses pengkopian DNA untuk

menghasilkan transkrip RNA komplemennya /
RNA transcript.
 Ini adalah merupakan tahap pertama dari proses
ekspresi dari setiap gen.
 RNA yang dihasilkan, apabila RNA ni pengkode
protein, akan mengalami splicing, poliadenilasi dan
transportasi ke sitoplasma.
 Setelah itu, melalui proses translasi akan
menghasilkan molekul protein yang diinginkan.
Catatan: uracil (U) pada RNA adalah
berpasangan dengan adenine (A) dari DNA.
 Untai DNA yang berperan sebagai pencetak/
template disebut: "template strand", "minus
strand", or "antisense strand".
 Sedangkan untai DNA yang lain disebut:
"non-template strand", "coding strand", "plus
strand", or "sense strand".
 Karena antara DNA coding strand dan
RNA strand adalah komplemen, mereka
memiliki sekuen yang sama kecuali T
pada DNA coding strand diganti dengan
U pada untai RNA.
Ilustrasi secara skematis proses transkripsi
(a) DNA sebelum transkripsi
(b) selama transkripsi, DNA membukasehingga
salah satu untai DNAnya dapat digunakan sebagai
template (pencetak) untuk mensintesis untai RNA
yang komplemen.
 Tahap-tahap utama proses transkripsi

 (i) Terjadinya ikatan antara enzim polimerase pada

situs inisiasi. Sekuen DNA yang menjadi penanda
inisiasi/ dimulainya transkripsi disebut promoter.

 (ii) Unwinding of the DNA double helix (pilinan double

heliks membuka). Enzim yang dapat embuka double
helix disebut helicase. Polymerases pada prokaryot
memiliki aktivitas sebagai helicase, sedangkan
polimerase pada eukaryot tidak memiliki aktivitas ini.
Membukanya DNA pada eukaryot dilakukan oleh
faktor transkripsi spesifik.
 (iii) Synthesis of RNA. RNA polimerases
menggunakan nucleoside triphosphates (NTPs)
untuk menyusun suatu untai RNA berdasarkan
sekuen pada DNA template.
 (iv) Termination. Antara Prokaryot dan eukaryot
terdapat perbedaan signal untuk terminasi
transkripsi ini:
 Transkripsi pada eukaryot lebih kompleks
dibandingkan pada prokaryot, salah satu
penyebabnya karena adanya histon pada
eukaryot yang dapat menghalangi akses
polimeras ke promoter.
 Hubungan gen dan protein

 Hampir semua gen mengkodekan informasi

pembuatan protein.
 Sekuen basa nitrogen pada DNA mengkodekan sekuen
asam amino pada protein.
MAKING MESSENGER RNA: CALLED
TRANSCRIPTION
Ilustrasi menggambarkan transkripsi DNA ke RNA
sampai terbentuknya protein
 DNA codes for the production of RNA.
 RNA codes for the production of
protein.
 Protein does not code for the
production of protein, RNA or DNA.
 Fungsi RNA polimerase
 Baik RNA- maupun
DNA-polymerase
dapat
menambahkan
nukleotida ke untai
yaang telah ada
untuk menjadikan
tambah panjang.
Perbedaanya:
RNA polimerase
dapat memulai
suatu untai baru,
tetapi DNA
polimerase tidak
dapat.
 The function of RNA polymerases
Nukleotida yang digunakan untuk memperpanjang untai RNA
yang sedang tumbuh adalah ribonucleoside triphosphates
(NTPs). Dua gugus phosphat dibebaskan sebagai
pyrophosphate (PPi) selama reaksi.
Pertambahan panjang selalu terjadi pada arah 5' ke 3‘.
Nukleotida pertama pada ujung 5’ tetap dengan gugus
phosphatnya.
 Elemen-elemen regulator gen

 Pengaturan transkripsi di mediasi oleh

interaksi antara faktor-faktor transkripsi dan
DNA binding sitenya. Terdapat empat macam
elemen ini:
1. Promoters
2. Enhancers
3. Silencers
4. Response elements
Gene organization. The transcription region consists of exons and
introns. The regulatory elements include promoter, response element,
enhancer and silencer (not shown). Downstream refers to the direction
of transcription, and upstream is opposite to the transcription
direction. The number increases along the direction of transcription,
with "+1" assigned for the initiation site. There is no "0" position. The
base pair just upstream of +1 is numbered "-1", not "0".
A typical gene
 1. Promoter
 Promoter adalah suatu sekuen DNA tempat dimana
proses transkripsi dimulai. Pada prokaryote,
sekuen dari suatu promoter dikenali oleh faktor
sigma (s) dari RNA polymerase. Pada eukaryote,
promoter dikenali oleh faktor transkripsi khusus
(specific transcription factors).
 Pada E. col memiliki 5 faktor sigma:
 Sigma 70: mengatur ekspresi hampir semua
gene.
 Sigma 32: mengatur ekspresi protein-protein
heat shock.
 Sigma 28: mengatur ekspresi operon flagellar
(terlibat dalam gerak sel).
 Sigma 38: mengatur ekspresi gen untuk
melawan stres eksternal.
 Sigma 54: mengatur ekspresi gen untuk
metabolisme nitrogen.
 Pada Eukaryot
 Terdapat perbedaan signifikan antara transkripsi gen
protein dan gen RNA.
 Elemn promotor paling umum pada gen protein eukaryot
adalah TATA box, yang terletak pada -35 sampai -
20. Promoter yang lain disebut initiator (Inr). Terdapat
sekuen konsensus pada initiator ini, yaitu:
PyPyAN(T/A)PyPy, dimana Py adalah pyrimidine (C atau T),
N = apa saja, dan (T/A) berarti T atau A. Basa nitrogen A
pada posisi ke tiga terletak pada +1 (the transcriptional start
site).
 TATA box dan initiator adalah merupakan elemen promoter
utama. Terdapat elemen-elemen lain yang sering terletak
dalam 200 bp dari transcriptional start site, misalnya CAAT
box dan GC box yang sering disebut sebagai elemen
promoter-proximal.
 Protein yang berinteraksi dengan initiator dan TATA box
dikenal dengan TATA-box binding protein (TBP), karena
TATA box ditemukan lebih awal dibanding initiator
 2. Enhancers
 Enhancer: adalah sekuen nukleotida tempat faktor
transkripsi berikatan, dan yang menyebabkan transkripsi
dari gen menjadi meningkat.
 Enhancer adalah elemen pengatur positif yang terletak
baik diarah upstream atau downstream dari
transcriptional initiation site. Namun demikian, umumnya
terletak upstream.
 Pada prokaryot, enhancer terletak sangat dekat dengan
promoter, tetapi pada eukaryot, enhancer jadi jauh
promoter.
 Suatu daerah enhancer dapat mengandung satu atau
lebih element yang dikenali oleh aktivator transkripsi.
 Enhancers bersifat "conditional" atau dapat dikatakan
bahwa enhancer ini meningkatkan transkripsi hanya
dalam kondisi tertentu, seperti misalnya ketika ada
hormon.
 3. Silencer

 Elemen yang sangat mirip dengan

enhancer, kecuali fungsinya yang
mengikat protein dan menghambat
transkripsi.
 4. Response elements

 Adalah sisi pengenalan dari faktor transkripsi

tertentu. Umumnya terletak dalam 1kb dari
transcriptional start site.
TRANSKRIPSI
 1. Inisiasi proses Transkripsi

 RNA polymerase dapat mengenali sisi awal dari

suatu gen, dengan demikian enzim ini
mengetahui dimana harus memulai mensintesis
mRNA.
 Daerah awal pengenalan berupa sekuen DNA
khusus yang berada pada sekuen awal suatu
gen yang disebut dengan promoter.
 Ini mrpkn suatu sekuen unidirectional (satu
arah) pada satu strand DNA yang memberitahu
RNA polymerase tempat mulai serta arah (pada
strand mana) sintesis.
2. Elongation (pemanjangan) Transkripsi

 RNA polymerase kemudian menambahkan

nukleotida untuk memperpanjang rantai mRNA
yang komplemen dengan strand DNA.
 RNA polymerase menempatkan rNTPs
(ribonucleic nucleotides triphosphates) dengan
cara yang sama seperti yang dilakukan DNA
polymerase dalam mengambi dan menempatkan
dNTPs. Namun demikian, karena sintesis ini
hanya berlangsung dalam untai tunggal dan
hanya berlangsung dalam arah 5' ke 3‘, maka
tidak perlu adanya fragmen Okazaki.
 Penting untuk diketahui bahwa sintesis RNA ini
berlangsung dalam satu arah (unidirectional)
3. Termination (pemberhentian) Transkripsi

 Bagaimana RNA polymerase mengetahui tempat

berhentinya?
 Sistem ini didasarkan pada sistem pada prokaryot.
Berhubung tidak ada inti pada prokaryot, ribosom can
dapat mulai mensintesis protein berdasarkan mRNA
segera setelah mRNA disintesis. Pada ujung akhir dari
suatu gen, sekuen mRNA membentuk suatu loop yang
memblock ribosom, sehingga ribosom kemudian
terlepas dr mRNA, dan inilah signal terminasi yang
dikenali oleh RNA polymerase. Segera setelah ribosom
lepas dari mRNA, RNA polymerase lepas dari DNA dan
proses transkripsi terhenti.
 RNA Processing

 RNA Processing: pre-mRNA --> mRNA

 Semua transkrip primer yang dihasilkan di
dalam nukleus, harus mengalami taham
pemrosesan untuk menghasilkan molekul RNA
yang fungsional untuk dikeluarkan ke
sitoplasma.
 RNA processing merupakan proses untuk
menghasilkan RNA yang dewasa (mature mRNA) bagi
gen protein, atau tRNA / rRNA fungsional dari primary
transcript.
 Pemrosesan pre-mRNA meliputi tahap-tahap:
 Capping – penambahan 7-methylguanylate (m7G) ke
ujung 5’
 Polyadenylation - penambahan poly-A ke ujung 3‘.

 Splicing – pembuangan intron dan menggabungkan/

menyambungkan exon.
The procedure of RNA processing for protein genes.
 5'-Capping
 Cap site: Two usages: In eukaryotes, the cap site is the position
in the gene at which transcription starts, and really should be
called the "transcription initiation site". The first nucleotide is
transcribed from this site to start the nascent RNA chain. That
nucleotide becomes the 5' end of the chain, and thus the
nucleotide to which the cap structure is attached (see "Cap"). In
bacteria, the CAP site (note the capital letters) is a site on the
DNA to which a protein factor (the Catabolite Activated Protein)
binds.
 Capping occurs shortly after transcription begins. The
chemical structure of the "cap" is shown in the following figure,
where m7G is linked to the first nucleotide by a special 5'-5'
triphosphate linkage. In most organisms, the first nucleotide is
methylated at the 2'-hydroxyl of the ribose. In vertebrates, the
second nucleotide is also methylated.
5’-capping, Modifications at the 5' end.
 3'-Polyadenylation
 A stretch of adenylate residues are added to the 3' end. The poly-A
tail contains ~ 250 A residues in mammals, and ~ 100 in yeasts.
Polyadenylation at the 3' end. The major signal for the 3' cleavage is
the sequence AAUAAA. Cleavage occurs at 10-35 nucleotides
downstream from the specific sequence. A second signal is
located about 50 nucleotides downstream from the cleavage
site. This signal is a GU-rich or U-rich region.
 RNA splicing
 RNA splicing is a process that removes introns
and joins exons in a primary transcript. An
intron usually contains a clear signal for
splicing (e.g., the beta globin gene). In some
cases (e.g., the sex lethal gene of fruit fly), a
splicing signal may be masked by a regulatory
protein, resulting in alternative splicing. In rare
cases (e.g., HIV genes), a pre-mRNA may
contain several ambiguous splicing signals,
resulting in a few alternatively spliced mRNAs.
 Splicing signal
 Most introns start from the sequence GU and end with
the sequence AG (in the 5' to 3' direction). They are
referred to as the splice donor and splice acceptor
site, respectively. However, the sequences at the two
sites are not sufficient to signal the presence of an
intron. Another important sequence is called the
branch site located 20 - 50 bases upstream of the
acceptor site. The consensus sequence of the
branch site is "CU(A/G)A(C/U)", where A is conserved
in all genes.
 In over 60% of cases, the exon sequence is (A/C)AG at
the donor site, and G at the acceptor site.
 Figure 5-A-4. The consensus sequence for
splicing. Pu = A or G; Py = C or U.
 Splicing mechanism
 The detailed splicing mechanism is quite complex. In short, it
involves five snRNAs and their associated proteins. These
ribonucleoproteins form a large (60S) complex, called
spliceosome. Then, after a two-step enzymatic reaction, the intron
is removed and two neighboring exons are joined together. The
branch point A residue plays a critical role in the enzymatic reaction.

•Schematic drawing for the formation of the spliceosome during RNA

splicing. U1, U2, U4, U5 and U6 denote snRNAs and their associated
proteins. The U3 snRNA is not involved in the RNA splicing, but is involved
in the processing of pre-rRNA.
RNA Processing
 Summary of the steps
 several protein transcription factors bind to promoter sites,
usually on the 5' side of the gene to be transcribed
 RNA polymerase, binds to the complex of transcription factors ,
working together, they open the DNA double helix
 RNA polymerase proceeds down one strand moving in the 3' ->
5' direction as it does so, it assembles ribonucleotides (supplied
as triphosphates, e.g., ATP) into a strand of RNA
 each ribonucleotide is inserted into the growing RNA strand
following the rules of base pairing. Thus for each C encountered
on the DNA strand, a G is inserted in the RNA; for each G, a C;
and for each T, an A. However, each A on the DNA guides the
insertion of the pyrimidine uracil (U, from uridine triphosphate,
UTP). There is no T in RNA.
 synthesis of the RNA proceeds in the 5' -> 3' direction.
 as each nucleoside triphosphate is brought in to add to the 3'
end of the growing strand, the two terminal phosphates are
removed
 Types of RNA
 Several types of RNA are synthesized:
 messenger RNA (mRNA). This will later be
translated into a polypeptide.
 ribosomal RNA (rRNA). This will be used in the
building of ribosomes: machinery for synthesizing
proteins by translating mRNA.
 transfer RNA (tRNA). RNA molecules that carry
amino acids to the growing polypeptide.
 small nuclear RNA (snRNA). DNA transcription of
the genes for mRNA, rRNA, and tRNA produces
large precursor molecules ("primary transcripts")
that must be processed within the nucleus to
produce the functional molecules for export to the
cytosol. Some of these processing steps are
mediated by snRNAs.
 Types of RNA
 Ribosomal RNA (rRNA)
 There are 4 kinds. In eukaryotes, these are
 18S rRNA. One of these molecules, along
with some 30 different protein molecules, is
used to make the small subunit of the
ribosome.
 28S, 5.8S, and 5S rRNA. One each of these
molecules, along with some 45 different
proteins, are used to make the large subunit
of the ribosome.
 The name given each type of rRNA reflects
the rate at which the molecules sediment in
the ultracentrifuge. The larger the number, the
larger the molecule (but not proportionally).
 Types of RNA
 Transfer RNA (tRNA)
 There are some 32 different kinds of tRNA in a
typical eukaryotic cell.
 each is the product of a separate gene
 they are small (~4S), containing 73-93 nucleotides
 many of the bases in the chain pair with each other
forming sections of double helix
 the unpaired regions form 3 loops
 each kind of tRNA carries (at its 3' end) one of the
20 amino acids (thus most amino acids have more
than one tRNA responsible for them)
 at one loop, 3 unpaired bases form an anticodon
 base pairing between the anticodon and the
complementary codon on a mRNA molecule brings
the correct amino acid into the growing
polypeptide chain.
 Types of RNA

 Messenger RNA (mRNA)

 Messenger RNA comes in a wide range of sizes
reflecting the size of the polypeptide it encodes.
Most cells produce small amounts of thousands
of different mRNA molecules, each to be
translated into a peptide needed by the cell.
 Many mRNAs are common to most cells,
encoding "housekeeping" proteins needed by
all cells (e.g. the enzymes of glycolysis). Other
mRNAs are specific for only certain types of
cells. These encode proteins needed for the
function of that particular cell (e.g., the mRNA
for hemoglobin in the precursors of red blood
cells).
 Types of RNA

 Small Nuclear RNA (snRNA)

 Approximately a dozen different genes for
snRNAs, each present in multiple copies, have
been identified.
 The snRNAs have various roles in the
processing of the other classes of RNA. For
example, several snRNAs are part of the
spliceosome that participates in converting pre-
mRNA into mRNA by excising the introns and
splicing the exons.
 The RNA polymerases
 The RNA polymerases are huge multi-subunit
protein complexes. Three kinds are found in
eukaryotes.
 RNA polymerase I (Pol I). It transcribes the rRNA
genes for the precursor of the 28S, 18S, and
5.8S molecules. (and is the busiest of the RNA
polymerases)
 RNA polymerase II (Pol II). It transcribes the
mRNA and snRNA genes.
 RNA polymerase III (Pol III). It transcribes the 5S
rRNA genes and all the tRNA genes.
However, the "Central Dogma" has had to be
revised a bit. It turns out that you CAN go back
from RNA to DNA, and that RNA can also make
copies of itself. It is still not possible to go from
Proteins back to RNA or DNA, and no known
mechanism has yet been demonstrated for
proteins making copies of themselves.
2. Synthesizing Proteins from the
Instructions of DNA

 Genetic information flows in a cell from:

 DNA ->RNA-> Protein
 In a prokaryotic cell, this process happens
at the same time:
However, in an eukaryotic cell, the
transcription & translation occur in
different places:
3. The Genetic Code
The Genetic Code uses three bases to
specify each amino acid
 4. RNA: Intermediary in Protein
Synthesis
 Why would the cell want to have an intermediate
between DNA and the proteins it encodes?
 · The DNA can then stay pristine and protected, away
from the caustic chemistry of the cytoplasm.

 · Gene information can be amplified by having many

copies of an RNA made from one copy of DNA.

 · Regulation of gene expression can be effected by

having specific controls at each element of the
pathway between DNA and proteins. The more
elements there are in the pathway, the more
opportunities there are to control it in different
circumstances.
 What is RNA?
 RNA has the same primary structure
as DNA. It consists of a sugar-
phosphate backbone, with
nucleotides attaches to the 1' carbon
of the sugar. The differences
between DNA and RNA are that:

 1. RNA has a hydroxyl group on the

2' carbon of the sugar (thus, the
difference between deoxyribonucleic
acid and ribonucleic acid).

2. Instead of using the nucleotide
thymine, RNA uses another
nucleotide called uracil:
In addition, because the RNA molecule
is not restricted to a rigid double
helix, it can form many different
tertiary structures. Each RNA
molecule, depending on the sequence
of its bases, can fold into a stable
three-dimensional structure.
 The Genetic Code
 How does an mRNA specify amino acid
sequence? The answer lies in the genetic
code. It would be impossible for each
amino aciud to be specified by one
nucleotide, because there are only 4
nucleotides and 20 amino acids. Similarly,
two nucleotide combinations could only
specify 16 amino acids. The final
conclusion is that each amino acid is
specified by a particular combination of
three nucleotides, called a codon: