EKSPRESI GEN
SUSANTI
EVY SETYOWATI
RAHARJA KUNCARA
RNA
Ada 3 tipe RNA:
RNAd membawa kode genetik ke ribosom
RNAr komponen utama ribosom
RNAt membawa asam amino ke ribosom
Rantai RNAd.
Struktur RNAt.
tRNA
The genetic code is read during translation
via adapter molecules, tRNAs, that have 3base anticodons complementary to
codons in mRNA.
"Wobble" during reading of the mRNA
allows some tRNAs to read multiple codons
that differ only in the 3rd base.
There are 61 codons specifying 20 amino
acids. Minimally 31 tRNAs are required for
translation, not counting the tRNA that
codes for chain initiation. Mammalian cells
produce more than 150 tRNAs.
RNA structure:
Most RNA molecules have
secondary structure,
consisting of stem & loop
domains.
A
:
U
U
:
A
A
:
U
stem
C
:
G
C UG
C
:
U
G
U
C U
loop
anticodon loop
tRNA
acceptor
stem
anticodon loop
tRNA
acceptor
stem
The appropriate
amino acid is
attached to the
ribose of the
terminal adenosine
(A, in red) at the 3'
end.
The anticodon
loop is at the
anticodon
Phe
tRNA
acceptor
stem
#46
(m7G)
#22
G
Tertiary base
Phe
pairs in tRNA
#13
C
#46
(m7G)
#22
G
#13
C
Tertiary base
Phe
pairs in tRNA
anticodon
tRNA
Phe
acceptor
stem
H
C
O
C
O
O
Amino acid
NH3+
H
C
CH2
ATP
Adenine
OH
H
OH
O
O
NH2
Aminoacyl-AMP
CH2
O
H
Adenine
OH
H
OH
PPi
H
C
O
C
O
O
CH2
NH2
Aminoacyl-AMP
In step 2, the
2' or 3' OH of
the terminal
adenosine of
tRNA attacks
the amino acid
carbonyl C
atom.
OH
H
OH
tRNA
AMP
tRNA
Adenine
O
O
CH2
H
H
Adenine
H
H
OH
O
C
HC
NH3+
(terminal 3nucleotide
of appropriate tRNA)
Aminoacyl-tRNA
tRNA
O
O
(terminal 3nucleotide
of appropriate tRNA)
CH2
H
H
Adenine
H
H
OH
HC
NH3+
Aminoacyl-tRNA
Class I aaRSs:
Identity elements usually include residues of
the anticodon loop & acceptor stem.
Class I aaRSs aminoacylate the 2'-OH of
adenosine at their 3' end.
Class II aaRSs:
Identity elements for some Class II enzymes
do not include the anticodon domain.
Class II aaRSs tend to aminoacylate the 3'OH of adenosine at their 3' end.
Proofreading/quality control:
Some Aminoacyl-tRNA Synthetases are known to
have separate catalytic sites that release by
hydrolysis inappropriate amino acids that are
misacylated or mis-transferred to tRNA.
E.g., the aa-tRNA Synthetase for isoleucine (IleRS) a
small percentage of the time activates the closely
related amino acid valine to valine-AMP.
After valine is transferred to tRNAIle, to form ValtRNAIle, it is removed by hydrolysis at a separate
active site of IleRS that accommodates Val but not
the larger Ile.
In some bacteria, editing of some misacylated tRNAs
is carried out by separate proteins that may be
evolutionary precursors to editing domains of aatRNA Synthetases.
Structure of tRNAs. (a) The primary structure of yeast alanine tRNA (tRNAAla), the first such sequence determined. This
molecule is synthesized from the nucleotides A, C, G, and U, but some of the nucleotides, shown in red, are modified
after synthesis: D = dihydrouridine, I = inosine, T = thymine, Y = pseudouridine, and m = methyl group. Although the exact
sequence varies among tRNAs, they all fold into four base-paired stems and three loops. The partially unfolded molecule
is commonly depicted as a cloverleaf. Dihydrouridine is nearly always present in the D loop; likewise, thymidylate,
pseudouridylate, cytidylate, and guanylate are almost always present in the TYCG loop. The triplet at the tip of the
anticodon loop base-pairs with the corresponding codon in mRNA. Attachment of an amino acid to the acceptor arm
yields an aminoacyl-tRNA. (b) Computergenerated three-dimensional model of the generalized backbone of all tRNAs.
Note the L shape of the molecule
A tRNA molecule. In this series of diagrams, the same tRNA moleculein this case a tRNA specific for the amino acid
phenylalanine (Phe)is depicted in various ways. (A) The cloverleaf structure, a convention used to show the complementary
base-pairing (red lines) that creates the double-helical regions of the molecule. The anticodon is the sequence of three
nucleotides that base-pairs with a codon in mRNA. The amino acid matching the codon/anticodon pair is attached at the 3
end of the tRNA. tRNAs contain some unusual bases, which are produced by chemical modification after the tRNA has been
synthesized. For example, the bases denoted Y (for pseudouridine) and D (for dihydrouridine) are derived from uracil. (B and
C) Views of the actual L-shaped molecule, based on x-ray diffraction analysis. Although a particular tRNA, that for the amino
acid phenylalanine, is depicted, all other tRNAs have very similar structures. (D) The linear nucleotide sequence of the
molecule, color-coded to match A, B, and C.
Structure of a tRNA-splicing endonuclease docked to a precursor tRNA. The endonuclease (a foursubunit enzyme) removes the tRNA intron (blue). A second enzyme, a multifunctional tRNA ligase (not
shown), then joins the two tRNA halves together
Processing of an Escherichia coli pre-tRNA. The example shown results in synthesis of tRNAtyr. The tRNA sequence in the
primary transcript adopts its base-paired cloverleaf structure and two additional hairpin structures form, one on either side of the
tRNA. Processing begins with the cut by ribonuclease E or F forming a new 3 end just upstream of one of the hairpins.
Ribonuclease D, which is an exonuclease, trims seven nucleotides from this new 3 end and then pauses while ribonuclease P
makes a cut at the start of the cloverleaf, forming the 5 end of the mature mRNA. Ribonuclease D then removes two more
nucleotides, creating the 3 end of the mature molecule. With this tRNA the 3-terminal CCA sequence is present in the RNA and is
not removed by ribonuclease D. With some other tRNAs this sequence has to be completely or partly added by tRNA
nucleotidyltransferase. Abbreviation: RNase, ribonuclease
Processing of tyrosine pre-tRNA involves four types of changes. A 14-nucleotide intron (blue) in the
anticodon loop is removed by splicing. A 16-nucleotide sequence (green) at the 5 end is cleaved by RNase P. U
residues at the 3 end are replaced by the CCA sequence (red) found in all mature tRNAs. Numerous bases in
the stem-loops are converted to characteristic modified bases (yellow). Not all pre-tRNAs contain introns that are
spliced out during processing, but they all undergo the other types of changes shown here. D = dihydrouridine; Y
= pseudouridine.
Mechanism of splicing in pre-tRNA. First, the pre-tRNA is cleaved at two places, on each side of the intron, thereby excising the
intron. The cleavage mechanism generates a 2,3-cyclic phosphomonoester at the 3 end of the 5 exon. The multistep
reaction joining the two exons requires two nucleoside triphosphates: a GTP, which contributes the phosphate group (yellow) for the
3 5 linkage in the finished tRNA molecule; and an ATP, which forms an activated ligase-AMP intermediate. The 2phosphate on the 5 exon is removed in the final step.
The structure of the aminoacyl-tRNA linkage. The carboxyl end of the amino acid forms an ester bond to
ribose. Because the hydrolysis of this ester bond is associated with a large favorable change in free energy, an
amino acid held in this way is said to be activated. (A) Schematic drawing of the structure. The amino acid is
linked to the nucleotide at the 3 end of the tRNA (see Figure 6-52). (B) Actual structure corresponding to boxed
region in (A). These are two major classes of synthetase enzymes: one links the amino acid directly to the 3-OH
group of the ribose, and the other links it initially to the 2-OH group. In the latter case, a subsequent
transesterification reaction shifts the amino acid to the 3 position. As in Figure 6-56, the R-group indicates the
side chain of the amino acid.
Amino acid activation. The two-step process in which an amino acid (with its side chain denoted by R) is
activated for protein synthesis by an aminoacyl-tRNA synthetase enzyme is shown. As indicated, the energy of
ATP hydrolysis is used to attach each amino acid to its tRNA molecule in a high-energy linkage. The amino acid
is first activated through the linkage of its carboxyl group directly to an AMP moiety, forming an adenylated amino
acid; the linkage of the AMP, normally an unfavorable reaction, is driven by the hydrolysis of the ATP molecule
that donates the AMP. Without leaving the synthetase enzyme, the AMP-linked carboxyl group on the amino acid
is then transferred to a hydroxyl group on the sugar at the 3 end of the tRNA molecule. This transfer joins the
amino acid by an activated ester linkage to the tRNA and forms the final aminoacyl-tRNA molecule.
Hydrolytic editing. (A) tRNA synthetases remove their own coupling errors through
hydrolytic editing of incorrectly attached amino acids. As described in the text, the
correct amino acid is rejected by the editing site. (B) The error-correction process
performed by DNA polymerase shows some similarities; however, it differs so far as
the removal process depends strongly on a mispairing with the template
The promoters of 5S rRNA and tRNA genes are downstream of the transcrip-tion initiation site.
Transcription of the 5S rRNA gene is initiated by the binding of TFIIIA, followed by the binding of
TFIIIC, TFIIIB, and the polymerase. The tRNA promoters do not contain a binding site for TFIIIA, and
TFIIIA is not required for their transcription. Instead, TFIIIC initiates the transcription of tRNA genes
by binding to promoter sequences, followed by the association of TFIIIB and polymerase. The TATAbinding protein (TBP) is a subunit of TFIIIB .
Whole
Ribosome
70S
Small
Subunit
30S
16S RNA
21 proteins
Rat
cytoplasm
80S
40S
18S RNA
33 proteins
Large
Subunit
50S
23S & 5S
RNAs
31 proteins
60S
28S, 5.8S, &5S
RNAs
49 proteins
5S rRNA
crown view
displayed as
ribbons & sticks.
PDB 1FFK
tRNA
EF-G
small subunit
mRNA
location
Small
ribosomal
subunit of a
thermophilic
bacterium:
rRNA in
monochrome;
proteins in
varied colors.
spacefill display
PDB 1FJF
ribbons
PDB 1FFK
Large
Ribosome
Subunit
PDB 1FFK
Large
Ribosome
Subunit
Protein
synthesis takes
place in a cavity
within the
ribosome,
between small &
large subunits.
Nascent
polypeptides
emerge through a
tunnel in the
large subunit.
The tunnel lumen
is lined with rRNA
PDB 1FFK
Pengertiaan splicingRNA
Splicing
RNA
merupakan
pemotongan
intron
untuk
menggabungkan exon dalam peristiwa
transkripsi. Sebelum membahas lebih
jauh tentang splicing, saya akan
mengulang penjelasan tentang intron
dan exon pada gen makhluk hidup.
Bagaimana intron
dipotong?
INISIASI
Tahap inisiasi terjadi karena adanya tiga komponen yaitu mRNA, sebuah tRNA
yang memuat asam amino pertama dari polipeptida, dan dua sub unit
ribosom.
mRNA yang keluar dari nukleus menuju sitoplasma didatangi oleh ribosom,
kemudian mRNA masuk ke dalam celah ribosom. Ketika mRNA masuk ke
ribosom, ribosom membaca kodon yang masuk. Pembacaan dilakukan
untuk setiap 3 urutan basa hingga selesai seluruhnya. Sebagai catatan
ribosom yang datang untuk mebaca kodon biasanya tidak hanya satu,
melainkan beberapa ribosom yang dikenal sebagai polisom membentuk
rangkaian mirip tusuk satu, di mana tusuknya adalah mRNA dan daging
adalah ribosomnya. Dengan demikian, proses pembacaan kodon dapat
berlangsung secara berurutan. Ketika kodon I terbaca ribosom (misal
kodonnya AUG), tRNA yang membawa antikodon UAC dan asam amino
metionin datang. tRNA masuk ke celah ribosom.
Ribosom di sini berfungsi untuk memudahkan perlekatan yang spesifik antara
antikodon tRNA dengan kodon mRNA selama sintesis protein. Sub unit
ribosom dibangun oleh protein-protein dan molekul-molekul RNA ribosomal.
Elogasi
Pada tahap elongasi dari translasi, asam amino-asam amino ditambahkan satu per
satu pada asam amino pertama (metionin). Ribosom terus bergeser agar mRNA
lebih masuk, guna membaca kodon II. Misalnya kodon II UCA, yang segera
diterjemahkan oleh tRNA berarti kodon AGU sambil membawa asam amino serine.
Di dalam ribosom, metionin yang pertama kali masuk dirangkaikan dengan serine
membentuk dipeptida.
Ribosom terus bergeser, membaca kodon III. Misalkan kodon III GAG, segera
diterjemahkan oleh antikodon CUC sambil membawa asam amino glisin. tRNA
tersebut masuk ke ribosom. Asam amino glisin dirangkaikan dengan dipeptida
yang telah terbentuk sehingga membentuk tripeptida. Demikian seterusnya proses
pembacaan kode genetika itu berlangsung di dalam ribobom, yang diterjemahkan
ke dalam bentuk asam amino guna dirangkai menjadi polipeptida.
Kodon mRNA pada ribosom membentuk ikatan hidrogen dengan antikodon molekul
tRNA yang baru masuk yang membawa asam amino yang tepat. Molekul mRNA
yang telah melepaskan asam amino akan kembali ke sitoplasma untuk mengulangi
kembali pengangkutan asam amino. Molekul rRNA dari sub unit ribosom besar
berfungsi sebagai enzim, yaitu mengkatalisis pembentukan ikatan peptida yang
menggabungkan polipeptida yang memanjang ke asam amino yang baru tiba.
Terminasi
68
Ekspresi Gen
69
Animasi Translasi
70
72
promoter
operator
-35
-10
TTGACA
TATAAT (Pribnow box)
73
75
(allolactose)
(cant bind)
(lac repressor)
o
76
-gal
Glucose
&
Galactose
78
Repressor
complex
co-repressor (tryptophan)
Biosynthetic
Operons
(Trp Operon)
79
effector
Activator
inactive
complex
80
hepatocyte
skin
82
TFIID
84
Penerapan
Merekayasa promoter yang
berbeda di depan coding
sequences yang diinginkan, dapat
menghasilkan:
a. Regulasi yang berbeda
b. Meningkatkan laju ekspresi
c. Mengubah waktu ekspresi
d. Ekspresi terjadi di jaringan yang
berbeda
e. Ekspresi pada organisme yang
85
berbeda
Animasi Regulasi
Operon Lac
Operon
86
Signal Transduksi
87
signal transduction refers to any process by which a cell converts one kind of
signal or stimulus into another, most often involving ordered sequences of
biochemical reactions inside the cell, that are carried out by enzymes, activated
by second messengers resulting in what is thought of as a "signal trandusction
pathway". Such processes are usually rapid, lasting on the order of milliseconds
in the case of ion flux, minutes for the activation of protein and lipid mediated
kinase cascades, or hours and days in terms of gene expression. In many signal
transduction processes, the number of proteins and other molecules participating
in these events increases as the process emanates from the initial stimulus,
resulting in a "signal cascade" and often results in a relatively small stimulus
eliciting a large response. This is referred to as amplification of the signal.
In bacteria and other single-cell organisms, the variety of a signal transduction a
processes of which the cell is capable influences how many ways it can react and
respond to its environment. In multicellular organisms , a multitude of different
signal transduction processes are required for coordinating the behavior of
individual cells to support the function of the organism as a whole. As may be
expected, the more complex the organism, the more complex the repertoire of
signal transduction processes the organism must possess. Thus, sensing of both
the external and internal environment at the cellular level, relies on signal
transduction. Many disease processes such as diabetes, heart disease,
autoimmunity and cancer arise from defects in signal transduction pathways,
further highlighting the critical importance of signal transduction to biology as
well as medicine.
88