1. The document discusses gene expression which involves the transcription of DNA into RNA and the subsequent processing of RNA into mature messenger RNA, transfer RNA, or ribosomal RNA. 2. Transcription is the first step where RNA polymerase makes a complementary RNA copy of a DNA sequence. This RNA transcript then undergoes processing to become a functional mature RNA molecule. 3. The key stages of RNA processing discussed are 5' capping, polyadenylation at the 3' end, and splicing to remove introns and join exons.

1. Ekspresi Gen
1. Transkripsi
Drs. Sutarno, MSc., PhD.

3. 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.

4. Tahap-tahap utama dalam ekspresi gen-gen
pengkode protein.
The Central Dogma of Molecular Biology:

5. 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.

6.  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.

7. 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.

8. 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".

9.  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.

10. 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.

11. 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.

12.  (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.

13. Hubungan gen dan protein
 Hampir semua gen mengkodekan informasi
pembuatan protein.
 Sekuen basa nitrogen pada DNA mengkodekan sekuen
asam amino pada protein.

14. MAKING MESSENGER RNA: CALLED
TRANSCRIPTION

15. Ilustrasi menggambarkan transkripsi DNA ke RNA
sampai terbentuknya protein

16.  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.

17. 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.

18. 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.
Pertamahan panjang selalu terjadi pada arah 5' ke 3‘.
Nukleotida pertama pada ujung 5’ tetap dengan gugus
phosphatnya.

19. 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

20. 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".

21. A typical gene

22. 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.

23.  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

24. 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.

25. 3. Silencer
 Elemen yang sangat mirip dengan enhancer, kecuali
fungsinya yang mengikat protein dan menghambat
transkripsi.

26. 4. Response elements
 Adalah sisi pengenalan dari faktor transkripsi tertentu.
Umumnya terletak dalam 1kb dari transcriptional start
site.

27. TRANSKRIPSI

28. 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.

29. 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)

30. 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.

31. 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.

32.  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.

33. The procedure of RNA processing for protein genes.

34. 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.

35. 5’-capping, Modifications at the 5' end.

36. 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.

37. 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.

38. 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.

39. RNA Processing

40. 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

41. 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.

42. 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).

43. 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.

44. 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).

45. 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.

46.  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.

47. 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.

48. 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:

49. However, in an eukaryotic cell, the
transcription & translation occur in
different places:

50. 3. The Genetic Code

52. The Genetic Code uses three bases to
specify each amino acid

53. 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.

54. 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: