Ekspresi Gen

1. TranskripsiDrs. 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 polymerasesNukleotida 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. Promoters2. Enhancers3. Silencers4. 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: