TRANSKRIPSI Agustina Setiawati, M.Sc., Apt Replikasi Transkripsi Translasi Protein RNA DNA Agustina Setiawati, M.Sc., Apt

REFERENSI/PUSTAKA Albert, B., Bray, D., Lewis, J., Rarr, M., Roberts, K. and Watson, J. O., 1994, Molecular Biology of The Cell, 3rd Ed., Garland Publishing, Inc., New York. Lodish, H., Arnold, B., Zipursky, S. L., Matsudara, P., David, B. and Darnell, J. E., 2000, Molecular Cell Biology, W. H. Freeman and Company, London.

Pendahuluan DOGMA SENTRAL

TRANSKRIPSI TRANSKRIPSI = SINTESIS RNA (mRNA, tRNA, rRNA) Prekursor : ATP CTP GTP UTP Polimerase RNA Urutan RNA ditentukan DNA Salah satu untai DNA sebagai cetakan Untai RNA tumbuh dengan arah 5’-3’ Polimerase mulai sintesis tanpa primer

Complementary base pairing CCCTTTGGGAAA DNA hydrogen bonding GGGAAACCCUUU RNA TB 15

RNA polymerase gene,or operon complementary RNA DNA template TB 41

POLIMERASE RNA (465.000) Holoenzim (') Enzim inti (‘) Subunit Jumlah Berat mol α2 2 37000 β 1 151000 β’ 155000 α70 50000

Struktur RNA polymerase ' holoenzyme ' core enzyme TB 52

Langkah pengabungan holo enzim a a2 a2b a2bb’ = core enzyme aI b b’ aII CORE ENZYME Sequence-independent, nonspecific transcription initiation + vegetative (principal s) s70 heat shock (for emergencies) s32 nitrogen starvation (for emergencies) s54 SIGMA SUBUNIT interchangeable, promoter recognition

aI b b’ aII s70 RNAP HOLOENZYME -s70 Promoter-specific transcription initiation In the Holoenzyme: ' binds DNA template  binds NTPs  and  ' together make up the active site  subunits appear to be essential for assembly and for activation of enzyme by regulatory proteins. They also bind DNA. s recognizes promoter sequences on DNA

Promoters Bagian DNA dimana RNA polimerase menempel untuk memulai transkripsi upstream region downstream region transcribed region gene dsDNA promoter transcription start site termination site TB 43

Tahap-tahap transkripsi Inisiasi Elongasi Terminasi

Transkripsi pd prokariot - INISIASI Tdr atas 2 tahap : Holoenzim mengenal & menempel pd promoter spesifik Pbtk kompleks terbuka (bag untai ganda yg mengelokor) & proses inisiasi baru blgs. Penempelan RNA polimerase pd promoter spesifik yaitu untai 10 nukleotida pd sisi 5’ (rantai -10, dikenal sbg Pribnow Box yg berisi rantai TATAAT & rantai -35 adl TTGACA)

PROMOTER PROKARIOT & EUKARIOT

Elongasi Perpanjangan rantai RNA

PERPANJANGAN TRANSKRIPSI

TB TERMINASI TRANSKRIPSI Terminator : DNA region that mediates the termination of transcription. Bebas Rho-dependent  Protein Rho UUUU RNA termination site gene dsDNA region where terminators are usually found TB 46 20

RNA polymerase terikat pada loop TERMINASI bebas Terbentuknya rantai RNA dengan bentuk hair pin (stem dan loop) yang kaya akan G-C pada stem dan U pada loop. RNA polymerase terikat pada loop

TERMINASI tergantung faktor Memerlukan protein rho (faktor ρ) dg BM 46000 & bfgs mbtk heksamer yg menempel pd ss RNA sepanjang 72 pasangan basa (setiap subunit protein ρ akan menempel pada 12 nukleotida) Aktivitas ATPase protein ρ akan memungkinkan protein ρ bergerak sepanjang hibrid DNA-RNA dg jalan menarik RNA menjauhi kompleks transkripsi.

Monosistronik & polisistronik Molekul mRNA eukariot tdr atas rangkaian2 nukleotida yg dpt ditranslasi & tdk dapat ditranslasi. Polipeptida plg banyak hanya tdr atas 500 asam amino yg dpt ditranslasi, dikenal sbg mRNA monosistronik krn hanya mampu mbtk tdk lbh dr 1 mcm polipeptida. Molekul mRNA prokariosit dpt mentranskripsi lebih dr 1 macam polipeptida, dsbt mRNA polisistronik. Contoh : mRNA polisistronik E. coli pbtk triptofan dg pjg 7000 rangkaian basa dpt mbtk 5 mcm enzim.

Polisistronik

Monosistronik

Eukaryotic Transcription DNA Cytoplasm Nucleus Nuclear pores RNA Transcription G AAAAAA RNA Processing mRNA Export G AAAAAA

RNA polymerase di eukariot

Binding of the general transcription factors TFIID TBP TAF A B E H F The next series of figures shows the sequential building of the preinitiation complex. It starts with the binding of TBP to the TATA box. -25 +1 TFIID (a multisubunit protein) binds to the TATA box to begin the assembly of the transcription apparatus the TATA binding protein (TBP) directly binds the TATA box TBP associated factors (TAFs) bind to TBP TFIIA, TFIIB, TFIIE, TFIIF, TFIIH, TFIIJ assemble with TFIID

Binding of RNA polymerase II TBP TAF A B E H F The binding of the general transcription factors then promotes binding of RNA polymerase II. The TF's also recruit histone acetylase to the promoter to help destablize (relax) nucleosomes, which are present on the DNA (but not shown). RNA pol II RNA polymerase II (a multisubunit protein) binds to the promoter region by interacting with the TFII’s TFs recruit histone acetylase to the promoter

Binding of specialized TFs TBP TAF A B E H F J +1 Transcription factors bind their respective DNA elements and also interact with proteins at the promoter. All of this is to promote the formation of a stable preinitiation complex so that RNA polymerase II can catalyze the formation of the first phosphodiester bond. RNA pol II transcription factors binding to other promoter elements and transcription elements interact with proteins at the promoter and further stabilize (or inhibit) formation of a functional preinitiation complex this process is called “transactivation”

Formation of a stable preinitiation complex TBP TAF A B E H F J +1 RNA pol II Formation of the preinitiation complex involves DNA bending, since many transcription elements are at a considerable distance from the promoter. the stability and frequency with which complexes are formed determines the rate of initation of transcription the rate of initiation of transcription is of major importance in determining the abundance of an mRNA species

CTD Initiation of transcription and promoter clearance TBP TAF A B E H J +1 RNA pol II CTD P Once the preinitiation complex has formed, TFIIH phosphorylates RNA polymerase II on its carboxy terminal domain. This destabilizes the interaction between RNA polymerase II and the transcription factors, allowing it to move down the gene. P P RNA pol II is phosphorylated by TFIIH on the carboxy terminal domain (CTD), releasing it from the preinitiation complex and allowing it to initiate RNA synthesis and move down the gene

Transkripsi Eukariotik

A “Simple” Eukaryotic Gene Transcription Start Site 5’ Untranslated Region 3’ Untranslated Region Introns 3’ 5’ Exon 2 Exon 3 Int. 2 Exon 1 Int. 1 Promoter/ Control Region Exons Terminator Sequence RNA Transcript 3’ 5’ Exon 2 Exon 3 Exon 1 Int. 2 Int. 1

Processing Eukaryotic mRNA 3’ Untranslated Region 5’ Untranslated Region Protein Coding Region 3’ 5’ Exon 2 Exon 3 Int. 2 Exon 1 Int. 1 5’ G 5’ Cap 3’ AAAAA 3’ Poly A Tail Exon 2 Exon 3 Exon 1 Int. 2 Int. 1 Setelah selesai disintesis, mRNA mengalami proses-proses sebagai berikut : Capping pada ujung 5’ Tailing pada ujung 3’ Penghilangan Intron (Splicing)

mRNA processing

Self splicing

mRNA Capping Melindungi mRNA dari degradasi eksonuklease 5’-3 Inisiasi transkripsi

Structure of the 5’ cap Triphosphate linkage 2’ ribose methylations 7mG = 7-methyl guanosine Triphosphate linkage 2’ ribose methylations All eukaryotic mRNAs have a 5' cap, which is synthesized co-transcriptionally, that is, immediately following initiation of transcription. It contains a 7-methyl guanosine residue that is coupled to the 5' nucleotide through a 5'-5' triphosphate bond. In addition to the methyl group on the guanine base, there are two other methylations that are on the 2' ribose sugars of the first two nucleotides.

CPSF (cleavage/polyadenylation specificity factor) AAAAAAA…..

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