struktur gen dan ekspresinya
TRANSCRIPT
GenStruktur dan Ekspresinya
Suatu unit DNA dalam kromosom yang menyandi suatu RNA
Apakah yang disebut sebagai Gen?
Yang menghasilkan protein, tRNA atau rRNA, disebut sebagai gen struktural
Yang mengontrol kapan dan bagaimana suatu gen lain diekspresikan disebut sebagai gen regulator
Yang menghasilkan protein untuk kehidupan sel disebut sebagai housekeeping genes termasuk di dalamnya yang terlibat sintesis protein
Yang lain, gen spesiifik jaringan, yang hanya diekspresikan di sel atau jaringan tertentu, misalnya gen penyandi insulin hanya diekspresikan di sel β pankreas
Beberapa bentuk gen:
Apapun fungsinya, semuanya tersusun atas daerah penyandi yang menghasilkan molekul RNA
Gen:
Struktur khas gen mikroba prokaryottcatgtcctggggcttggttacggaggctatcaaaatattggaccnttcgtgccagcttaccagctctggaaaatactcagtatc
cgctatgtacgagatatccccaaggctagtttttaatctaaagcctatagttgttgggtcgctgtgctgggatggagttattatcatctcctcatttcctagcctaaacctgtccccaggattaggagcatggacttcctctaatgcctccaagtggtacttgctcagggctggggtatgatcctcgtccccataaaccacgcttctagaacctattagggttcccctcttcttagtwaccccataggtcatcccytcaacgatcacytcggcatcgttgcagtgatcggtgtgtctatgcgagatgaagagaacatctatcttcctggggtctagcttatatctaatcatcctaactagcgctccaggcccagggtccacaaagatatttttgcttgccttgatgaagaatccacccgtagatcttacttgagttatcgtcacgaacctgcccccaccggcacccaggaacgtaatctctatcatttttagtcccgaaattaaagtgcgaggcttatgcttttaaggatgtatggcgaaaggtgaagtttattagaagttagaatctaaagatttcagattgggtgggggtaatgataatcgatgctgattacataacggaagatggcaagccgataataaggatattcaaaaaggaaaagggagagtttaaggtagaatacgataggacgtttagaccctacatttatgctcttttaaaggatgattcggccatagatgaggttaagaagataaccgccgagaggcacggaaagatagtcaggataaccgaggttgagaaagtccagaagaaattcctaggaaggccaatagaagtctggaagctctatcttgagcatccccaggatgttccagccataagagagaagataagggaacatccagctgtagttgatatatttgaatacgacataccctttgcgaagcgctacctcatagacaagggattgactccaatggaggggaacgaggagctaacgtttctagccgttgatatagaaacattgtaccatgaaggagaggagttcgggaaagggccaataataatgatcagctacgccgacgaggaaggggccaaggtgataacttggaagagcatagacttaccttacgttgaagtggtttcgagcgagagggagatgataaagaggctcgtgaaggtaattagagagaaagatcccgacgtgataataacgtacaatggtgataatttcgactttccgtacctcttaaagagggctgaaaagctcggaataaagctcccccttggaagggacaatagcgagccgaaaatgcagaggatgggggattcattagccgtagagataaagggcagaatacacttcgatttattccccgccataagaagaacgatcaaccttccaacatacaccctcgaaacggtttatgaggttatatttggaaagtctaaggagaaagtctatgcccatgagatagctgaggcctgggaaaccgggaaagggctagagagggtagctaagtattcaatggaagatgcgaaggtaacctctgagctcggaaaggagttcttcccgatggaagcccagctagcaggctcgttggccatccagtttgggacgtttcaaggtcgagcaccggaaacctcgttgagtggtttctccttacgaaggcctacgagagaaatgagctcgcgcccaataaaccggacgagagggaatacgagagaaggctaagagagagctatgaagggggttacgttaacgagccagagaagggattgtgggaagggatagtcagcttagactttaggtccctatatccctctataattataactcacaacgtctcaccagacactttgaatagagaaaattgcaaggaatatgacgttgccccccaagtggggcacagattctgcaaggatttcccaggattcataccaagcttactgggtaacctactggaggagagacaaaagataaaaaagaggatgaaagaaagtaaagatcccgtcgagaagaaactccttgattacagacagagagctataaaaatacttgcaaacagctattatggctattatggatatgcaaaggccagatggtactgtaaagagtgtgcagagagcgtaaccgcatggggaaggcagtacatagacctggttaggagggaacttgagagcagaggatttaaagttctctacatagacacagatggcctctacgcaacgattcctggagccaagcatgaggaaataaaagagaaggcattgaagttcgtcgagtacataaactccaagttacctgggcttcttgaattggaatacgaaggtttctacgcgagagggttcttcgtgacgaagaaaaagtacgcactaatcgacgaggaaggaaagatagttacgagggggctcgaaatagtaaggagagattggagtgaaatagcaaaggagacccaggccaaggttctcgaggcaatactcaagcacggtaacgttgatgaggccgtaaaaatagtaaaggaggttacagaaaaactcagtaaatatgaaataccacccgaaaagcttgtaatttatgagcagataacgaggcctctgagcgagtataaagcgataggccctcacgttgcagtagctaaaaggctcgcagcgaagggagtaaaagttaagccagggatggttatcggttacatagttttgargggagacgggccaataagcaagagggccatagctatagaggagttcgatcccaaaaagcataagtacgatgccgaatactacatagagaaccaagttctgccagcggtggagaggatattgagagcatttggttatcgcaaagaagatttgaggtatcaaaaaactaaacaagtgggcctcggagcatggcttaagttctaga
Panjang ORF = 2991 nukleotida
Struktur khas gen eukaryot>hg16_knownGene_BC009483 range=chr5:131895859-131902650 (IRF1 fragment)
GTGCGCCCGAGCCCCGCCGAACCGAGGCCACCCGGAGCCGTGCCCAGTCCACGCCGGCCGTGCCCGGCGGCCTTAAGAACCCGGCAACCTCTGCCTTCTTCCCTCTTCCACTCGGAGTCGCGCTCCGCGCGCCCTCACTGCAGCCCCTGCGTCGCCGGGACCCTCGCGCGCGACCGCCGAATCGCTCCTGCAGCAGAGgtgagtacgcctttgaggcgcggggcaccggcggcgtcgaataaaaggcgcgcggggcaccaggaagtggggggtcgaaagctccaggctggagactcgccggcgcgcggcgttgcccgggcctccgcgcgggctccggggggcgccggaggagctgcgagccgcgggccgcggcgcggggagggcgggacgcggcgtggaccgcccacccggacgaggctgccggcgcccggcagctttcgcagatctgcgtgcgcgcagccgccaggggcctgtaggtggcccgctatgttcgtcccgcgcatccacacgccgtgccggggaccgagtgtcagcccacgcgtgggcgcccagtgctcccggctttcggcggtcccagctccgcgcccaggcgacaggttttgggctccctgtgctggtggcaagggctggcttactgcccaggtggctggagggaatcgtgacctacggagactgcgggaagaggcgccacaggtgttccttgggccacttctccagaggaggggaaaccgggccggaagggttagcgtcctggtcttagcgttgtgggcgctgtggctgtcaggaaggcgtagaatggattcaggggggcgggagggggctgttcagggtgacggctagccctttgctagctagtggttacaactcaagtcaagggaatttcttcttggcatcaagcaaaagaagtccctcccttcccaaaggatttgaattttgagcgaaaagttctgaaattagggtatctgtgcattttgtctcttttcctgcatatgaatcctgaagccatcacttgcatgcctgtctcctccagagactggctgggaggggctgaaggaaggggcaaaagcatttttgcctaagatgctgaaaaaatttggagagcagttttattccagcgcagctcccctccgcactgagtgtagtacctagcagctggctgaggtgaggggagggtaactaagtgacctcgggtggggcaggtcactgcccaggtactgttcaacagattccagactggagcctctgtgttctctttacagCCAACATGCCCATCACTCGGATGCGCATGAGACCCTGGCTAGAGATGCAGATTAATTCCAACCAAATCCCGGGGCTCATCTGGATTAATAAAgtgagtgtaactctttgggttttcctgccactgttttaacccatgtacttctggagggaccaaagcttcagatgcagctcaaaaagggaagtgataacgggacaagcaggtgtttctcccagtgggtcctgcatgcagggagtgtgcacggcccagcctgggcctcacttgcatgactcctgccttcttcccttcttgaggtagggcacccacctgaaggcacttccagtttccagcagcaagactttccagcatctgcagagctggagttctgctctcctctaagcgagacccttacaaacatacacagcactctgcagggctccaatcgaacaaatagaagactgagaagtggatgctgctgggcagaaacgtgcctggcttagcagaggacaaacgagttaatcttgcaccagtcactctggcccaagaagcctatagctggtgcacttggggcaacatagaccctatagacttagtagcaatgatagtattcata
Ekson(Panjang rerata ~ 120nucleotides)
Introns
Rata-rata, hanya 5%Dari hasil transkripsi yang menyandi protein.
Promoter regionPromoter region• TATA boxTATA box• CAAT box (in mammals)CAAT box (in mammals)• GC box (GGGCGGG)GC box (GGGCGGG)
Initiation codonInitiation codon Stop codonStop codon
PolyadenylationPolyadenylationsignalsignalAATAAAATAA
Exon 1Exon 1 Exon 2Exon 2 Exon 3Exon 3 Exon 4Exon 4
Intron 1Intron 1 Intron 2Intron 2 Intron 3Intron 35’5’ 3’3’
EukaryotEukaryot
Struktur Gen
Initiation codonInitiation codon Stop codonStop codon
Promoter regionPromoter region• Shine-Dalgarno box (AGGAGG)Shine-Dalgarno box (AGGAGG)• Pribnow box (TATAAT)Pribnow box (TATAAT)• -35 site (TTGACA)-35 site (TTGACA) ProkaryotProkaryot
5’5’ 3’3’
ORF
TerminatorTerminator
TerminatorTerminator
Struktur Gen
Promoter: urutan basa yang merupakan tempat bagi mesin-mesin sel untuk memulai transkripsi, biasanya terletak sebelum atau di dalam suatu gen
Terminator: urutan basa yang memberikan tanda bagi mesin-mesin sel untuk mengakhiri transkripsin, biasanya di akhir gen
Prokaryote
Start and stop
Operon
Operon: Suatu potongan DNA yang mengandung dua atau lebih gen yang fungsinya terkait, dan transkripsinya dikendalikan oleh satu promoter
Sistem Operon pada jasad Prokaryot
Prokaryotic promoters
Promoter determines:1. Which strand will serve as a template.2. Transcription starting point.3. Strength of polymerase binding.
RNA polymerase subunit for promoter recognition is called sigma-factor
Different variations (7 for E. coli) Consensus binding sequences (Table 6.2 in textbook)
The E. coli Promoter - consensus sequence Promoters sequences can vary tremendously. RNA polymerase recognizes hundreds of different
promoters
5’-TTGACA— 16-18 —TATAAT— 6-8 — NAC--NATG...– 3’3’-AACTGT— 16-18 —ATATTA— 6-8 — NTG--NTAC...– 5’
pppAC--NAUG...– 3’ Met...
Transcriptional start site
-1 +1
Translationalstart site
~+40
Pribnow-boxTATA-box-10 region
-35 region“RNA pol.
recognition site”
Begin of RNA transcript
Begin of coding sequence
Promoter regionRNA polymerase binding
Consensus (average) seq. for the – 35 region
Promoters strength Weak promoters: low degree of similarity with consensus TATA-box and the
-35 region, or spacing b/n them is not 17 nts transcribed about once every 10 min. in E. coli
Strong promoters: high degree of identity with TATA box & -35 region may be transcribed as high as every 2 sec. in E. coli Other factors
Strong promoters look more like the consensus:
The recA promoter is a strong promoter:
TTGATA -- 16 -- TATAATTTGACA -- 17 -- TATAAT
Weak promoters look less like the consensus:
The araBAD promoter is a weak promoter:
CTGACG -- 18 -- TACTGTTTGACA -- 17 -- TATAAT
Shine-Dalgrano box Suatu urutan nukleotida (konsensus = AGGAGG) yang berada
di daerah tak tertranslasi yang lebih awal dari suatu gen (5'-) penghasil mRNA jasad prokaryot.
Urutan basa Shine-Dalgarno berfungsi sebagai tempat berikatannya ribosom [Ribosome Binding Site (RBS) yang berikatan dengan ujung 3’ dari 16S rRNA (penyusun ribosom)]
Biasanya tersusun oleh 4-18 nukleotida yang merupakan bagian start codon suatu gen
Initiation codonInitiation codon Stop codonStop codon
Promoter regionPromoter region• Shine-Dalgarno box (AGGAGG)Shine-Dalgarno box (AGGAGG)• Pribnow box (TATAAT)Pribnow box (TATAAT)• -35 site (TTGACA)-35 site (TTGACA) ProkaryoteProkaryote
5’5’ 3’3’
ORF
TerminatorTerminator
Shine Dalgarno
Open Reading Frame (ORF) Suatu potongan DNA yang berpotensi
menyandi suatu protein Identifikasi suatu ORF adalah indikasi
pertama untuk mengetahui fungsi suatu potongan DNA
Initiation codonInitiation codon Stop codonStop codon
Promoter regionPromoter region• Shine-Dalgarno box (AGGAGG)Shine-Dalgarno box (AGGAGG)• Pribnow box (TATAAT)Pribnow box (TATAAT)• -35 site (TTGACA)-35 site (TTGACA) ProkaryoteProkaryote
5’5’ 3’3’
ORF
TerminatorTerminator
Ekson: urutan basa yang digunakan untuk menyandi protein
Intron: urutan basa yang tidak digunakan untuk menyandi protein dan terletak di antara ekson Intron akan dipotong/dibuang
Ekson & Intron
Promoter regionPromoter region• TATA boxTATA box• CAAT box (in mammals)CAAT box (in mammals)• GC box (GGGCGGG)GC box (GGGCGGG)
Initiation codonInitiation codon Stop codonStop codon
PolyadenylationPolyadenylationsignalsignalAATAAAATAA
Exon 1Exon 1 Exon 2Exon 2 Exon 3Exon 3 Exon 4Exon 4
Intron 1Intron 1 Intron 2Intron 2 Intron 3Intron 35’5’ 3’3’
EukaryotEukaryot
mRNAs (pro and eu)
Translation (procaryote)
RNA polymerase (procaryote) Phage –
one polypeptide (11 ,000), 200 nucleotides /sec,
E. coli – Five polypeptides two chains (mol. mass, 36,512) one chain (150,619)) one ’ chain (155,162) one chain (10,105) include two zinc atoms Additional factor, sigma (factor, (70,236) 40 nucleotides/ sec
The structure of a bacterial RNA polymerase
Subunits and sigma factor
Eukaryotic Gene Promoters Contain AT rich concensus sequence located –19 to –27 bp from transcription start (TATA box)
Site where RNA polymerase II binds
Possible distant regions acting as enhancers or silencers (even more than 50 kb).
More complex mechanism than prokaryotes
Eukaryote RNA polymerases 3 types of RNA polymerases are employed
in transcription of genes: RNA polymerase I transcribes rRNA RNA polymerase II transcribes all genes
coding for polypeptides RNA polymerase III transcribes small
cytoplasmatic RNA, such as tRNA.
Eukaryote gene structure vs. prokaryote gene structure
No operons Capping at 5’ end and polyadenylation at 3’ end
Transport of mRNA out of nucleus Effects stability and efficiency of translation
Introns Alternative splicing CpG islands around promoter regions
CpG tends to methylate and mutate Conservation implies function
Eukaryote
AUG … UAG 3’ UTR
5’ UTR
Green=ORF(open reading frame)
Eukaryote
5’ - CAP
Transcription termination
• Intrinsic (rho-independent) termination• Rho-dependent termination
Intrinsic (rho-independent) termination
Observed either in prokaryote or eukaryote
Rho-independent termination
Rho-dependent termination
Rho• 419 aa• Hexamer• ATPase activity• helicase
Rho-dependent termination
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