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

See You …….