Gen Struktur dan Ekspresinya

Apakah yang disebut sebagai Gen?

Suatu unit DNA dalam kromosom yang menyandi suatu RNA

Beberapa bentuk 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 pankreas

Gen:
Apapun fungsinya, semuanya tersusun atas daerah penyandi yang menghasilkan molekul RNA

Struktur khas gen mikroba prokaryot

tcatgtcctggggcttggttacggaggctatcaaaatattggaccnttcgtgccagcttaccagctctggaaaatactcagtatccgctatgtacgagatatccccaaggctagtttt taatctaaagcctatagttgttgggtcgctgtgctgggatggagttattatcatctcctcatttcctagcctaaacctgtccccaggattaggagcatggacttcctctaatgcctccaa gtggtacttgctcagggctggggtatgatcctcgtccccataaaccacgcttctagaacctattagggttcccctcttcttagtwaccccataggtcatcccytcaacgatcacytc ggcatcgttgcagtgatcggtgtgtctatgcgagatgaagagaacatctatcttcctggggtctagcttatatctaatcatcctaactagcgctccaggcccagggtccacaaagat atttttgcttgccttgatgaagaatccacccgtagatcttacttgagttatcgtcacgaacctgcccccaccggcacccaggaacgtaatctctatcatttttagtcccgaaattaaagt gcgaggcttatgcttttaaggatgtatggcgaaaggtgaagtttattagaagttagaatctaaagatttcagattgggtgggggtaatgataatcgatgctgattacataacggaaga tggcaagccgataataaggatattcaaaaaggaaaagggagagtttaaggtagaatacgataggacgtttagaccctacatttatgctcttttaaaggatgattcggccatagatga ggttaagaagataaccgccgagaggcacggaaagatagtcaggataaccgaggttgagaaagtccagaagaaattcctaggaaggccaatagaagtctggaagctctatcttga gcatccccaggatgttccagccataagagagaagataagggaacatccagctgtagttgatatatttgaatacgacataccctttgcgaagcgctacctcatagacaagggattga ctccaatggaggggaacgaggagctaacgtttctagccgttgatatagaaacattgtaccatgaaggagaggagttcgggaaagggccaataataatgatcagctacgccgacg aggaaggggccaaggtgataacttggaagagcatagacttaccttacgttgaagtggtttcgagcgagagggagatgataaagaggctcgtgaaggtaattagagagaaagatc ccgacgtgataataacgtacaatggtgataatttcgactttccgtacctcttaaagagggctgaaaagctcggaataaagctcccccttggaagggacaatagcgagccgaaaatg cagaggatgggggattcattagccgtagagataaagggcagaatacacttcgatttattccccgccataagaagaacgatcaaccttccaacatacaccctcgaaacggtttatga ggttatatttggaaagtctaaggagaaagtctatgcccatgagatagctgaggcctgggaaaccgggaaagggctagagagggtagctaagtattcaatggaagatgcgaaggt aacctctgagctcggaaaggagttcttcccgatggaagcccagctagcaggctcgttggccatccagtttgggacgtttcaaggtcgagcaccggaaacctcgttgagtggtttct ccttacgaaggcctacgagagaaatgagctcgcgcccaataaaccggacgagagggaatacgagagaaggctaagagagagctatgaagggggttacgttaacgagccaga gaagggattgtgggaagggatagtcagcttagactttaggtccctatatccctctataattataactcacaacgtctcaccagacactttgaatagagaaaattgcaaggaatatgac gttgccccccaagtggggcacagattctgcaaggatttcccaggattcataccaagcttactgggtaacctactggaggagagacaaaagataaaaaagaggatgaaagaaagt aaagatcccgtcgagaagaaactccttgattacagacagagagctataaaaatacttgcaaacagctattatggctattatggatatgcaaaggccagatggtactgtaaagagtgt gcagagagcgtaaccgcatggggaaggcagtacatagacctggttaggagggaacttgagagcagaggatttaaagttctctacatagacacagatggcctctacgcaacgatt cctggagccaagcatgaggaaataaaagagaaggcattgaagttcgtcgagtacataaactccaagttacctgggcttcttgaattggaatacgaaggtttctacgcgagagggtt cttcgtgacgaagaaaaagtacgcactaatcgacgaggaaggaaagatagttacgagggggctcgaaatagtaaggagagattggagtgaaatagcaaaggagacccaggcc aaggttctcgaggcaatactcaagcacggtaacgttgatgaggccgtaaaaatagtaaaggaggttacagaaaaactcagtaaatatgaaataccacccgaaaagcttgtaatttat gagcagataacgaggcctctgagcgagtataaagcgataggccctcacgttgcagtagctaaaaggctcgcagcgaagggagtaaaagttaagccagggatggttatcggttac atagttttgargggagacgggccaataagcaagagggccatagctatagaggagttcgatcccaaaaagcataagtacgatgccgaatactacatagagaaccaagttctgccag cggtggagaggatattgagagcatttggttatcgcaaagaagatttgaggtatcaaaaaactaaacaagtgggcctcggagcatggcttaagttctaga

Panjang ORF = 2991 nukleotida

Struktur khas gen eukaryot

>hg16_knownGene_BC009483 range=chr5:131895859-131902650 (IRF1 fragment) GTGCGCCCGAGCCCCGCCGAACCGAGGCCACCCGGAGCCGTGCCCAGTCC ACGCCGGCCGTGCCCGGCGGCCTTAAGAACCCGGCAACCTCTGCCTTCTT CCCTCTTCCACTCGGAGTCGCGCTCCGCGCGCCCTCACTGCAGCCCCTGC GTCGCCGGGACCCTCGCGCGCGACCGCCGAATCGCTCCTGCAGCAGAGgt gagtacgcctttgaggcgcggggcaccggcggcgtcgaataaaaggcgcg cggggcaccaggaagtggggggtcgaaagctccaggctggagactcgccg gcgcgcggcgttgcccgggcctccgcgcgggctccggggggcgccggagg agctgcgagccgcgggccgcggcgcggggagggcgggacgcggcgtggac cgcccacccggacgaggctgccggcgcccggcagctttcgcagatctgcg tgcgcgcagccgccaggggcctgtaggtggcccgctatgttcgtcccgcg catccacacgccgtgccggggaccgagtgtcagcccacgcgtgggcgccc agtgctcccggctttcggcggtcccagctccgcgcccaggcgacaggttt tgggctccctgtgctggtggcaagggctggcttactgcccaggtggctgg agggaatcgtgacctacggagactgcgggaagaggcgccacaggtgttcc ttgggccacttctccagaggaggggaaaccgggccggaagggttagcgtc ctggtcttagcgttgtgggcgctgtggctgtcaggaaggcgtagaatgga ttcaggggggcgggagggggctgttcagggtgacggctagccctttgcta gctagtggttacaactcaagtcaagggaatttcttcttggcatcaagcaa aagaagtccctcccttcccaaaggatttgaattttgagcgaaaagttctg aaattagggtatctgtgcattttgtctcttttcctgcatatgaatcctga agccatcacttgcatgcctgtctcctccagagactggctgggaggggctg aaggaaggggcaaaagcatttttgcctaagatgctgaaaaaatttggaga gcagttttattccagcgcagctcccctccgcactgagtgtagtacctagc agctggctgaggtgaggggagggtaactaagtgacctcgggtggggcagg tcactgcccaggtactgttcaacagattccagactggagcctctgtgttc tctttacagCCAACATGCCCATCACTCGGATGCGCATGAGACCCTGGCTA GAGATGCAGATTAATTCCAACCAAATCCCGGGGCTCATCTGGATTAATAA Agtgagtgtaactctttgggttttcctgccactgttttaacccatgtact tctggagggaccaaagcttcagatgcagctcaaaaagggaagtgataacg ggacaagcaggtgtttctcccagtgggtcctgcatgcagggagtgtgcac ggcccagcctgggcctcacttgcatgactcctgccttcttcccttcttga ggtagggcacccacctgaaggcacttccagtttccagcagcaagactttc cagcatctgcagagctggagttctgctctcctctaagcgagacccttaca aacatacacagcactctgcagggctccaatcgaacaaatagaagactgag aagtggatgctgctgggcagaaacgtgcctggcttagcagaggacaaacg agttaatcttgcaccagtcactctggcccaagaagcctatagctggtgca cttggggcaacatagaccctatagacttagtagcaatgatagtattcata

Ekson (Panjang rerata ~ 120 nucleotides)

Introns

Rata-rata, hanya 5% Dari hasil transkripsi yang menyandi protein.

Struktur Gen
Promoter region Shine-Dalgarno box (AGGAGG) Pribnow box (TATAAT) Prokaryot -35 site (TTGACA) Terminator 5 Initiation codon Promoter region TATA box CAAT box (in mammals) GC box (GGGCGGG) Exon 1 5 Intron 1 Initiation codon Intron 2 Intron 3 3

ORF

Stop codon

Eukaryot
Exon 2 Exon 3 Exon 4

Polyadenylation signal AATAA 3 Terminator

Stop codon

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

Sistem Operon pada jasad Prokaryot

Operon
Operon: Suatu potongan DNA yang mengandung dua atau lebih gen yang fungsinya terkait, dan transkripsinya dikendalikan oleh satu promoter

Prokaryotic promoters

1. 2. 3.

Promoter determines:
Which strand will serve as a template. Transcription starting point. 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

Promoter region RNA polymerase binding

-35 region RNA pol. recognition site

Pribnow-box TATA-box -10 region

Transcriptional Translational start site start site -1 +1 ~+40

5-TTGACA 16-18 TATAAT 6-8 NAC--NATG... 3 3-AACTGT 16-18 ATATTA 6-8 NTG--NTAC... 5

Consensus (average) seq. for the 35 region

pppAC--NAUG... 3 Met...
Begin of RNA transcript Begin of coding sequence

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 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:

Strong promoters look more like the consensus:

The recA promoter is a strong promoter:

TTGATA -- 16 -- TATAAT TTGACA -- 17 -- TATAAT

Weak promoters look less like the consensus:

The araBAD promoter is a weak promoter:

CTGACG -- 18 -- TACTGT TTGACA -- 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

Promoter region Shine-Dalgarno box (AGGAGG) Pribnow box (TATAAT) Prokaryote -35 site (TTGACA)

Terminator
5 Initiation codon 3

ORF

Stop codon

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
Promoter region Shine-Dalgarno box (AGGAGG) Pribnow box (TATAAT) Prokaryote -35 site (TTGACA) Terminator 5 Initiation codon 3

ORF

Stop codon

Ekson & Intron

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

Polyadenylation signal AATAA 3

Promoter region TATA box CAAT box (in mammals) Eukaryot GC box (GGGCGGG) Exon 1 Exon 2 Exon 3 5

Exon 4

Intron 1 Initiation codon

Intron 2

Intron 3
Stop codon

mRNAs (pro and eu)

Translation (procaryote)

RNA polymerase (procaryote)

Phage one polypeptide (11 ,000), 200 nucleotides /sec, E. coli Five polypeptides two a chains (mol. mass, 36,512) one b chain (150,619)) one b chain (155,162) one w chain (10,105) include two zinc atoms Additional factor, sigma (s) 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

Eukaryote

AUG 5 UTR

UAG

3 UTR

Green=ORF(open reading frame)

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 .