GENE TRANSCRIPTION

DNA RNA RNA: always single stranded Only 1 strand of DNA act as a template for transcription RNA synthesis: 5 3 Steps: Initiation, Elongation, Termination Required components: - Template ssDNA
RNA polymerase Promoter element Upstream DNA sequences, Enhancer element Transcription factors, activator

GENE TRANSCRIPTION IN PROKARYOTES

1 type of RNA polymerase in E.coli: holoenzyme (core enzyme+ subunit) Core enzyme: 2, , , () subunits

subunit: Initiation factor Promoter: 10 dan 35 region

GENE TRANSCRIPTION IN PROKARYOTES

Separation of double helix DNA (17 base pairs: ~2 helix turns) Open promoter complex Synthesis of RNA chain starts with pppA or pppG subunit dissociates after addition of the 10th nucleotide (position +10) Elongation: NusA protein DNA-RNA hybrid: ~ 12 nucleotides Termination: (rho) factor, oligo (dA-rU)

DNA

RNA

PROTEIN

GENE TRANSCRIPTION IN EUKARYOTES

RNA polymerase enzymes: monomer and 3 types:

RNA pol I: 18S, 5,8S dan 28S rRNA RNA pol II: mRNA dan UsnRNA except U6snRNA RNA pol III: tRNA, 5S rRNA, U6snRNA

DNA control element:

Promoter & Enhancer element

Transcription factor

DNA Control Elements

Eucaryotic Genes:
Letak dan jenis bervariasi tergantung enzim RNA pol.

RNA polimerase Genes:

TATA box (around 25)

Basal promoter element

Upstream promoter element Enhancer element

distance dependent

distance or orientation independent

PROMOTER ELEMENT
Contoh: Promotor gen SV40
Situs Inisiasi

Transcription Factor
Protein 2 Types: - DNA binding protein - Non DNA binding protein DNA binding protein as a transcription factor has 2 domains: - DNA binding domain - Transcription activation domain

TRANSCRIPTION FACTOR
Stimulation of Gene Transcription: helps RNA polymerase binding to promoter or affects reaction rate of transcription initiation catalyzed by RNA polymerase which bound to promoter

TRANSCRIPTION FACTOR

Identification of DNA-binding protein: - Mobility-shift assay - DNase-footprinting assay 4 structural types of transcription factor: - Helix-turn-helix (Homeodomain) - Zincfinger - Leucine zipper - Helix-loop-helix

Motif Helix-Turn-Helix

Motif Zinc-finger

Motif Leucine zipper

Motif Helix-Loop-Helix

A. Footprint Assay
Ekstrak Inti sel Fragmen restriksi atau Oligonukleotida

B. Band shift Assay

Fragmen restriksi atau Oligonukleotida Tanda Radioaktif Ekstrak Inti sel

Tanda Radioaktif

Tanpa Protein

Ikatan Sp1 pada Kotak GC

DNA Tanpa Tambahan protein Elektroforesis

Autoradiografi

Ikatan spesifik Dengan Sp1

Kompleks DNA-protein Fragmen DNA bebas

Terlindung dari pemotongan

Elektroforesis Autoradiografi

ENHANCER ELEMENT
Far from start point of transcription, at 5 region (upstream) Does not act as a promoter, but enhances the promoter function by interaction with DNA binding protein Only active in the specific cell type Regulated by several inducing signals