Differential Gene Expression

Transcription

1 Developmental Biology Biology 4361 Differential Gene Expression October 13, 2005

2 core transcription initiation site 5 promoter 3 TATAT +1 upstream downstream

3 Basal transcription factors (eukaryotes)

4 TFIID the TFIID complex binds to the TATA box through its TBP subunit TATA TFIIA +1 transcription initiation site TFIID is stabilized by TFIIA TFIIA TFIIB TFIIH TFIIB and TFIIH join the complex at the TATA box RNA polymerase II a complex of RNA pol II, TFIIE and TFIIF is positioned by TFIIB and its carboxyterminal domain is bound by TFIID TFIIE TFIIF carboxyterminal domain (CTD) CTD the CTD is phosphorylated by TFIIH and is released by TFIID; transcription begins RNA transcript

5 Enhancers cis acting regulatory elements bind specific transcription factors differ from promoters: 1) need a promoter to work 2) can work at a distance 3) can work in reverse orientation

6 Specific Transcription Factors Specific regulators activate repress multiple domains combinatorial competitive silencing

7 Transcription factor engrailed (After Pabo and Sauer 1992.) The homeodomain of the Engrailed protein binds to a particular site in the DNA. Helix 3 contacts the base pairs in the major groove, while the amino terminal portion of the homeodomain enters the minor groove. (After Pabo and Sauer 1992.)

8 Transcription factor families helix turn helix homeodomain zinc finger leucine zipper basic helix loop helix

9 estrogen receptor zinc finger domain

10 helix loop helix domain leucine zipper domain

11 homeodomain

12 Enhancers and transcription factors combinatorial transcription factors NOTE enhancers can act as repressors/silencers Figure 16.4

13 Figure 16.7 Transcription factor competition

14 Transcription factor regulation of gene expression activate/repress activate/repress competitive

15 Regulation of transcription factors Environmental activators heat shock genes metallothionein cytochrome p450s Inactive precursors phosphorylation/dephosphorylation Multimerization Transport from cytoplasm Ligand binding e.g. hormones

16 Transcription factor domains DNA binding activation dimerization homodimers heterodimers ligand binding allosteric control

17 Figure Transcription factor regulation steroid hormone receptors

18 Chromatin structure acetylation state of histones controls DNA binding Regulation: acetyltransferases deacetylases

19 Chromatin configuration John H. Frenster heterochromatin euchromatin

20 nrna processing promoter exon 1 intron 1 exon 2 intron 2 termination region 7 methyl Gppp primary transcript gene transcription, 5 capping cleavage signal AAUAA cap 7 methyl Gppp cleavage factor 3 cleavage, polyadenylation poly(a)polymerase AAAAAAA(n) 7 methyl Gppp splicing exon 1 exon 2 AAAAAAA(n) mature mrna, ready to be transported to the cytoplasm and translated

21 α Tropomyosin alternative splicing striated muscle striated muscle myoblast smooth muscle neuroblast/muscle hepatoma brain

22 Translational Control mrna half life determines translation number stability avoid digestion 5 cap, 3 UTR essential controls poly(a) tail short no translation/no digestion length = translation number

23 Methods In situ hybridization gene fusion reporter gene construction

24 in situ hybridization cdna production Figure 15.11

25 in situ hybridization single stranded cdna of known sequence fluorescence tag hybridize * radioactive tag FISH fluorescence in situ hybridization labeled hybrid DNA molecule autoradiography *

26 Reporter genes LacZ bacterial galactosidase Fig.6. Expression of Bicoid (A), Hunchback (B), zerknüllt (C) and transgenic Clogmia hunchback in Drosophila embryos (D). Clogmiahunchback regulatory sequence drives a lacz reporter gene on the dorsal side of the embryo.