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Regulation of eukaryotic genes Gene silencing Enhancers Activators Functional domains of activators Idea for another extra credit project Explore DNA binding domains of proteins. 1. Go to a web site with a Chime tutorial, e.g. GAL4 or Cro 2. Or use Kinemages 3. Write a roughly 2 page report on how a particular protein recognizes a DNA sequence States of eukaryotic genes • Inactive: – Closed chromatin – Open chromatin, but repressors or lack of activators keep frequency of initiation low. – Open chromatin, transcription has initiated, but polymerases will not elongate. • Active: – Open chromatin, basal transcription: requires TATA + Inr – Open chromatin, activated transcription: requires enhancer or upstream activator sequences Silent and open chromatin Transcription initiation and pausing Basal and activated transcription Silencing Mechanism Silencer • Cis-acting sequences that cause a decrease in gene expression • Similar to enhancer but has an opposite effect on gene expression • Gene repression - inactive chromatin structure (heterochromatin) • Examples – Telomeric silencing – a or genes - silent loci of mating type switching in yeast Silencer binding proteins • Silencer binding protein serve as anchors for expansion of repressed chromatin • Rap1 protein binds to silencer elements • SIR proteins (Silent Information Regulators) • Nucleates assembly of multi-protein complex – hypoacetylated N-terminal tails of histones H3 and H4 – methylated N-terminal tail of H3 (Lys 9) • Experiments: Condensed chromatin – Resistant to DNaseI digestion – Delete silencer - genes are derepressed Gene Silencing Silencing Mechanism Enhancers • Cis-acting sequences that cause an increase in expression of a gene • Act independently of position and orientation with respect to the gene. • Can act to: – Increase the rate of initiation at a promoter – Increase the fraction of cells in which a promoter is active SV40 Control region • Origin of replication • Promoter and upstream activator sequences for early transcription • Promoter for late transcription • Enhancer SV40 map Many regulatory DNA sequences in SV40 control region Stimulation of transcription by enhancer is independent of orientation and position SV40: Early Late wt T-Ag + pos T-Ag + orien T-Ag + Enhancer Enh- T-Ag - Enhancers also regulate cellular genes Enhancer contains multiple binding sites for transcriptional activators SV40: Early Late Enhancer T-Ag wt A C B high level deletion C B low level revertant C C B high level An enhanson Enhancers can occur in a variety of positions with respect to genes Enhancer Upstream Enhancer P Transcription unit Adjacent Downstream Internal Distal Ex1 Ex2 Activator proteins Modular nature of activator proteins • DNA binding domain: recognition and binding to specific DNA sequences • Multimerization domain: allows formation of homo- or hetero-multimers • Activation domain: – Needed for increase in expression of responding gene – Targets are still under investigation • General transcription factors • Histone modifying enzymes • Nucleosome remodeling complexes, etc Modular structure of GAL4 1 98 148 196 N DNA Activation binding Dimerization 768 881 C Activation GAL80 binding Induction by galactose exposes an activation surface • In the presence of galactose, GAL4 activates several genes whose products are required for galactose metabolism. • GAL4 binds to a DNA sequence called UASG. • In the absence of galactose, GAL80 blocks GAL4 activation. • Binding of the sugar causes GAL80 to move. • This exposes the activation domain of GAL4. Induction of GAL4 Domain swap experiments show the domains are interchangeable • Fuse an DNA-binding domain (DBD) from one transcription factor to the activation domain (AD) of a different one. – DBD from LexA (E. coli) – AD from GAL4 (yeast) • Now a target gene can be placed under control of the DNA binding site for the first factor – GAL1 gene with oLex (LexA binding sites) can be activated by the fusion protein. • Basis for 2-hybrid screen for any interacting proteins Domain swap experiments: Diagram 1 Domain swap experiments: Diagram 2 Two Hybrid Screens (Interaction Cloning), part 1 Two Hybrid Screens (Interaction Cloning), part 2