Welcome to Class 21! Introductory Biochemistry! Lecture 21: Outline and Objectives l Regulation of Gene Expression in Prokaryotes! l transcriptional regulation! l principles! l lac operon! l trp attenuation! l Regulation of Gene Expression in Eukaryotes! l transcriptional regulation! l transactivators & co-activators! l coordinate regulation! l post-transcriptional regulation! l alternative splicing! 1!
Information Pathways! Now,! Regulation!! Gene Expression! in Prokaryotes and Eukaryotes! 2!
Regulation of Transcription! Constitutive! constant, unregulated transcription! Induction! increased transcription! Repression! decreased transcription! RNA Polymerase Binds to DNA at Promoters! Some E. coli promoter sequences:! Figure 26-5! Strong promoters match the consensus well! Weak promoters diverge from consensus! 3!
Promoter Specificity! can be regulated:! σ subunit mediates! promoter recognition! E. coli RNA Polymerase! α2ββʹ ω! catalytic core! σ! σ! σ70 recognizes most E. coli promoters:! Figure 28-2! σ32 recognizes heat shock promoters:! Figure 28-3! Promoter Specificity! can be regulated:! σ subunit mediates! promoter recognition! E. coli RNA Polymerase! α2ββʹ ω! catalytic core! σ! σ! 4!
Affinity of RNA Polymerase is also Regulated! by Binding of Proteins to or near Promoters! Repressors: impede access of RNA polymerase! Activators: enhance access of RNA polymerase! Figure 28-2! Negative Regulation! of Transcriptional Initiation! Figure 28-4! 5!
Positive Regulation! of Transcriptional Initiation! Figure 28-4! Prokaryotic Genes are Regulated! in Units called Operons! Figure 28-6! 6!
The lac Operon:! Encodes genes required for use of lactose as a carbon source! A=beta-galactoside transacetylase! Figure 28-7,8! The lac Operon is Negatively Regulated! by lac Repressor! repressor binds! in the absence! of allolactose! Transcription is Repressed! lac I = Repressor gene! Figure 28-7, 5 th ed! Operator = Repressor binding site! 7!
The lac Operon is Negatively Regulated! by lac Repressor! repressor binds! DNA in the! absence of! inducer! Transcription! is Repressed! allolactose! repressor does not! bind DNA in the! presence of inducer! Transcription! is Induced! The lac Operon is also Subject to! Positive Regulation! The lac promoter-operator region:! Figure 28-10! Lactose is a secondary sugar--! Glucose is the preferred carbon source!! How is glucose used preferentially when both are present?! 8!
The lac Operon is also Subject to! Positive Regulation! Figure 28-17b! The promoter is sub-optimal! Activation is necessary for high levels of transcription! Positive Regulation of the lac Operon! Is Achieved through CRP! Figure 28-17a! CRP (camp receptor protein)! CRP binds camp! CRP-cAMP binds to CRP site! [camp] inversely related to [glucose] because glucose inhibits adenylate cyclase! stimulates transcription! CRP is a! glucose sensor! 9!
The lac Operon:! lactose absent! Figure 28-17! The lac Operon:! lactose present! Figure 28-17! 10!
Translation is Co-Transcriptional! in Prokaryotes! The trp Operon! trp repressor binds! to the trp operator! But that s not all!! Figure 28-18! 11!
The Leader Region is Essential! for Attenuation of the trp Operon! a trp! sensor! Figure 28-19! When tryptophan levels are high--! Transcription is terminated! Figure 28-19! When tryptophan levels are low--! Transcription proceeds! 12!
Attenuation in other operons! Operon Leader Sequence trp MKAIFVLKGWWRTS phea MKHIPFFFAFFFTFP his MTRVQFKHHHHHHHPD! thr MKRISTTITTTITITTQNGAG! leu MSHIVRFTGLLLLNAFIVRGRPVGGIQH! ilv MTALLRVISLVVISVVVIIIPPCGAALGRGKA! trp Operon Movie! 13!
Lecture 21: Outline and Objectives l Regulation of Gene Expression in Prokaryotes! l transcriptional regulation! l principles! l lac operon! l trp attenuation! l Regulation of Gene Expression in Eukaryotes! l transcriptional regulation! l transactivators & co-activators! l coordinate regulation! l post-transcriptional regulation! l alternative splicing! Potential Points of Gene Regulation! In Eukaryotes:! Figure 28-1! 14!
A Consensus RNA Polymerase II Promoter! Figure 26-8! Contain multiple regulatory sequences! Upstream activating sequences (UASs in yeast)! Enhancers (in higher eukaryotes)! Can be very far upstream!! Transcriptional Regulation in Eukaryotes! Promoter access is restricted! DNA is compacted into chromatin! Condensed chromatin is inaccessible! Chromatin must be remodeled! Figure 24-31! 15!
Chromatin Remodeling Enzymes! Transcriptional Regulation in Eukaryotes! Activation of transcription! Basal machinery can assemble at promoters! unregulated! low transcription rate! Activators induce expression of genes! regulated! induce high rate! Multiple activators insure specificity! Repression of transcription! Eukaryotic genome is too large to synthesize a different repressor for every gene! Repressors are redundant to chromatin masking of promoters! 16!
Transcriptional Activation in Eukaryotes! Requires Three Classes of Proteins:! basal transcription factors! unregulated! recruit RNAP to promoters! transactivators! sensitive to regulation! bind to DNA (at enhancers)! co-activators! non-dna binding! mediate interactions between RNAP and transactivators! Basal! Transcription! Factors:! recruit RNAP! to promoters! BUT, the rate of! basal transcription! is very low! Figure 26-9a! 17!
Transcriptional Activation in Eukaryotes! facilitate! looping! Coactivators:! interact with both! RNAP II and! Transactivators!! transactivators bound to enhancers! act at a distance at enhancers! Figure 28-28a! Repression can Occur! by Interference with Activation! Figure 28-28b! 18!
Transcriptional activation! Figure 28-29! Genes of Galactose Metabolism! are Coordinately Regulated! 19!
Activation of galactose genes! Figure 28-30! Transactivators have a Modular Structure! activation! domains! DNA binding domains! Figure 28-31! 20!
Transcriptional Regulation! by Steroid Hormone Receptors! Figure 12-30! Steroid Hormone Receptors:! DNA binding domain! transactivation domain! ligand binding domain! Figure 28-33! The Hormone/Receptor Complex is a Transactivator! 21!
Transcriptional Regulation! by Extracellular Signals! extracellular signals! are often read out! through a cascade! of Phosphorylation! How can! Phosphorylation! Modulate! Transcription?! Phosphorylation can Regulate Transcription! Factor Activity through Several Mechanisms! 2 common points of regulation are:! NLS can be masked! 22!
Potential Points of Gene Regulation! In Eukaryotes:! Figure 28-1! Alternative RNA Processing! Figure 26-19! Can produce multiple mrnas from a single primary transcript! 23!
Alternative RNA Processing! Figure 26-20! Can generate multiple proteins from a single gene! Potential Points of Gene Regulation! Allow for:! Global Control! Coordinate Control! Gene-specific Control! Figure 28-1! 24!