Gene Regulation. Chapter 16. Levels of Gene Regulation. Bacterial Gene Regulation. Genes vs. Regulatory Elements. Unicellular flexibility

Transcription

1 Gene Regulation Chapter 16 Control of Gene Expression (Part 2) Unicellular flexibility Genes turned on and off in response to environment Multicellular specialization Genes for one cell type are not expressed in other cell types Levels of Gene Regulation Gene Structure Transcription mrna processing Regulation of mrna stability Translation Post translational protein modification Figure 16.1 Genes vs. Regulatory Elements Structural genes: Metabolism, structure, biosynthesis Regulatory genes: Affect transcription or translation DNA binding proteins Regulatory elements: Not transcribed Affect gene expression Bacterial Gene Regulation Functionally related genes often clustered Can be transcribed together on same mrna Operon: Group of bacterial structural genes that are transcribed together. includes promoters and regulatory elements 1

2 Modes of Transcriptional Control Negative Regulatory protein acts as repressor Bind to DNA and inhibits transcription 2 Classes of Operon Inducible Transcription is normally OFF Modulator turns transcription ON Positive Regulatory protein acts as activator Binds to DNA and stimulates transcription Repressible Transcription is normally ON Modulator turns transcription OFF An Example: The lac Operon of E. coli Figure 16.6 Involved in lactose metabolism in E. coli Lactose: Disaccharide Doesn t diffuse across membrane easily Enzymes: Β-Galactosidase Permease Transacetylase F Cells Cells containing an F plasmid with some bacterial genes. Figure

3 Partial Diploids Conjugation between an F Cell and an F- cell can result in cells with 2 copies of some genes Partial Diploids will come in really handy for studying gene expression! (Chapter 16) These are called Partial Diploids or merozygotes lac Mutations Partial Diploid strains of E. coli: 2 copies of lac operon Bacterial chromosome Plasmid Cis acting mutations: Control expression of genes on the same piece of DNA only Trans acting mutations: Control expression of genes on other DNA molecules Genotypes of Partial Diploids Bacterial Chromosome / Plasmid Examples: lacz - lacy + / lacz + lacy - Structural mutation of lacz gene on bacterial chromosome Structural mutation of lacy gene on plasmid laci + lacz - / laci - lacz + Figure Chromosome Plasmid Chromosome Plasmid 3

4 Figure Types of Mutations I lacis lacz+ / laci+ lacz+ Structural gene Mutations Affect structure of enzymes, not regulation The wild type is Trans Dominant Regulator gene Mutations Constitutive: lac enzymes produced constantly (in regular E. coli) In partial diploids, laci+ is Trans Dominant lacis encodes a superrepressor Figure Types of Mutations II Constitutive! Operator mutations lacoc indicates a mutation in the DNA sequence of the operator Repressor cannot bind to operator lacoc is cis dominant and constitutive Figure Figure Constitutive! Cis acting! 4

5 Figure Cis acting! Types of Mutations III Promoter mutations lacp - indicates a mutation in the DNA sequence of the promoter RNA polymerase cannot bind to promoter lacp - is cis dominant laci + lacp - laco c lacz + lacy - / laci - lacp + laco + lacz - lacy + What is the ENZYMATIC ACTIVITY? Lactose Absent Lactose Present B-Gal Permease B-Gal Permease???? Use - for no activity and + for activity laci + lacp - laco c lacz + lacy - / laci - lacp + laco + lacz - lacy + What is the ENZYMATIC ACTIVITY? Lactose Absent Lactose Present B-Gal Permease B-Gal Permease A) B) C) D) laci + lacz - / laci - lacz + Figure 16.9 Chromosome Plasmid Chromosome Plasmid 5

6 Catabolite Repression Glucose is the preferred food source for E. coli When glucose is available: Genes for metabolism of other sugars are repressed Catabolite Repression CAP and camp Catabolite Acitvator Protein Binds to DNA upstream of lac promoter RNA polymerase won t bind efficiently to lac promoter unless CAP is first bound to DNA Cyclic AMP (adenosine-3,5 -cyclic monophosphate) CAP can t bind to DNA without camp Concentration of camp inversely proportional to glucose concentration This is POSITIVE CONTROL because CAP is an ACTIVATOR Figure trp Operon Controls biosynthesis of tryptophan Negative repressible operon Figure Attenuation Another form of transcriptional control for the trp operon. Transcription is initiated but terminates prematurely. 6

7 Figure Figure Look Familiar? Rho-independent Termination Rho-independent Termination 1) Two inverted repeats in the DNA sequence are transcribed 2) A string of ~6 Adenines follows the second inverted repeat 3) The inverted repeats form a hairpin structure pausing the polymerase 4) The A-U bonds break and the RNA molecule separates from the template Figure Check out the online animation for the lac operon and attenuation Antisense RNA RNA regulator of gene expression Antisense RNA Small RNA molecules complementary to certain sequences on mrnas. Inhibit translation Example: ompf gene of E. coli Important in cellular osmoregulation Increased osmolarity turns on micf micf produces Antisense RNA 7

8 Figure Figure Ribosome cannot bind Eukaryotic Gene Regulation No operons in Eukaryotes Chromatin affects gene expression Activators are more common Many mechanisms at many levels Eukaryotic Gene Regulation Eukaryotic Gene Regulation Gene Structure Transcription mrna processing Regulation of mrna stability Translation Post translational protein modification Gene Regulation: Gene Structure (Chromatin) DNAaseI Hypersensitivity DNAase I digests DNA Doesn t work when DNA tightly bound to histones Transcriptionally active genes DNAaseI hypersensitive sites Regions near transcriptionally active genes where DNA configuration is more open DNA binding proteins? 8

9 Gene Regulation: Gene Structure (Chromatin) cont. Histone acetylation Facilitates transcription DNA methylation Cytosine bases methylated Associated with transcription repression CpG islands: GC CG Gene Regulation: Transcriptional Control Transcriptional activators Stabilize basal transcription apparatus (BTA) Often interact with BTA through coactivators Stimulate transcription Repressors May bind to regulatory promoter May bind to silencers ENHANCERS AND INSULATORS Figure Enhancers affect transcription at distant promoters Alpha chain of Tcell receptor: enhancer is 69,000 bp downstream of promoter Enhancers can stimulate any promoter in its vicinity Insulators (boundary elements) limit the effect of enhancers RESPONSE ELEMENTS Response elements DNA regulatory elements which are bound by transcriptional activator proteins. Example: Metallothionein Response elements to heavy metals Eukaryotic Genes may be activated by several different response elements Multiple Response Elements (MREs) allow the same gene to be activated by different stimuli. 9

10 Response elements to a particular stimulus can be associated with multiple genes, allowing a single stimulus to activate multiple genes. Gene Regulation: Messenger RNA Processing Alternative Splicing: SR Proteins: often regulate splicing Example: T-antigen gene of mammalian virus SV40 Splicing Factor 2 (SF2) is a type of SR Protein Another Example: Sex determination in Drosophila. Gene Regulation: RNA Stability Variation in mrna Stability Stability of mrna affected by: 5 Cap Poly (A) tail 5 UTR Coding region 3 UTR Variation in Protein Production Gene Regulation: Translation and Posttranslational Control Availability of Translational Apparatus: Ribosomes, aminoacyl trnas, initiation factors, elongation factors. Less available: slower translation. Proteins binding to 5 UTR Posttranslational modification Trimming, acetylation, addition of phosphates, carboxyl groups, etc. RNA Interference (RNA Silencing) Double stranded RNA initiates a cascade that degrades complementary mrna. May have evolved as a defense against double stranded RNA viruses. Very handy for artificially regulating gene expression Model organisms Genetically engineered organisms 10

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