1. Regulation of Gene Expression in Prokaryotes Dr. Jason R Mayberry Castle View High School

2. The Need for Regulation of Gene Expression Gene Expression Regulation: factors that determine under what circumstances the information in genes are used to produce functional proteins. Why wouldn’t cells express all their genes all the time? Cell Cycle:Different genes are needed at different times in the cell’s cycle (e.g. for DNA Replication or for Mitosis) Energy Efficiency:Energy is wasted on producing proteins that aren’t always needed. Respond to the Environment:Cell Behavior often depends on which proteins are active at any given point in time; producing different proteins allows for different responses to environmental conditions. Cell Specialization in Multicellular Organisms:Different cell types in multicellular organisms have different functions, and therefore require a different set of proteins. 1 S G2 G1 M 2 3 4

3. Different Opportunities for Gene Expression Regulation Signal Eukaryotes Prokaryotes Chromatin Chromatin modification: DNA unpacking DNA Gene available for transcription Transcription Exon RNA Primary transcript Intron RNA processing Tail mRNA in nucleus Cap Transport to cytoplasm NUCLEUS CYTOPLASM mRNA in cytoplasm Degradation of mRNA Translation Polypeptide Protein processing Active protein Degradation of protein Transport to cellular destination Cellular function (such as enzymatic activity or structural support)

4. Prokaryote Genes • Regulatory Gene • Codes for a protein Activator or Repressor that binds to a specific Activation site or Operator, respectively. • May be produced in an Active or Inactive form. Prokaryote chromosome • Operon • Codes for a set of enzymes that participate in a metabolic pathway Activation Site Binds to Active Activators Promoter Binds to RNA Polymerase OperatorBinds to Active Inhibitors Gene 1 Gene 2 …Gene n • Prokaryote Genes do NOT have: • Enhancers • Introns/Exons • Spatial separation of Transcription and Translation with mRNA Processing

5. Operon Transcription and Translation Products Regulatory Gene Operon A P …Gn O G1 G2 transcription Polycistronic mRNA:one transcript with separate start/stop codons for multiple genes G1 G2 …Gn mRNA Concurrent Translation (begins before transcription is complete) Multiple protein products, all functioning in the same metabolic pathway Proteins Protein 1 Protein 2 Protein n

7. Two Types of Operon Negative Regulation Repressible Operon Inducible Operon Repressor is made in an inactive form Repressor is made in an active form Off By Default On By Default Repressor binds to operator, blocking transcription Repressor does NOT bind to operator; transcription proceeds A corepressor binds to the Repressor, activating it so it binds to the operator and blocks transcription. An Inducer binds to Repressor, inactivating it and allowing transcription to occur

8. Two Types of Operon Negative Regulation Repressible Operon Inducible Operon On By Default Off By Default Nutrient Missing P1 P2 P3 Inducer inactivates Repressor Nutrient Present Corepressor activates repressor • Usually produce enzymes that make necessary end products (anabolic) • End product is often the corepressor • Only turned off when sufficient product is available. • Usually produce enzymes that break down nutrients (catabolic) • Nutrient is often the inducer • Only turned on when sufficient nutrient is available.

9. Positive Operon Regulation Inducible Operon Repressoris active by default Activatoris inactive by default Off By Default Inducerinactivates Repressor Low Level Transcription Coactivatoractivates the activator High Level Transcription

10. Repressible Operon Inducible Operon Repressor is made in an inactive form Activatoris made in an inactive form Repressor is made in an active form On By Default Off By Default Repressor binds to operator, blocking transcription P1 P2 P3 Repressor does NOT bind to operator; transcription proceeds followed by translation and anabolic ativity Low Level Transcription An Inducer binds to Repressor, inactivating it and allowing transcription to occur When corepressor concentration is high, it binds to the Repressor, activating it; activated repressor binds to the operator and blocks transcription. High Level Transcription Coactivatoractivates the Activator

11. The Tytophan (trp) Operon Regulatory gene trppromoter Promoter E trpA trpR trpE trpD trpC trpB trp operator RNA polymerase 3′ Polypeptide subunits that make up enzymes for tryptophan synthesis 5′ Inactive trp repressor E D C B A Tryptophan absent, repressor inactive, operon on trpR trpA trpE trpD trpC trpB 3′ 5′ Active trp repressor Tryptophan Tryptophan present, repressor active, operon off

12. The Lactose (lac) operon Regulatory gene Promoter Operator lacA lacI lacY lacZ 3′ 5′ RNA polymerase Active lac repressor Lactose absent, repressor active, operon off lacA lacI lacZ lacY RNA polymerase 3′ 5′ Transacetylase Permease β-Galactosidase Inactive lac repressor Allolactose Enzymes for using lactose Lactose present, repressor inactive, operon on

13. Positive Regulation of Lac Operon Operator When Lactose is present and glucose is scarce (cAMP level high):abundant lac mRNA is synthesized lacI lacZ RNA polymerase binds and transcribes CRP-binding site Active CRP cAMP Inactive lac repressor Inactive CRP Allolactose *CRP = cAMP receptor/reactive protein Operator When Lactose present, and glucose present (cAMP level low):little lac mRNA is synthesized lacZ lacI CRP-binding site RNA polymerase less likely to bind Inactive CRP Inactive lac repressor

14. Two modes of Negateive Feedback Precursor Genes that encode enzymes 1, 2, and 3 Feedback inhibition trpE Enzyme 1 trpD Regulation of gene expression Enzyme 2 trpC Propos a Mechanism of Action by which tryptophan could inhibit Enzyme 1. Propos a Mechanism of Action by which tryptophan could inhibit gene expression. – trpB – Enzyme 3 trpA 1) Regulation of enzyme production Tryptophan 2) Regulation of enzyme activity

15. Review of Regulation of Enzyme Activity

16. Regulating Enzymes Anarchy: a state of disorder due to absence or nonrecognition of authority. Anarchy in our cells is bad… …we need proteins to do what they do when we need them to do it. …Protein activity must be regulated. The activity of enzymes can be regulated in many ways: 1) Production (Gene Expression) vs degradation (Proteolysis) 2) Producing an inactive form that later gets cleaved to produce active form 3) Competitive Inhibition 4) Allosteric Regulation 5) Phosphorylation vs Dephosphorylation Details on the next many slides

17. Regulating Proteins The activity of enzymes can be regulated in many ways: 1) Gene Expression (Protein Production) vs. Proteolysis (protein degradation) Regulated Gene Expression X Crystallin Targeted Proteolysis Albumin

18. Regulating Proteins • The activity of enzymes can be regulated in many ways: 2) Producing an inactive form (a Pro-Enzyme) that later gets cleaved to produce active form Pro-InsulinPro = inactive form Insulin C-Peptide (non-functional)

19. Regulating Proteins The activity of enzymes can be regulated in many ways: 3) Competitive Inhibition (b) Cooperativity: Substrate Would-be substrate Competitive inhibitor Stabilized active form Inactive form When proteins have multiple active sites. Cooperativity occurs if binding of substrate to each site, induces a conformational shift that improves the binding (fit) at as-yet unoccupied sites. When a molecule other than the usual substrate binds to the substrate’s active site, thus preventing enzyme function.

20. Regulating Proteins The activity of enzymes can be regulated in many ways: 4) Allosteric Regulation Noncompetitive inhibition Allosteric Activation vs Inhibition Would-be substrate Activator active site Stabilized active form Noncompetitive inhibitor Inactive/low-active form Allosteric Regulation: When a molecule binds to a protein away from the active site, inducing a conformational change that inactivates the active site. Inhibitor Stabilized inactive form

21. Regulating Proteins The activity of enzymes can be regulated in many ways: 5) Phosphorylation vs Dephosphorylation (a specific type of allosteric regulation) • Phosphorylation of a protein induces a conformational change in the protein that can either activate or inactive it. • Kinase: type of enzyme that phosphorylates other enzymes • To be Kinased: to be phosphorylated. Inactive/Active Active/Inactive