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Prokaryotic Regulation:. Bacteria do not need the same enzymes and other proteins all of the time. - They need only: 1. The enzymes required to break down the nutrients available to them or
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Prokaryotic Regulation: • Bacteria do not need the same enzymes and other proteins all of the time. - They need only: 1. The enzymes required to break down the nutrients available to them or 2. The enzymes required to synthesize whatever metabolites are absent under the present circumstances.
Prokaryotic Regulation:The Operon Model (Jacob & Monod 1961) • An operon consists of three components: 1.Promoter • DNA sequence where RNA polymerase first attaches • Short segment of DNA 2. Operator • DNA sequence where active repressor binds • Short segment of DNA
Prokaryotic Regulation:The Operon Model (Jacob & Monod 1961) 3. Structural Genes • One to several genes coding for enzymes of a metabolic pathway • Translated simultaneously as a block • Long segment of DNA A regulator gene is located outside of the operon. It codes for a repressor that controls whether the operon is active or not.
Repressible Operons:The trp Operon - Normally turned ON • If tryptophan (an amino acid) is ABSENT: • Repressor is unable to attach to the operator (expression is normally “on”) • RNA polymerase binds to the promoter • Transcription & translation occur • Enzymes for synthesis of tryptophan are produced • Tryptophan will be produced by E. coli
Repressible Operons:The trp Operon - Genes repressed • If tryptophan IS present enzymes are not needed and following occurs: • Tryptophan combines with repressor, causing it to change shape, thus acting as a corepressor • Repressor becomes functional • Blocks transcription & synthesis of enzymes and tryptophan is NOT produced
Operon usually ON, must be turned OFF Repressor Transcription bound ? occurs? NO -------> YES YES -------> NO *** Corepressors are frequently the products in the pathway. In this case, tryptophan is the corepressor. Summary of repressible trp operon
Inducible Operons:The lac Operon - Normally turned OFF • When E. coli is denied glucose & is given lactose instead, it immediately begins to make three enzymes needed for the metabolism of lactose. • These enzymes are encoded by three structural genes which are adjacent to one another on the chromosome. They are controlled by one regulator gene that codes for a one repressor.
Inducible Operons:The lac Operon - Normal OFF state • If lactose (a sugar that can be used for food) is absent: • Repressor attaches to the operator • RNA polymerase cannot bind to promoter • Transcription of structural genes is blocked • Enzymes needed to digest lactose NOT made
Inducible Operons:The lac Operon - Induced state • If lactose IS present: • It combines with repressor and renders it unable to bind to operator by causing shape of repressor to change • RNA polymerase binds to the promoter • Transcription of genes occurs • The three enzymes necessary for lactose catabolism are produced • Lactose will be digested by enzymes
Operon usually OFF, must be turned ON Repressor Transcription bound ? occurs? YES -------> NO NO -------> YES *** Inducers are frequently the reactants in the pathway. In this case, the lactose is the inducer. Summary of inducible lac operon
The lac Operon - Further control • E. coli preferentially break down glucose. Thus, they have a way to ensure that the lac operon is only turned on maximally when glucose is absent. • This involves use of cyclic AMP which is abundant when glucose is absent. - Cyclic AMP binds to a molecule called catabolite activator protein (CAP).
The lac Operon - Further control (2) • The cAMP-CAP complex then binds to a CAP binding site next to the lac operonpromoter. • • When CAP binds to DNA, the DNA bends. • - This exposes the promoter to RNA polymerase which is now better able to bind to the promoter.
The lac Operon - Further control (2) • When glucose IS present: • There is little cAMP in the cell - CAP is not activated by cAMP - lac operon does NOT function maximally and cell will preferentially use glucose as its food source.
Animations for the Operons • http://highered.mcgraw-hill.com/olc/dl/120080/bio27.swf Trp Operon http://highered.mcgraw-hill.com/olc/dl/120080/bio26.swf lac Operon
Eukaryotic Regulation • A variety of mechanisms to control gene expression: • Five primary levels of control: • Nuclear levels • Chromatin Packing • Transcriptional Control • Posttranscriptional Control • Cytoplasmic levels • Translational Control • Posttranslational Control
Regulation of Gene Expression:Levels of Control in Eukaryotes
Chromatin Structure • Eukaryotic DNA associated with histone proteins • Together make up chromatin • As seen in the interphase nucleus • Nucleosomes: • DNA wound around balls of eight molecules of histone proteins • Looks like beads on a string • Each bead a nucleosome • The levels of chromatin packing determined by degree of nucleosome coiling
Chromatin Packing • Euchromatin • Loosely coiled DNA • Appears lightly stained in micrographs • Transcriptionally active - capable of being transcribed • Heterochromatin • Tightly packed DNA • Appears darkly stained in micrographs • Transcriptionally inactive
Chromatin Packing • Barr Bodies • Females have two X chromosomes, but only one is active • Other is tightly packed along its entire length • Inactive X chromosome is called a Barr body • Inactive X chromosome does not produce gene products
Transcriptional Control • Transcription controlled by DNA-binding proteins called transcription factors • Bind to a promoter adjacent to a gene • Transcription activators bind to regions of DNA called enhancers. Might be brought near region of promoter by hairpin loops in DNA. • Always present in cell, but most likely have to be activated before they will bind to DNA
Transcriptional Control (2) • Transposons are specific DNA sequences that have the ability to move within and between chromosomes. • Their movement to a new location sometimes alters neighboring genes by decreasing their expression - Thus, they can act like regulator genes - They also can be a source of mutations.
Posttranscriptional Control • Posttranscriptional control operates within the nucleus on the primary mRNA transcript • Given a specific primary transcript: • Excision of introns can vary • Splicing of exons can vary • Thus, differing versions of the mRNA transcript might leave the nucleus
Posttranscriptional Control • Posttranscriptional control may also control speed of mRNA transport from nucleus to cytoplasm • Will affect the number of transcripts arriving at rough ER • And therefore the amount of gene product realized per unit time
Translational Control • Translational control determines degree to which mRNA is translated into a protein product • Presence of 5′ cap and the length of poly-A tail on 3′ end can determine whether translation takes place and how long the mRNA is active - Example: Long life of mRNA in RBCs that code for hemoglobin attributed to presence of 5’ cap and 3’ poly-A tail
Posttranslational Control • Some proteins are not immediately active after synthesis. • Some need to be activated - Folding and breaking into chains must occur in bovine insulin before it is active • Some are degraded quickly - Cyclin proteins that control cell cycle
Animations for Eukaryotic Control • http://highered.mcgraw-hill.com/olc/dl/120080/bio28.swf Control of gene expression in eukaryotes http://highered.mcgraw-hill.com/olc/dl/120080/bio31.swf Transcription Complex and Enhancers
Effect of Mutations onProtein Activity • A mutation is a permanent change in the sequence of bases in DNA. • Effects on proteins can range from no effect to complete inactivity • Germ-line mutations • Occur in sex cells; can be passed on to future generations • Somatic mutations • Occur in body cells; can’t be passed on to future generations • Can lead to development of cancer
Effect of Mutations onProtein Activity • Point Mutations • Involve change in a single DNA nucleotide • Changes one codon to a different codon • Could change one amino acid for another • Effects on protein vary: • Drastic - completely nonfunctional • Reduced functionality • Unaffected
Effect of Mutations onProtein Activity • Frameshift Mutations • One or two nucleotides are either inserted or deleted from DNA • Can lead to completely new codon order • Protein can rendered nonfunctional • Normal : THE CAT ATE THE RAT • After deletion: THE ATA TET HER AT • After insertion: THE CCA TAT ETH ERA T
Nonfunctional Proteins • Examples of nonfunctional proteins: • Hemophilia due to the transposon Alu • Phenylketonuria (PKU) due to faulty code for one enzyme • Cystic fibrosis due to inheritance of faulty code for a chloride ion channel • Androgen insensitivity due to a faulty receptor for androgens (male sex hormones)
Carcinogenesis • Development of cancer involves a series of mutations: • •Proto-oncogenes – Stimulate cell cycle but are usually turned off. Can mutate and become oncogenes which are turned on all the time. • •Tumor suppressor genes – inhibit cell cycle • Mutation in oncogene and tumor suppressor gene: • Stimulates cell cycle uncontrollably • Leads to tumor formation
Causes of Mutations • Spontaneous Errors: • Happen for no apparent reason • Example of spontaneous germ-line mutation is achondroplasia, a type of dwarfism Replication Errors: • DNA polymerase proofreads new strands • Generally corrects errors -1 in 1,000,000,000 replications error occurs
Causes of Mutations • Environmental Mutagens • A mutagen is an environmental agent that increases the chances of a mutation • Carcinogens - Mutagens that increase the chances of cancer -Many agricultural & industry chemicals -Many drugs • Tobacco smoke chemicals • Radiation (X-rays, gamma rays, UV)
