1. Chapter 5

2. Topics1. Overview of gene expressioni.Operonsii. The transcription processiii.The translation process2. Regulation of transcriptioni.Induction and transcriptionii. Positive controlsiii.Attenuation i. Global control3. Catabolite repression 3. 1.Overview of gene expression Operons Transcription process Translation 4. Gene expression The processes that cells and viruses use to regulatethe way that the information in genes is turnedinto gene products. Gene regulation is essential for viruses, prokaryotesand eukaryotes . It increases the versatility and adaptability ofan organism by allowing the cell to express proteinwhen needed. 5. Switching Genes On & Off All the cells in our body contain identical set of genes How do cells become so different? Express different subsets of genes Each cell switches genes on or off depending on current need Needs to be strictly controlled expression of a gene in a wrong amount, wrong time or wrong celltype can lead to deleterious phenotype (cell death, cancer) 6. Gene control in eukaryotes Gene expression in eukaryotes is controlled by avariety of mechanisms: those that prevent transcription those that prevent expression after the protein hasbeen produced. 7. Regulation Occurs at Many Levels1. Transcriptional control2. Post-transcriptional control3. Transport to cytoplasm4. mRNA stability5. Translational control6. Post-translational control 8. Gene Control in Prokaryotes Prokaryotes have two levels of gene control. Transcriptional mechanisms control the synthesisof mRNA and translational mechanisms control the synthesis ofprotein after mRNA has been produced. 9. Operons Operons are groups of genes that function to produceproteins needed by the cell. Two (2) kinds: 1. Structural genes - code for proteins needed for thenormal operation of the cell. For example, they may be proteins needed for the breakdown of sugars. The structural genes are grouped together and a single mRNA molecule is produced during their transcription.2. Regulator genes - code for proteins that regulate other genes. Operons have not been found in eukaryotes 10. Operon Figure 8.12 11. Transcription DNA is transcribed to make RNA (mRNA, tRNA, andrRNA) Transcription begins when RNA polymerase binds tothe promoter sequence Transcription proceeds in the 5 3 direction Transcription stops when it reaches theterminator sequence 12. The Process of Transcription Figure 8.7 13. The Process of Transcription Figure 8.7 14. RNA Processing in Eukaryotes Figure 8.11 15. Translation mRNA is translated incodons (three nucleotides) Translation of mRNA beginsat the start codon: AUG Translation ends atnonsense codons: UAA,UAG, UGA Figure 8.2 16. The Genetic Code 64 sense codons on mRNAencode the 20 amino acids The genetic code isdegenerate tRNA carries thecomplementary anticodonFigure 8.2 17. The Process of Translation Components needed to begin translations come together.Figure 8.9 18. The Process of Translation On the assembled ribosome, at tRNA carrying the first amino acid in paired with the start codon on the mRNA. The place where this firsts tRNA sits is called the p site. A tRNA carrying the second amino acid approaches. Figure 8.9 19. The Process of Translation The second codon of the mRNA pairs with a tRNA carrying the second amino acids joins to the seconds by a peptide bond. This attaches the polypeptide to the tRNA in the p site.Figure 8.9 20. The Process of TranslationThe ribosome moves alongthe mRNA until thesecond tRNA is in the psite. The next codon to betranslated is brought intothe a site. The firsts tRNAnow occupies the e site. Figure 8.9 21. The Process of Translation The second amino acid is paired with the start codon on the mRNA. Is release from the e site. Figure 8.9 22. The Process of Translation The ribosome continues to move along the mRNA and new amino acids are added to the polypeptide Figure 8.9 23. The Process of Translation When the ribosome reaches a stop codon, the polypeptide is released.Figure 8.9 24. The Process of Translation Finally, the last tRNA isreleased ,and theribosome comes apart.The released polypeptideforms a new protein.Figure 8.9 25. RegulationTopics: Induction :i.Induction: lac operon andii. repression: trp operon Positive and negative controls Attenuation Global controls 26. Induction and repression Constitutive genes are expressed at a fixed rate Other genes are expressed only as needed such as: Inducible genes Repressible genes 27. Enzyme induction Metabolites or substrates can turn on inactive genesso that they are transcribed. In the process of enzyme induction, the substrate, ora compound structurally similar to the substrate, evokes the formation of enzyme(s) which are usually involved in the degradation of the substrate. 28. Inducible enzymes: Enzymes that are synthesized asa result of genes being turned on. Inducer: The substance that activates genetranscription. Inducible enzymes are produced only in response tothe presence of a their substrate (produced onlywhen needed). So that energy is not wasted to synthesize unneededenzymes. 29. Induction of lac operons The best case of enzyme induction involves the enzymes oflactose degradation in E. coli. Only in the presence of lactose does the bacterium synthesizethe enzymes that are necessary to utilize lactose as a carbonand energy source for growth. Two enzymes are required for the initial breakdown oflactose: lactose permease, which actively transports the sugar into the cell, and beta galactosidase, which splits lactose into glucose plus galactose. The genes for these enzymes are contained within the lactoseoperon (lac operon) in the bacterial chromosome 30. Mechanism of lac operon The lac operon is an example of an inducible operonbecause the structural genes are normally inactive. They are activated when lactose is present. 31. The region of DNA where the repressor protein binds isthe operator site. The promoter site is a region of DNA where RNApolymerase can bind. The entire unit (promoter, operator, and genes) isan operon. The operator acts like a switch that can turn severalgenes on or off at the same time. 32. In order for E.coli to digest lactose, it requires threetypes of enzymes A, B and C. Hence it requires gene A, Band C. These are the structural genes. In normal condition, the genes do not function becausea repressor protein is active and bound to the DNApreventing transcription. 33. When lactose is present, it acts as an inducer by bindingto the repressor protein, thus preventing it from attachingto the operator. RNA polymerase can then bind to the operator andtranscribe the mRNA molecule. Three different proteins are synthesized. 34. When all of the lactose in the cell has beencatabolized, the repressor protein binds to the operatorand shuts down the operon. The repressor protein is produced by a regulator gene. 35. Functional and regulatorycomponents of the lac operon Lac IRegulatory gene that encodes for the lac Repressor protein that is concerned with regulating the synthesis of thestructural genes in the operon. Lac I is adjacent to the Promoter site of the operon. An active repressor binds to aspecific nucleotide sequence in the operator region and thereby blocks binding of RNAp to the promoter to initiatetranscription. The lac repressor is inactivated by lactose, and is active in the absence of lactose. OOperator specific nucleotide sequence on DNA to which an active Repressor binds. PPromoter specific nucleotide sequence on DNA to which RNA polymerase binds to initiate transcription. (Thepromoter site of the lac operon is further divided into two regions, an upstream region called the CAP site, and adownstream region consisting of the RNAp interaction site. The CAP site is involved in catabolite repression of the lacoperon.). If the Repressor protein binds to the operator, RNAp is prevented from binding with the promoter andinitiating transcription. Under these conditions the enzymes concerned with lactose utilization are not synthesized. Lac Z, Y and A Structural Genes in the lac operon. Lac Z encodes for Beta-galactosidase; Lac Y encodes the lactose permease; Lac Aencodes a transacetylase whose function is not known.lac lactose, the inducer molecule. When lactose binds to the Repressor protein, the Repressor is inactivated; the operon isdepressed; the transcription of the genes for lactose utilization occurs. 36. Repression It is the regulatory mechanism that inhibits geneexpression and decrease the synthesis of enzymes Usually a response to the overabundance of an end-product of a metabolic pathway Repression is mediated by regulatory proteins calledrepressors Repressors block the ability of RNA polymerase to initiate transcription from the repressed genes 37. Repressive operon Repressible operons are the opposite of inducibleoperons. Transcription occurs continuously and the repressorprotein must be activated to stop transcription. Example is tryptophan. It is an amino acids required by an E.coli 38. Repressible operon (trp operon)Genes that code for proteins that produce tryptophan arecontinuously transcribed.Figure 8.13 39. If tryptophan is present in the environment E. coli doesnot need to synthesize it and the tryptophan-synthesizinggenes should be turned off. This occurs when tryptophan binds with the repressorprotein, activating it. 40. Unlike the repressor discussed with the lac operon, thisrepressor will not bind to the DNA unless it is activatedby binding with tryptophan. Tryptophan is therefore a co-repressor. 41. The trp operon encodes the genes for the synthesis oftryptophan. This cluster of genes regulated by a repressor that bindsto the operator sequences. The activity of the trp repressor for binding the operatorregion is enhanced when it binds tryptophan known as acorepressor. Since the activity of the trp repressor is enhanced in thepresence of tryptophan, the rate of expression ofthe trp operon is graded in response to the level oftryptophan in the cell. Expression of the trp operon is also regulatedby attenuation. 42. Structural RepressorGenesInducible Inactive Active (inhibits)OperonsRepressible Active Inactive (inhibitsOperonswhen activated) 43. Tryptophan in negative feedback inhibition Tryptophan can inhibit the first enzyme in the synthesispathway. The presence of high levels of tryptophan inhibits theactivity of the enzyme as shown in the biosynthesispathway below. 44. Positive & negative controls Most gene expression is controlled at the level oftranscription Regulation of gene expression by proteins can be eitherpositive or negative. Regulation in prokaryotes is usually negative while it ispositive in eukaryotes. 45. The trp and lac operons discussed above are examples ofnegative control because a repressor blocks transcription. In one case (lac operon) the repressor is active andprevents transcription. In the other case (trp) the repressor is inactive and mustbe activated to prevent transcription. 46. Structural genesRepressor or regulatorNegative control Inducible operon Inactive Active (inhibits)(an active repressorinhibits Repressible operon Active Inactive (inhibitstranscription) when activated)Positive control (an active regulator Inactive Inactive (promotespromotes transcription)when activated) 47. Positive control mechanisms require the presence of anactivator protein before RNA polymerase will attach. The activator protein itself must be bound to an inducermolecule before it attaches to mRNA. 48. Attenuation The attenuator plays an important regulatory rolein prokaryotic cells because of the absence ofthe nucleus in prokaryotic organisms. The attenuator refers to a specific regulatory sequencethat, when transcribed into RNA, forms hairpin structuresto stop transcription when certain conditions are not met 49. CATABOLITE REPRESSION Many inducible operons are not only controlled by theirrespective inducers and regulatory genes, but they arealso controlled by the level of glucose in theenvironment. The ability of glucose to control the expression of anumber of different inducible operons is calledCATABOLITE REPRESSION. 50. When levels of glucose (a catabolite) in the cell are high, amolecule called cyclic AMP is inhibited from forming. But when glucose levels drop, ATP phosphates are releaseduntil at last forming cAMP:ATP --> ADP + Pi --> AMP + Pi --> cAMP cAMP binds to a protein called CAP (catabolite activatorprotein), which is then activated to bind to the CAP bindingsite. This activates transcription, perhaps by increasing the affinityof the site for RNA polymerase. This phenomenon iscalled catabolite repression. 51. Lactose present, no Lactose + glucose present glucose Figure 8.15 52. Catabolite Repression(a) Growth on glucose or lactose alone(b) Growth on glucose and lactosecombined During lag time, intracellular cyclic AMP increases, the lac operon is transcribed, more lactose is transported into the cell.Figure 8.14 53. End of chapter 5: Regulation of gene expression