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WELCOME

WELCOME. Dr.G.Presanna Kumar BOTANY NSS COLLEGE PANDALAM. Regulation of Gene Expression In Prokaryotes. 2 categories of Genes Constitutive genes (House keeping genes) – Non constitutive (Inducible genes/ Regulated genes)-.

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WELCOME

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  1. WELCOME Dr.G.Presanna Kumar BOTANY NSS COLLEGE PANDALAM

  2. Regulation of Gene ExpressionIn Prokaryotes • 2 categories of GenesConstitutive genes (House keeping genes) –Non constitutive (Inducible genes/ Regulated genes)-

  3. A gene is a nucleotide sequence that carries the information needed to produce a specific RNA or protein product

  4. Constitutive Gene Expression E. coli 1.Constitutive genes (House keeping genes) –Expressed always in almost all cells. Most E.coli genes maintain a constant rate of synthesis.Not subjected to regulation-Product always required eg. Enzymes of Glycolysis / Kreb’s cycle coding sequence promoter

  5. 2.Non constitutive (Inducible genes/ Regulated genes)- Not expressed always- Activity regulated by various molecular signals Inducer – increases expression of these genes when a specific inducer is present.The synthesis of galactosidase is induced by its substrate, lactose in the medium. coding sequence promoter operator operon in E. coli

  6. The three enzymes are Operon- Unit of gene expression in Prokaryotes lac operon in E.coli –Jacob and Monad 1961 It consists of linear sequence of genes- 1.Structuralgenes: Code for polypeptide synthesis(Enzymes, transport proteins,hormones etc.)3 in lac operon and 5 in trp operon 2.Control genes :Control the activity of str.genes.Ther are 3types of con. genes. a)Regulator gene:It supresses the activity of str.gene by producing a protein called repressor.Reprsr.binds on the operator for supressing str.genes

  7. Genes of the lac Operon • Genes are grouped: • lacZ = b-galactosidase • lacY = galactoside permease • lacA = galactoside transacetylase • All 3 genes are transcribed together producing 1 mRNA, a (polycistronic mRNA) that starts from a single promoter Polycistronic mRNA- An mRNA coding for more than one protein

  8. DNA lacl lacz lacY lacA RNApolymerase 3 mRNA 5 -Galactosidase Permease Transacetylase Protein Inactiverepressor Allolactose(inducer) Operon- When the coding sequences of 2 or more genes are transcribed into a single mRNA molecule starting from a single promoter, the genes are said to form an operon.Many bacterial genes are organised into operons while this kind of organisation is absent in eukaryotes Lactose present, repressor inactive → lac operon

  9. Inducer of the lac Operon This is an inducible operon, meaning gene expression ß-galactosidase is stimulated by the presence of a co-inducer, lactose. • Inducer (one molecule) of lac operon binds the repressor • The inducer is allolactose, an alternative form of lactose

  10. Promoter Regulatorygene Operator DNA lacl lacZ NoRNAmade RNApolymerase mRNA Activerepressor Protein The lac operon: regulated synthesis of inducible enzymes 3 5 Lactose absent, repressor active →

  11. Proposed CAP-cAMP Activation of lac Transcription • The CAP-cAMP dimer binds to its target site on the DNA • The aCTD (a-carboxy terminal domain) of polymerase interacts with a specific site on CAP • Binding is strengthened between promoter and polymerase • CAP-cAMP bends its target DNA by about 100° when it binds

  12. Positive Control of Transcription: CAP Absence of the lac repressor is essential but not sufficient for effective transcription of the lac operon. The activity of RNA polymerase also depends on the presence of another DNA-binding protein called catabolite activator protein or CAP. Like the lac repressor, CAP has two types of binding sites: One binds the nucleotide cyclic AMP; the other binds a sequence of 16 base pairs upstream of the promoter However, CAP can bind to DNA only when cAMP is bound to CAP. so when cAMP levels in the cell are low, CAP fails to bind DNA and thus RNA polymerase cannot begin its work, even in the absence of the repressor. So the lac operon is under both negative (the repressor) and positive (CAP) control.

  13. The cell will only turn on the lac operon at a high level if: 1.  lactose is present AND 2. glucose is absent!     So there must also be a monitor for glucose in the cell.  That monitor is a small molecule called cAMP.  When glucose goes up, cAMP goes down.  When glucose goes down, cAMP goes up.  cAMP can bind to a protein called CRP (the cAMP receptor protein).  When cAMP is bound to CRP, CRP is active.  When cAMP is NOT bound to CRP, CRP is INactive.     How does this relate to the lac operon?  Well, if CRP gets activated by cAMP (high cAMP = low glucose), it can bind to a site on the DNA next to the lac promoter and can help the RNA polymerase to bind tightly to the promoter.  This stimulates transcription of the lac operon.     In other words, low glucose = high cAMP = active CRP = stimulated transcription of the lac operon!

  14. Promoter CAP-binding site Operator RNA polymerase can bindand transcribe Control of the lac operon by catabolite activator protein (CAP) DNA lacl lacZ ActiveCAP cAMP Inactive lac repressor InactiveCAP Lactose present, glucose scarce (cAMP level high) →

  15. Promoter DNA lacl lacZ CAP-binding site Operator RNA polymerase can’t bind InactiveCAP Inactive lac repressor The lac Operon: +Lactose; +Glucose Binding of CAP -cAMP helps RNA polymerase form an open promoter complex Lactose present, glucose present (cAMP level low) →

  16. The lac Operon • The lac operon was the first operon discovered • Contains 3 genes coding for E. coli proteins that permit the bacteria to use the sugar lactose • Galactoside permease which transports lactose into the cells (across the plasma membrane ) • b-galactosidasecuts the lactose into galactose and glucose.Once induced by the presence of lactose, the quantity of beta-galactosidase in the cells rises from virtually none to almost 2% of the weight of the cell. • Galactoside transacetylase whose function is unclear

  17. Positive Control of lac Operon • Positive control of lac operon by a substance sensing lack of glucose that responds by activating lac promoter • The concentration of nucleotide, cyclic-AMP, rises as the concentration of glucose drops

  18. Tryptophan operon • Regulated by two control mechanisms • Repressor binding to the operator • Attenuation: premature termination of transcription The trp operon is a repressor operon, meaning gene expression of the operon is repressed by the presence of the co-repressor, tryptophan.

  19. The trp Operon • The E. coli trp operon contains the genes for the enzymes the bacterium needs to make the amino acid tryptophan • The trp operon codes for anabolic enzymes, those that build up a substance • Anabolic enzymes are typically turned off by a high level of the substance produced • This operon is subject to negative control by a repressor when tryptophan levels are elevated • trp operon also exhibits attenuation

  20. trp Trp operon controls the production of the amino acid tryptophan r. Trp Repressor Gene (always “on”) promotor 5 Genes needed for tryptophan production rp. RNA Pol. binding site o. Operator r. rp. o. Gene 1 Gene 1 Gene 3 Gene 4 Gene 5 RNA polymerase mRNA transcription unit mRNA for trp repressor Enz 1 Enz 2 Enz 3 Enz 4 Enz 5 Trp repressor protein translated in its “inactive” form

  21. trp operon Promoter RNA polymerase Start codon Stop codon Polypeptides that make up enzymes for tryptophan synthesis The trp operon: regulated synthesis of repressible enzymes Promoter Genes of operon trpR trpD trpC trpB trpE trpA DNA Operator Regulatory gene 3 mRNA 5 mRNA 5 C E D B A Inactiverepressor Protein Tryptophan absent, repressor inactive →

  22. How is the trp operon turned off once enough trp is made? DNA No RNA made RNA polymerse, therefore is physically blocked from transcribing genes for trp Active trp repressor can now bind to operator mRNA Active repressor Protein Tryptophan present, repressor active → Tryptophan (corepressor)

  23. Attenuation: Tryptophan Operon • Inverted repeat in mRNA of trp operon • Stem loop forms • 2 conformations: 3,4 stem loop and 2,3 stem loop • 3,4 stem loop = transcription pause and reach termination signal • 2,3 stem loop = do not have a termination signal

  24. Mechanism of Attenuation Figure 9.27

  25. Mechanism of Attenuation Figure 9.27

  26. Tryptophan’s Role in Negative Control of the trp Operon • Five genes code for the polypeptides in the enzymes of tryptophan synthesis • The trp operator lies wholly within the trp promoter • High tryptophan concentration is the signal to turn off the operon • Presence of tryptophan helps the trp repressor bind to its operator

  27. The leader sequence:two trp codons and a stop codon

  28. Negative Control of the trp Operon • Without tryptophan no trp repressor exists, just the inactive protein, aporepressor • If aporepressor binds tryptophan, changes conformation with high affinity for trp operator • Combine aporepressor and tryptophan to have the trp repressor • Tryptophan is a corepressor

  29. Riboswitches • Small molecules can act directly on the 5’-UTRs of mRNAs to control their expression • Regions of 5’-UTRs capable of altering their structures to control gene expression in response to ligand binding are called riboswitches

  30. Riboswitches • RNA domains in an mRNA molecule that can bind small molecules to control translation of mRNA • Located at 5′ end of mRNA • Binding results from folding of RNA into a 3-D structure • Similar to a protein recognizing a substrate • Riboswitch control is analogous to negative control • Found in some bacteria, fungi, and plants

  31. Regulation by Riboswitch Figure 9.25

  32. Riboswitch Action • Region that binds to the ligand is an aptamer • An expression platform is another module in the riboswitch which can be: • Terminator • Ribosome-binding site • Another RNA element that affects gene expression • Operates by depressing gene expression • Some work at the transcriptional level • Others can function at the translational level

  33. Model of Riboswitch Action • FMN binds to aptamer in a riboswitch called the RFN element in 5’-UTR of the ribD mRNA • Binding FMN, base pairing in riboswitch changes to create a terminator • Transcription is attenuated • Saves cell energy as FMN is a product of the ribD operon

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