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Hebrews 1:1-2 1 God, who at sundry times and in divers manners spake in time past unto the fathers by the prophets, 2 Hath in these last days spoken unto us by his Son, whom he hath appointed heir of all things, by whom also he made the worlds; ©1999 Timothy G. Standish Controlling Gene Expression Timothy G. Standish, Ph. D. ©1999 Timothy G. Standish All Genes Can’t be Expressed At The Same Time Some genes are needed for the function of all cells all the time. These genes are called constitutive genes and are expressed by all cells. Other genes are only needed by certain cells or at specific times. The expression of these inducible genes is tightly controlled in most cells. For example, beta cells in the pancreas make the protein insulin by expressing the insulin gene. If neurons expressed insulin, problems would result. ©1999 Timothy G. Standish Operons Are Groups Of Genes Expressed By Prokaryotes The genes grouped in an operon are all needed to complete a given task Each operon is controlled by a single control sequence in the DNA Because the genes are grouped together, they can be transcribed together then translated together ©1999 Timothy G. Standish The Lac Operon Genes in the lac operon allow E. coli bacteria to metabolize lactose Lactose is a sugar that E. coli is unlikely to encounter, so it would be wasteful to produce the proteins needed to metabolize it unless necessary Metabolizing lactose for energy only makes sense when two criteria are met: – Other more readily metabolized sugar (glucose) is unavailable – Lactose is available ©1999 Timothy G. Standish The Lac Operon - Parts The lac operon is made up of a control region and four genes The four genes are: – LacZ - b-galactosidase - An enzyme that hydrolyzes the bond between galactose and glucose – LacY - Codes for a permease that lets lactose across the cell membrane – LacA - Transacetylase - An enzyme whose function in lactose metabolism is uncertain – Repressor - A protein that works with the control region to control expression of the operon ©1999 Timothy G. Standish The Lac Operon - Control The control region is made up of two parts: Promoter – These are specific DNA sequences to which RNA Polymerase binds so that transcription can occur – The lac operon promoter also has a binding site for another protein called CAP Operator – The binding site of the repressor protein – The operator is located downstream (in the 3’ direction) from the promoter so that if repressor is bound RNA Polymerase can’t transcribe ©1999 Timothy G. Standish The Lac Operon: When Glucose Is Present But Not Lactose Come on, let me through Hey man, I’m constitutive Repressor CAP Binding Repressor mRNA RNA Pol. Promoter Operator LacZ LacY LacA Repressor No way Jose! Repressor CAP ©1999 Timothy G. Standish The Lac Operon: When Glucose And Lactose Are Present Great, I can transcribe! Hey man, I’m constitutive Repressor CAP Binding RNA Pol. Promoter Operator X Repressor mRNA Repressor Repressor LacZ LacY RNA LacA Pol. Repressor This lactose has bent me out of shape CAP Some transcription occurs, but at a slow rate ©1999 Timothy G. Standish The Lac Operon: When Lactose Is Present But Not Glucose Hey man, I’m constitutive Repressor CAP Binding CAP Bind to me Polymerase Yipee…! RNA Pol. Promoter Operator cAMP X Repressor mRNA LacZ RNA LacA Pol. LacY Repressor CAP cAMP Repressor Repressor This lactose has bent me out of shape cAMP CAP ©1999 Timothy G. Standish The Lac Operon: When Neither Lactose Nor Glucose Is Present Hey man, I’m constitutive Repressor CAP Binding CAP Bind to me Polymerase RNA Pol. Alright, I’m off to the races . . . Come on, let me through! Promoter Operator LacZ LacY LacA Repressor cAMP Repressor mRNA Repressor STOP Right there Polymerase CAP cAMP cAMP CAP ©1999 Timothy G. Standish The Trp Operon Genes in the trp operon allow E. coli bacteria to make the amino acid tryptophan Enzymes encoded by genes in the trp operon are all involved in the biochemical pathway that converts the precursor chorismate to tryptophan. The trp operon is controlled in two ways: – Using a repressor that works in exactly the opposite way from the lac operon repressor – Using a special attenuator sequence ©1999 Timothy G. Standish The Tryptophan Biochemical Pathway COO- Glutamine Glutamate + Pyruvate COO- CH2 5-Phosphoribosyla-Pyrophosphate NH2 COO- HO H O C H Anthranilate synthetase (trpE and D) Chorismate OH OH -2O PO 3 CH2 C C C -OOC OH PPi Anthranilate synthetase -2O P 3 O CH2 Anthranilate H CH2 C N-(5’-Phosphoribosyl)Anthranilate isomerase Indole- H Enol-1-oH H C 3’-glycerol phosphate synthetase N Carboxyphenylamino H H -1-deoxyribulose phosphate Glyceraldehyde- Tryptophan synthetase (trpB and A) H 3-phosphate Serine H2O -OOC C C HN N-(5’H Phosphoribosyl) -anthranilate OH H H N-(5’-Phosphoribosyl)-anthranilate OH isomerase Indole-3’-glycerol OH OH phosphate synthetase (trpC) CO2+H2O -2O PO 3 O -OOC C H C H N H Indole-3-glycerol phosphate CH2 NH3+ Tryptophan synthetase N H Indole N H Tryptophan ©1999 Timothy G. Standish The Trp Operon: When Tryptophan Is Present Hey man, I’m constitutive Repressor RNA Pol. Foiled Again! Promo. Operator Lead. Aten. trpE trpD trpC trpB trpA Repressor Trp Repressor mRNA STOP Right there Polymerase Repressor Trp ©1999 Timothy G. Standish The Trp Operon: When Tryptophan Is Absent Hey man, I’m constitutive RNA RNA Operator Repressor Promo. Lead. Aten. trpE trpD trpC trpBPol.trpA Pol. Repressor mRNA I need tryptophan Repressor needs his little buddy tryptophan if I’m to be stopped Repressor ©1999 Timothy G. Standish Attenuation The trp operon is controlled both by a repressor and attenuation Attenuation is a mechanism that works only because of the way transcription and translation are coupled in prokaryotes Therefore, to understand attenuation, it is first necessary to understand transcription and translation in prokaryotes ©1999 Timothy G. Standish Transcription And Translation In Prokaryotes 5’ 3’ 3’ 5’ RNA Pol. Ribosome mRNA Ribosome 5’ ©1999 Timothy G. Standish The Trp Leader and Attenuator Met-Lys-Ala-Ile-Phe-ValAAGUUCACGUAAAAAGGGUAUCGACA-AUG-AAA-GCA-AUU-UUC-GUALeu-Lys-Gly-Trp-Trp-Arg-Thr-Ser-STOP CUG-AAA-GGU-UGG-UGG-CGC-ACU-UCC-UGA-AACGGGCAGUGUAUU 1 2 CACCAUGCGUAAAGCAAUCAGAUACCCAGCCCGCCUAAUGAGCGGGCUUUU 3 4 Met-Gln-Thr-Gln-Lys-Pro UUUU-GAACAAAAUUAGAGAAUAACA-AUG-CAA-ACA-CAA-AAA-CCG trpE . . . Terminator ©1999 Timothy G. Standish The mRNA Sequence Can Fold In Two Ways 1 1 2 2 3 3 4 4 Terminator hairpin ©1999 Timothy G. Standish The Attenuator When Starved For Tryptophan 5’ 3’ 3’ Help, I need Tryptophan RNA Pol. 2 Ribosome 5’ 3 4 1 ©1999 Timothy G. Standish The Attenuator When Tryptophan Is Present 5’ 3’ 3’ Ribosome 5’ 2 RNA Pol. 3 4 1 ©1999 Timothy G. Standish Control Of Expression In Eukaryotes Some of the general methods used to control expression in prokaryotes are used in eukaryotes, but nothing resembling operons is known Eukaryotic genes are controlled individually and each gene has specific control sequences preceding the transcription start site In addition to controlling transcription, there are additional ways in which expression can be controlled in eukaryotes ©1999 Timothy G. Standish Eukaryotes Have Large Complex Genomes The human genome is about 3 x 109 base pairs or ≈ 1 m of DNA Because humans are diploid, each nucleus contains 6.3 x 109 base pairs or ≈ 2 m of DNA That is a lot to pack into a little nucleus! ©1999 Timothy G. Standish Eukaryotic DNA Must be Packaged Eukaryotic DNA exhibits many levels of packaging The fundamental unit is the nucleosome, DNA wound around histone proteins Nucleosomes arrange themselves together to form higher and higher levels of packaging. ©1999 Timothy G. Standish Highly Packaged DNA Cannot be Expressed The most highly packaged form of DNA is “heterochromatin” Heterochromatin cannot be transcribed, therefore expression of genes is prevented Chromosome puffs on some insect chomosomes illustrate where active gene expression is going on ©1999 Timothy G. Standish It Only a Subset of Genes is Expressed at any Given Time takes lots of energy to express genes Thus it would be wasteful to express all genes all the time By differential expression of genes, cells can respond to changes in the environment Differential expression, allows cells to specialize in multicelled organisms. Differential expression also allows organisms to develop over time. ©1999 Timothy G. Standish A “Simple” Eukaryotic Gene Transcription Start Site 5’ 5’ Untranslated Region Introns Exon 1 Int. 1 Promoter/ Control Region 3’ Untranslated Region Exon 2 3’ Int. 2 Exon 3 Exons Terminator Sequence RNA Transcript ©1999 Timothy G. Standish Eukaryotic mRNA 5’ Untranslated Region 5’ G Exon 1 Exon 2 3’ Untranslated Region Exon 3 AAAAA 3’ Protein Coding Region 5’ Cap RNA processing achieves three things: 3’ Poly A Tail Removal of introns Addition of a 5’ cap Addition of a 3’ tail This signals the mRNA is ready to move out of the nucleus and may control its lifespan in the cytoplasm ©1999 Timothy G. Standish Enhancers DNA Many bases 5’ 3’ Enhancer 5’ Promoter TF Transcribed Region 3’ TF 5’ TF TF RNA RNA Pol. Pol. 5’ 3’ RNA ©1999 Timothy G. Standish ©1999 Timothy G. Standish