1. Controlling Gene Expression

2. 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.

3. 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

4. 3’3’ 5’5’ 5’5’ 3’3’ Transcription And Translation In Prokaryotes Ribosome 5’5’ mRNA RNA Pol.

5. 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 key to understanding all operons is to consider the action of the repressor protein.

6. The Trp Operon: When Tryptophan Is Absent Repressor Promo. trpDtrpBLead. Operator trpAtrpCtrpEAten. RNA Pol. Repressor mRNA Hey man, I’m constitutive RNA Pol.

7. The Trp Operon: When Tryptophan Is Present STOP Right there Polymerase Trp Repressor Promo. trpDtrpBLead. Operator trpAtrpCtrpEAten. RNA Pol. Foiled Again! Repressor mRNA Hey man, I’m constitutive

8. 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

9. 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 hydrolizes 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

10. 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 down stream (in the 3’ direction) from the promoter so that if repressor is bound RNA Polymerase can’t transcribe

11. The Lac Operon: No Lactose RepressorPromoter LacYLacALacZ Operator CAP Binding RNA Pol. Repressor mRNA Hey man, I’m constitutive Come on, let me through No way Jose!

12. The Lac Operon: When Lactose is Present RepressorPromoter LacYLacALacZ Operator CAP Binding Repressor mRNA Hey man, I’m constitutive Lac Repressor X RNA Pol. RNA Pol. Great, I can transcribe! Some transcription occurs, but at a slow rate This lactose has bent me out of shape

13. Inducible v. Repressible Systems The Trp operon is a REPRESSIBLE operon because the PRODUCT of the operon binds allosterically to a regulatory protein (the repressor) activating it and causing it to block transcription – turn OFF the operon. The lac Operon is an INDUCIBLE operon because the SUBSTRATE of the operon binds allosterically to a regulatory protein (the repressor) inactivating it, thus preventing it from binding to the operator and thus allowing transcription – turning ON the operon. Both of the situations described above are showing Operons under NEGATIVE control because both operons are switched OFF by the ACTIVE form of the repressor protein.

14. NEGATIVE Regulation When the ACTIVE FORM of the regulatory protein SWITCHES OFF transcription, the regulation is said to be NEGATIVE. The trp operon is always under negative control However the lac operon can demonstrate both positive and negative regulation under certain circumstances.

15. POSITIVE Regulation When the ACTIVE form of a regulatory protein ACTIVATES transcription – turns the Operon ON the regulation is said to be positive The key to understanding the lac Operon under positive control is to realize that we need to ADD another regulatory protein and TWO MORE nutritional situations

16. Lac Operon Old and New Two previously discussed conditions are –Lactose Present and Lactose Absent But glucose is the preferred metabolite of the E.Coli so we need to consider the situation when glucose is present or absent. The levels of glucose and cAMP are inversly proportional –When glucose level is low, cAMP High and vice versa

17. cAMP and CRP CAP – cAMP Receptor Protein is an inactive protein that is activated by cAMP When active the cAMP-CRP complex binds to the CAP binding site Once bound to this site it causes a more “efficient” binding of RNA polymerase resulting in increased transcription rate Thus the definition of positive control – “the active form of a protein increases transcription” has been met

18. Overview Presenc e of Lactose YES On – but low rate NOYES Under these circumstances since Glucose is present, Lactose would not be the preferred food supply and cAMP levels low so no cAMP/CAP complex to enhance transcription. Therefore transcription would occur but at low levels. On + max rate! NO Under these circumstances, there is no Lactose, so there is no need for the enzymes that break it down to be transcribed. Off YESNO Again while the lack of glucose indicates the cAMP/CAP complex would enhance transcription, there is no Lactose so no need for the enzymes that break it down Off ExplanationAction of Operon Presenc e of Glucose When Glucose is absent cAMP levels are high. Thus cAMP would bind to CAP to form a cAMP/CAP complex which enhances binding of RNA Pol to the Promotor and enhances Transcription

19. The Lac Operon: When Lactose Is Present But Not Glucose RepressorPromoter LacYLacALacZ Operator CAP Binding Repressor mRNA Hey man, I’m constitutive CAP cAMP Lac Repressor X This lactose has bent me out of shape CAP cAMP CAP cAMP Bind to me Polymerase RNA Pol. RNA Pol. Yipee…!

20. The Lac Operon: When Neither Lactose Nor Glucose Is Present RepressorPromoter LacYLacALacZ Operator CAP Binding CAP cAMP CAP cAMP CAP cAMP Bind to me Polymerase RNA Pol. Repressor mRNA Hey man, I’m constitutive Repressor STOP Right there Polymerase Alright, I’m off to the races... Come on, let me through!

21. The Lac Operon: When Glucose Is Present But Not Lactose RepressorPromoter LacYLacALacZ Operator CAP Binding RNA Pol. Repressor mRNA Hey man, I’m constitutive Come on, let me through No way Jose! CAP

22. The Lac Operon: When Glucose And Lactose Are Present RepressorPromoter LacYLacALacZ Operator CAP Binding Repressor mRNA Hey man, I’m constitutive CAP Lac Repressor X RNA Pol. RNA Pol. Great, I can transcribe! Some transcription occurs, but at a slow rate This lactose has bent me out of shape

23. Summary Presenc e of Lactose YESNO YES NOYES NO ExplanationAction of Operon Presenc e of Glucose

24. 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

25. Eukaryotes Have Large Complex Geneomes The human genome is about 3 x 10 9 base pairs or ≈ 1 m of DNA Because humans are diploid, each nucleus contains 6 x 10 9 base pairs or ≈ 2 m of DNA That is a lot to pack into a little nucleus!

26. 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.

27. 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

28. Only a Subset of Genes is Expressed at any Given Time It 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.

29. DNA Cytoplasm Nucleus G AAAAAA Export Degradation etc. G AAAAAA Control of Gene Expression G AAAAAA RNA Processing mRNA RNA Transcription Nuclear pores Ribosome Translation Packaging Modification Transportation Degradation

30. Logical Expression Control Points DNA packaging Transcription RNA processing mRNA Export mRNA masking/unmasking and/or modification mRNA degradation Translation Protein modification Protein transport Protein degradation Increasing cost The logical place to control expression is before the gene is transcribed

31. A “Simple” Eukaryotic Gene Terminator Sequence Promoter/ Control Region Transcription Start Site 3’3’5’5’ RNA Transcript Introns Exon 2Exon 3 Int. 2 Exon 1 Int. 1 3’ Untranslated Region 5’ Untranslated Region Exons

32. Eukaryotic mRNA Protein Coding Region 3’ Untranslated Region5’ Untranslated Region Exon 2Exon 3Exon 1 AAAAA G 3’3’5’5’ 3’ Poly A Tail5’ Cap RNA processing achieves three things: 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 life span in the cytoplasm