1. 1 Gene regulation in Prokaryotes Bacteria were models for working out the basic mechanisms, but eukaryotes are different. Some genes are constitutive, others go from extremely low expression (“off”) to high expression when “turned on”. Many genes are coordinately regulated. –Operon: consecutive genes regulated, transcribed together; polycistronic mRNA. –Regulon: genes scattered, but regulated together.

2. 2 Rationale for Operon Many metabolic pathways require several enzymes working together. In bacteria, transcription of a group of genes is turned on simultaneously, a single mRNA is made, so all the enzymes needed can be produced at once. http://galactosaemia.com.hosting.domaindirect.com/images/metabolic-pathway.gif

3. 3 Proteins change shape http://omega.dawsoncollege.qc.ca/ray/genereg/operon3.JPG When a small molecule binds to the protein, it changes shape. If this is a DNA-binding protein, the new shape may cause it to attach better to the DNA, or “fall off” the DNA.

4. 4 Definitions concerning operon regulation Control can be Positive or Negative –Positive control means a protein binds to the DNA which increases transcription. –Negative control means a protein binds to the DNA which decreases transcription. Induction –Process in which genes normally off get turned on. –Usually associated with catabolic genes. Repression –Genes normally on get turned off. – Usually associated with anabolic genes.

5. 5 Structure of an Operon www.cat.cc.md.us 1.Structural genes: actual genes being regulated. 2.Promoter region: site for RNA polymerase to bind, begin transcription. 3.Operator region: site where regulatory protein binds. 4.Regulatory protein gene: need not be in the same area as the operon. Protein binds to DNA.

6. 6 Animations Look up Animations showing the effects of the lactose repressor on the lac operon. –As with translation, details will vary. For example, the lactose repressor protein is a tetramer. How many sites depict it this way? –Be wary of oversimplification.

7. 7 The Lactose Operon The model system for prokaryotic gene regulation, worked out by Jacob and Monod, France, 1960. The setting: E. coli has the genes for using lactose (milk sugar), but seldom sees it. Genes are OFF. –Repressor protein (product of lac I gene) is bound to the operator, preventing transcription by RNA polymerase. Green: repressor protein Purple: RNA polymerase

8. 8 Lactose operon-2 When lactose does appear, E. coli wants to use it. Lactose binds to repressor, causing shape change; repressor falls off DNA, allows unhindered transcription by RNA polymerase. Translation of mRNA results in enzymes needed to use lactose.

9. 9 Lactose operon definitions Control is Negative –When repressor protein is bound to the DNA, transcription is shut off. This operon is inducible –Lactose is normally not available as a carbon source; genes are “shut off” –In bacteria, many similar operons exist for using other organic molecules. –Proteins for transporting the sugar, breaking it down are produced.

10. 10 Repressible operons Operon codes for enzymes that make a needed amino acid (for example); genes are “on”. –Repressor protein is NOT attached to DNA –Transcription of genes for enzymes needed to make amino acid is occurring. The change: amino acid is now available in the culture medium. Enzymes normally needed for making it are no longer needed. –Amino acid, now abundant in cell, binds to repressor protein which changes shape, causing it to BIND to operator region of DNA. Transcription is stopped. This is also Negative regulation (protein + DNA = off).

11. 11 Repression picture Transcription by RNA polymerase prevented.

12. 12 Regulation can be fine tuned The more of the amino acid present in the cell, the more repressor-amino acid complex is formed; the more likely that transcription will be prevented.

13. 13 Positive regulation Binding of a regulatory protein to the DNA increases (turns on) transcription. –More common in eukaryotes. Prokaryotic example: the CAP-cAMP system –Catabolite-activating Protein –cAMP: ATP derivative, acts as signal molecule –When CAP binds to cAMP, creates a complex that binds to DNA, turning ON transcription. –Whether there is enough cAMP in the cell to combine with CAP depends on glucose conc.

14. 14 Positive regulation-2 Glucose is preferred nutrient source –Other sugars (lactose, etc.) are not. Glucose inhibits activity of adenylate cyclase, the enzyme that makes cAMP from ATP. When glucose is high, cAMP is low, less cAMP is available to bind to CAP. –CAP is “free”, doesn’t bind to DNA, genes not on. When glucose is low, cAMP is high –Lots of cAMP, so CAP-cAMP forms, genes on. Works in conjunction with induction.

15. 15 Cartoon of Positive Regulation

16. 16 Attenuation: fine tuning repression Attenuation occurs in prokaryotic repressible operons. Happens when transcription is on. Regulation at the level of translation Several things important: –Depends on base-pairing between complementary sequences of mRNA –Requires simultaneous transcription/translation –Involves delays in progression of ribosomes on mRNA

17. 17 Mechanism of attenuation- tryp operon

18. 18 Mech. of attenuation -2

19. 19 Attenuation-3