1. 31 Gene regulation in bacteria

2. Lecture Outline 11/18/05 Finish up from last time: Transposable elements (“jumping genes”) Gene Regulation in Bacteria –Transcriptional control –Cells adjust to their environment by turning genes on and off The operon concept –Repressors, Inducers, Operators, Promoters Repressible operons (e.g. trp) Inducible operons (e.g. lac)

3. Transposable elements Normal and ubiquitous –Prokaryotes- Genes transpose to/from cell’s chromosome, plasmid, or a phage chromosome. –Eukaryotes- Genes transpose to/from same or a different chromosome. Cause genetic changes –Chromosome breaks –Duplications –Knock-out genes

4. I’ll talk about 2 kinds: Insertion sequences Ac/Ds elements in corn A third major class: Retrotransposons –Uses RNA intermediate and reverse transcriptase –Most Important class in mammalian genomes

5. Insertion sequence (IS) elements: Simplest type of transposable element –Found in bacterial chromosomes and plasmids. –Encode only genes for mobilization and insertion. Inverted terminal repeats

6. Integration of an Insertion Element Don’t worry about the details, just the concept Staggered cut at target site Insert IS element Fill in the gaps IS element carries transposase gene Transposase recognizes terminal repeats

7. Transposons Have additional genes, such as those for antibiotic resistance (examples Tn3 (ampicillin), Tn10 (tetracycline) Figure 18.19b Inverted repeats Transposase gene Insertion sequence Antibiotic resistance gene Transposon 5353 5353

8. Barbara McClintock’s discovery of transposons in corn: Kernel color alleles/traits were “unstable”. McClintock concluded transposon called “Ds” inserted into the “C” gene for colored kernels Nobel prize, 1983

9. Transposon effects on corn kernel color. Ac activates Ds Two transposable elements in different sites Normal gene for purple kernels Ds element inserts into color gene and inactivates it Ac can make transposase Ds can move, but lacks enzyme

10. One method for Conservative Transposition “Cut and Paste” Transposable element is cut out by transposase and inserts in another location. No increase in the number of transposable elements- just a change in position From Griffiths, Intro to Genetic Analysis

11. One method for replicative transposition From Griffiths, Intro to Genetic Analysis

12. Gene regulation in bacteria But ALL organisms must adjust to changes in their environment and all have evolved numerous control mechanisms. E.coli bacteria eat whatever we eat!

13. Regulation of metabolism occurs at two levels: –Adjusting the activity of metabolic enzymes already present –Regulating the genes encoding the metabolic enzymes Figure 18.20a, b (a) Regulation of enzyme activity Enzyme 1 Enzyme 2 Enzyme 3 Enzyme 4 Enzyme 5 Regulation of gene expression Feedback inhibition Tryptophan Precursor (b) Regulation of enzyme production Gene 2 Gene 1 Gene 3 Gene 4 Gene 5 – –

14. Types of Regulated Genes Constitutive genes are always expressed –Tend to be vital for basic cell functions (often called “housekeeping genes”) Inducible genes are normally off, but can be turned on when substrate is present Common for catabolic enzymes (i.e. for the utilization of particular resources) Repressible genes are normally on, but can be turned off when the end product is abundant Common for anabolic enzymes

15. In bacteria, genes are often clustered into operons Operons have: 1.Several genes for metabolic enzymes 2.One promoter 3.An operator, or control site (“on-off” switch) 4.A separate gene that makes a repressor or activator protein that binds to the operator RO123PP

16. The trp Operon 5 genes: E, D, C, B, A Same order as enzymes for trp synthesis Controlled by a single promoter and operator

17. More Terminology Repressors and Activators are proteins that bind to DNA and control transcription. Co-repressors and Inducers: small “effector” molecules that bind to repressors or activators

18. Genes of operon Protein Operator Polypeptides that make up enzymes for tryptophan synthesis Regulatory gene RNA polymerase Promoter trp operon 5 3 mRNA trpD trpE trpCtrpB trpA trpR DNA mRNA ED C BA The trp operon: regulated synthesis of repressible enzymes Figure 18.21a 5 Tryptophan absent -> repressor inactive -> operon “on”

19. DNA mRNA Protein Tryptophan (corepressor) Active repressor No RNA made Tryptophan present -> repressor active -> operon “off”. Figure 18.21b Active repressor can bind to operator and block transcription

20. Tryptophan changes the shape of the repressor protein so it can bind DNA

21. The lac operon: regulated synthesis of inducible enzymes Figure 18.22a DNA mRNA Protein Active repressor RNA polymerase No RNA made lacZ lacl Regulatory gene Operator Promoter Lactose absent, repressor active, operon off. The lac repressor is innately active, and in the absence of lactose it switches off the operon by binding to the operator. (a) 5 3

22. mRNA 5' DNA mRNA Protein Allolactose (inducer) Inactive repressor lacl lacz lacYlacA RNA polymerase PermeaseTransacetylase  -Galactosidase 5 3 (b) Lactose present, repressor inactive, operon on. Allolactose, an isomer of lactose, derepresses the operon by inactivating the repressor. In this way, the enzymes for lactose utilization are induced. mRNA 5 lac operon Figure 18.22b

24. Positive Gene Regulation Both the trp and lac operons involve negative control of genes –because the operons are switched off by the active form of the repressor protein Some operons are also subject to positive control –Via a stimulatory activator protein, such as catabolite activator protein (CAP)

25. Promoter Lactose present, glucose scarce (cAMP level high): abundant lac mRNA synthesized. If glucose is scarce, the high level of cAMP activates CAP, and the lac operon produces large amounts of mRNA for the lactose pathway. (a) CAP-binding site Operator RNA polymerase can bind and transcribe Inactive CAP Active CAP cAMP DNA Inactive lac repressor lacl lacZ Figure 18.23a –In E. coli, when glucose is always the preferred food source –When glucose is scarce, the lac operon is activated by the binding of the catabolite activator protein (CAP) Positive Gene Regulation- CAP

26. When glucose is abundant, – CAP detaches from the lac operon, which prevents RNA polymerase from binding to the promoter Figure 18.23b (b) Lactose present, glucose present (cAMP level low): little lac mRNA synthesized. When glucose is present, cAMP is scarce, and CAP is unable to stimulate transcription. Inactive lac repressor Inactive CAP DNA RNA polymerase can’t bind Operator lacl lacZ CAP-binding site Promoter