31 Gene regulation in bacteria

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

7. Transposons Have additional genes, such as those for antibiotic resistance • (examples Tn3 (ampicillin), Tn10 (tetracycline) Transposon Antibioticresistance gene Insertion sequence Insertion sequence 5 3 5 3 Inverted repeats Transposase gene Figure 18.19b

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

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

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

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: • Several genes for metabolic enzymes • One promoter • An operator, or control site (“on-off” switch) • A separate gene that makes a repressor or activatorprotein that binds to the operator R P P O 1 2 3

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

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

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

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

21. Promoter Regulatorygene Operator DNA lacl lacZ NoRNAmade 3 RNApolymerase mRNA 5 Activerepressor Protein (a) Lactose absent, repressor active, operon off. The lac repressor is innately active, and inthe absence of lactose it switches off the operon by binding to the operator. Figure 18.22a • The lac operon: regulated synthesis of inducible enzymes

22. lac operon DNA lacl lacz lacY lacA RNApolymerase 3 mRNA 5 mRNA 5' mRNA 5 -Galactosidase Permease Transacetylase Protein Inactiverepressor Allolactose(inducer) (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. 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. Operator RNA polymerase can bindand transcribe Promoter DNA lacl lacZ CAP-binding site ActiveCAP cAMP Inactive lac repressor InactiveCAP (a) 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. Figure 18.23a Positive Gene Regulation- CAP • 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)

26. Promoter Operator DNA lacl lacZ CAP-binding site RNA polymerase can’t bind InactiveCAP Inactive lac repressor 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. (b) Figure 18.23b • When glucose is abundant, • CAP detaches from the lac operon, which prevents RNA polymerase from binding to the promoter