1. Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint 11-1 Chapter 11: Gene expression

2. Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint 11-2 Regulation of gene expression Some proteins are required continuously or often—the genes encoding them are expressed constitutively Many other proteins are required for specific purposes—the genes are inducible under particular conditions

3. Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint 11-3 Control points for expression Transcriptional regulation Post-transcriptional regulation –includes co-transcriptional control—RNA splicing –mRNA stability and transport Translation Post-translational regulation –protein stability –activation or inactivation e.g. cleavage or phosphorylation

4. Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint 11-4 Prokaryote gene expression Most gene regulation is transcriptional in prokaryotes Prokaryotic genes are located in functional groups Genes encoding enzymes in the same pathway are arranged in sequence under the control of a single promoter (cont.)

5. Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint 11-5 Prokaryote gene expression (cont.) The genes and their regulatory sequences are called an operon Transcription from the promoter produces multi- gene RNA transcripts Multiple polypeptides are translated from these RNAs

6. Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint 11-6 Induction Expression of inducible genes is either positive or negative at the level of transcription Positive regulation (induction) is often used in the catabolism of compounds for energy production— the presence of the molecule stimulates expression of genes in the pathway (Fig. 11.2 (a))

7. Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint 11-7 Repression Negative regulation (repression) may be used in anabolic or synthetic pathways, where the presence of the synthesised compound represses the expression of the genes in the pathway Thus the genes are only expressed in the absence of the compound (Fig. 11.2 (b))

8. Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint 11-8 Fig 11.2(b): Repression

9. Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint 11-9 Bacterial operons (Fig 11.3) An example is the lac operon, responsible for the metabolism of lactose to glucose and galactose

10. Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint 11-10 The lac operon The genes in the lac operon are induced in the presence of lactose in the culture media The lac operon is an example of negative regulation—the genes are repressed in the absence of lactose The repressor protein binds to lacO, the operator region of the lac promoter, and prevents transcription by blocking RNA polymerase binding to the promoter (cont.)

11. Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint 11-11 The lac operon (cont.) A metabolite of lactose called allolactose binds to a repressor protein and changes its shape, preventing binding to the lac promoter This is called an allosteric interaction and is a common molecular regulatory mechanism As lactose is used up, the free repressor protein can bind to the promoter and prevent transcription (cont.)

12. Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint 11-12 Fig 11.4: Regulation of the lac operon (a)

13. Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint 11-13 Fig 11.4: Regulation of the lac operon (b) (c)

14. Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint 11-14 The lac operon (cont.) The lac operon is also positively regulated When glucose is present as well as lactose, the lac operon is repressed to allow metabolism of glucose This is mediated by the catabolite activator protein (CAP) cAMP normally binds to CAP protein (Fig. 11.5) allowing transcription from the operon in the absence of repressor binding (cont.)

15. Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint 11-15 The lac operon (cont.) High levels of glucose lower cAMP concentration The CAP–cAMP complex cannot form and transcription is reduced even in the presence of lactose The double regulation of the lac operon ensures that it is only expressed at low glucose concentrations when lactose is present

16. Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint 11-16 Fig 11.5: Regulation of the lac operon in (a) low and (b) high glucose conditions (a) (b)

17. Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint 11-17 Fig 11.6: Expression of the lac operon

18. Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint 11-18 The trp operon This operon contains five genes for tryptophan biosynthesis under the control of a single promoter The absence of tryptophan leads to trp operon expression Regulatory events are similar to lac operon The trp repressor protein binds to the operator sequence only in the presence of tryptophan (cont.)

19. Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint 11-19 The trp operon (cont.) Tryptophan binds to the trp repressor protein, changing its conformation and allowing binding Transcription cannot proceed Low levels of tryptophan prevent repressor binding Transcription and synthesis of tryptophan proceed

20. Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint 11-20 Fig 11.7: Regulation of the trp operon

21. Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint 11-21 Gene regulation in eukaryotes Most eukaryotic genes are controlled at the level of transcription The mechanisms involve trans-acting regulatory proteins interacting with cis-acting control sequences on the DNA DNA control sequences include promoters, promoter-proximal elements, enhancers and silencers (cont.)

22. Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint 11-22 Gene regulation in eukaryotes (cont.) Eukaryotic promoters do not provide sufficient recognition signals for RNA polymerase II to initiate transcription in vivo. Core promoter elements must each be recognised by regulatory proteins (basal factors and coactivators) which bind to these sites RNA polymerase II is bound and initiated properly and transcription commences

23. Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint 11-23 Fig 11.8: Regulation of transcription in eukaryotic cells

24. Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint 11-24 Yeast GAL regulation The yeast Saccharomyces cerevisiae is a good model of eukaryote gene expression In the presence of a medium containing galactose, five genes are expressed Unlike the genes in an operon, these five genes are separate and transcribed independently The expression of the genes is controlled by both positive and negative protein regulators (cont.)

25. Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint 11-25 Yeast GAL regulation (cont.) The GAL4 (Gal4p) protein binds to an upstream DNA activator sequence (UAS) on all five genes and induces their expression The GAL80 protein (Gal80p) acts as a transcription repressor by binding to Gal4p, preventing the induction of transcription When galactose is present a metabolite dissociates the Gal80p from the Gal4p, allowing transcription to proceed

26. Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint 11-26 Fig 11.10: Regulation of galactose (GAL)-inducible genes in yeast

27. Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint 11-27 Enhancers and silencers Series of DNA sequences that are recognised and bound to by DNA-binding proteins called transcription factors Enhancers and silencers can, however, act over longer distances than promoters (up to 50kb), and may be positioned either upstream or downstream of the promoter they enhance Each enhancer or silencer element acts independently of others

28. Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint 11-28 Regulatory cascades Transcription factors bind to enhancer or silencer sequences They are often tissue-specific, allowing a complex pattern of gene expression Transcription factors are themselves gene products Coordinated expression of many genes can therefore be achieved by just a few genes Such regulatory cascades are important in the development of complex organisms

29. Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint 11-29 DNA conformation Coiling of DNA into nucleosomes during chromosome condensation inhibits transcription Acetylation of histone proteins binding the nucleosomes reduces the DNA binding strength DNA is not coiled so tightly and RNA polymerase II can bind to initiate transcription Enhancer-binding complexes often include histone-acetylating enzymes

30. Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint 11-30 RNA-mediated regulation Not all regulatory genes act by producing a protein product Small RNA molecules have been discovered that bind directly to mRNA and block their translation These ‘microRNAs’ provide another level of gene regulation

31. Copyright  2005 McGraw-Hill Australia Pty Ltd PPTs t/a Biology: An Australian focus 3e by Knox, Ladiges, Evans and Saint 11-31 Fig 11.11: Control of gene expression by microRNAs