Chapter 18~Regulaton of Gene Expression

1. Chapter 18~Regulaton of Gene Expression

2. Control of Prokaryotic (Bacterial) Genes

3. Bacterial metabolism • Bacteria need to respond quickly to changes in their environment • if they have enough of a product, need to stop production • why? waste of energy to produce more • how? stop production of enzymes for synthesis • if they find new food/energy source, need to utilize it quickly • why? metabolism, growth, reproduction • how? start production of enzymes for digestion STOP GO

4. - - = inhibition Remember Regulating Metabolism? • Feedback inhibition • product acts as an allosteric inhibitor of 1st enzyme in tryptophan pathway • but this is wasteful production of enzymes Oh, I remember thisfrom our Metabolism Unit!

5. - - - = inhibition Different way to Regulate Metabolism • Gene regulation • instead of blocking enzyme function, block transcription of genes for all enzymes in tryptophan pathway • saves energy by not wasting it on unnecessary protein synthesis Now, that’s a good idea from a lowly bacterium!

6. Gene regulation in bacteria • Cells vary amount of specific enzymes by regulating gene transcription • turn genes on or turn genes off • turn genes OFF exampleif bacterium has enough tryptophan then it doesn’t need to make enzymes used to build tryptophan • turn genes ON exampleif bacterium encounters new sugar (energy source), like lactose, then it needs to start making enzymes used to digest lactose STOP GO

7. Bacteria group genes together • Operon • genes grouped together with related functions • example: all enzymes in a metabolic pathway • promoter = RNA polymerase binding site • single promoter controls transcription of all genes in operon • transcribed as one unit & a single mRNA is made • operator = DNA binding site of repressor protein

8. So how can these genes be turned off? • Repressor protein • binds to DNA at operator site • blocking RNA polymerase • blocks transcription

9. RNA polymerase RNA polymerase repressor repressor enzyme1 1 enzyme2 2 enzyme3 3 enzyme4 4 promoter = repressor protein operator Operon model Operon: operator, promoter & genes they control serve as a model for gene regulation gene1 gene2 gene3 gene4 TATA DNA mRNA Repressor protein turns off gene by blocking RNA polymerase binding site.

10. trp RNA polymerase RNA polymerase repressor repressor repressor enzyme1 1 enzyme2 2 enzyme3 3 enzyme4 4 promoter repressor protein operator tryptophan trp trp trp trp trp trp trp trp trp tryptophan – repressor protein complex Repressible operon: tryptophan Synthesis pathway model When excess tryptophan is present, it binds to tryp repressor protein & triggers repressor to bind to DNA • blocks (represses) transcription gene1 gene2 gene3 gene4 TATA DNA mRNA trp conformational change in repressor protein! trp

11. Tryptophan operon What happens when tryptophan is present? Don’t need to make tryptophan-building enzymes Tryptophan is allosteric regulator of repressor protein

12. RNA polymerase RNA polymerase repressor repressor repressor enzyme1 1 enzyme2 2 enzyme3 3 enzyme4 4 promoter repressor protein operator lactose lac lac lac lac lac lac lac lactose – repressor protein complex Inducible operon: lactose Digestive pathway model When lactose is present, binds to lac repressor protein & triggers repressor to release DNA • induces transcription lac gene1 gene2 gene3 gene4 TATA DNA mRNA lac conformational change in repressor protein! lac

13. Lactose operon What happens when lactose is present? Need to make lactose-digesting enzymes Lactose is allosteric regulator of repressor protein

14. 1961 | 1965 Jacob & Monod: lac Operon • Francois Jacob & Jacques Monod • first to describe operon system • coined the phrase “operon” Jacques Monod Francois Jacob

15. Operon summary • Repressible operon • usually functions in anabolic pathways • synthesizing end products • when end product is present in excess,cell allocates resources to other uses • Inducible operon • usually functions in catabolic pathways, • digesting nutrients to simpler molecules • produce enzymes only when nutrient is available • cell avoids making proteins that have nothing to do, cell allocates resources to other uses

16. Positive gene control • occurswhen an activator moleculeinteracts directly with the genome to switch transcription on. • Even if the lac operon is turned on by the presence of allolactose, the degree of transcription depends on the concentrations of other substrates. • The cellular metabolism is biased toward the utilization of glucose.

17. Positive Gene Regulation • Some operons are also subject to positive control through a stimulatory protein, such as catabolite activator protein (CAP), an activator of transcription • When glucose (a preferred food source of E. coli) is scarce, CAP is activated by binding with cyclic AMP • Activated CAP attaches to the promoter of the lac operon and increases the affinity of RNA polymerase, thus accelerating transcription

18. Positive Gene Regulation • If glucose levels are low (along with overall energy levels), then cyclic AMP(cAMP) binds to cAMP receptor protein (CRP) which activates transcription. • If glucose levels are sufficient and cAMP levels are low (lots of ATP), then the CRP protein has an inactive shape and cannot bind upstream of the lac promotor.

19. Control of Eukaryotic Genes

20. The BIG Questions… • How are genes turned on & off in eukaryotes? • How do cells with the same genes differentiate to perform completely different, specialized functions?

21. Evolution of gene regulation • Prokaryotes • single-celled • evolved to grow & divide rapidly • must respond quickly to changes in external environment • exploit transient resources • Gene regulation • turn genes on & off rapidly • flexibility & reversibility • adjust levels of enzymes for synthesis & digestion

22. Evolution of gene regulation • Eukaryotes • multicellular • evolved to maintain constant internal conditions while facing changing external conditions • homeostasis • regulate body as a whole • growth & development • long term processes • specialization • turn on & off large number of genes • must coordinate the body as a whole rather than serve the needs of individual cells

23. Points of control • The control of gene expression can occur at any step in the pathway from gene to functional protein 1. packing/unpacking DNA 2. transcription 3. mRNA processing 4. mRNA transport 5. translation 6. protein processing 7. protein degradation

24. 1. DNA packing as gene control • Degree of packing of DNA regulates transcription • tightly wrapped around histones • no transcription • genes turned off • heterochromatin darker DNA (H) = tightly packed • euchromatin lighter DNA (E) = loosely packed H E

25. DNA methylation • Methylation of DNA blocks transcription factors • no transcription  genes turned off • attachment of methyl groups (–CH3) to cytosine • C = cytosine • nearly permanent inactivation of genes • ex. inactivated mammalian X chromosome = Barr body

26. Histone acetylation • Acetylation of histones unwinds DNA • loosely wrapped around histones • enables transcription • genes turned on • attachment of acetyl groups (–COCH3) to histones • conformational change in histone proteins • transcription factors have easier access to genes

27. Epigenetic Inheritance • Although the chromatin modifications just discussed do not alter DNA sequence, they may be passed to future generations of cells • The inheritance of traits transmitted by mechanisms not directly involving the nucleotide sequence is called epigenetic inheritance

28. 2. Transcription initiation • Control regions on DNA • promoter • nearby control sequence on DNA • binding of RNA polymerase & transcription factors • “base” rate of transcription • enhancer • distant control sequences on DNA • binding of activator proteins • “enhanced” rate (high level) of transcription

29. Model for Enhancer action • Enhancer DNA sequences • distant control sequences • Activator proteins • bind to enhancer sequence & stimulates transcription • Silencer proteins • bind to enhancer sequence & block gene transcription Turning on Gene movie

30. Transcription complex Activator Proteins • regulatory proteins bind to DNA at distant enhancer sites • increase the rate of transcription Enhancer Sites regulatory sites on DNA distant from gene Enhancer Activator Activator Activator Coactivator E F B RNA polymerase II H TFIID A Coding region T A T A Core promoter and initiation complex Initiation Complex at Promoter Site binding site of RNA polymerase

31. Fig. 18-9-3 Promoter Activators Gene DNA Distal control element Enhancer TATA box General transcription factors DNA-bending protein Group of mediator proteins RNA polymerase II RNA polymerase II Transcription initiation complex RNA synthesis

32. 3. Post-transcriptional control • Alternative RNA splicing • variable processing of exons creates a family of proteins

33. 4. Regulation of mRNA degradation • Life span of mRNA determines amount of protein synthesis • mRNA can last from hours to weeks RNA processing movie

34. 5. Control of translation • Block initiation of translation stage • regulatory proteins attach to 5' end of mRNA • prevent attachment of ribosomal subunits & initiator tRNA • block translation of mRNA to protein Control of translation movie

35. 6-7. Protein processing & degradation • Protein processing • folding, cleaving, adding sugar groups, targeting for transport • Protein degradation • ubiquitin tagging • proteasome degradation Protein processing movie

36. Ubiquitin 1980s | 2004 • “Death tag” • mark unwanted proteins with a label • 76 amino acid polypeptide, ubiquitin • labeled proteins are broken down rapidly in "waste disposers" • proteasomes Aaron Ciechanover Israel Avram Hershko Israel Irwin Rose UC Riverside

37. Proteasome • Protein-degrading “machine” • cell’s waste disposer • breaks down any proteins into 7-9 amino acid fragments • cellular recycling play Nobel animation

38. Concept 18.3: Noncoding RNAs play multiple roles in controlling gene expression • Only a small fraction of DNA codes for proteins, rRNA, and tRNA • A significant amount of the genome may be transcribed into noncoding RNAs • Noncoding RNAs regulate gene expression at two points: mRNA translation and chromatin configuration

39. NEW! RNA interference • Small interfering RNAs (siRNA) • short segments of RNA (21-28 bases) • bind to mRNA • create sections of double-stranded mRNA • “death” tag for mRNA • triggers degradation of mRNA • cause gene “silencing” • post-transcriptional control • turns off gene = no protein produced siRNA

40. Hot…Hotnew topicin biology Action of siRNA dicerenzyme mRNA for translation siRNA double-stranded miRNA + siRNA breakdownenzyme (RISC) mRNA degraded functionally turns gene off

41. Gene Regulation 7 6 protein processing & degradation 1 & 2. transcription - DNA packing - transcription factors 3 & 4. post-transcription - mRNA processing - splicing - 5’ cap & poly-A tail - breakdown by siRNA 5. translation - block start of translation 6 & 7. post-translation - protein processing - protein degradation 5 4 initiation of translation mRNAprocessing 2 1 initiation of transcription mRNA protection 4 mRNA splicing 3

42. Molecular Biology of Cancer • Oncogene •cancer-causing genes • Proto-oncogene •normal cellular genes • How? 1-movement of DNA; chromosome fragments that have rejoined incorrectly 2-amplification; increases the number of copies of proto-oncogenes • 3-proto-oncogene point mutation; protein product more active or more resistant to degradation • Tumor-suppressor genes •changes in genes that prevent uncontrolled cell growth (cancer growth stimulated by the absence of suppression)

43. Cancers result from a series of genetic changes in a cell lineage • The incidence of cancer increases with age because multiple somatic mutations are required to produce a cancerous cell • As in many cancers, the development of colon cancer is gradual