3. Lysogenic vs Lytic Life Cycle

4. 2007-2008 Prokaryotic (Bacterial) Genes

5. 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

6. 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 = inhibition - Now, that’s a good idea from a lowly bacterium! - -

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

8. 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

9. Animation http://bcs.whfreeman.com/thelifewire/conten t/chp13/1302001.html

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

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

12. operatorpromoter Operon model DNATATA RNA polymerase repressor = repressor protein Operon: operator, promoter & genes they control serve as a model for gene regulation gene1gene2gene3gene4 RNA polymerase Repressor protein turns off gene by blocking RNA polymerase binding site. 1234 mRNA enzyme1enzyme2enzyme3enzyme4

13. mRNA enzyme1enzyme2enzyme3enzyme4 operatorpromoter Repressible operon: tryptophan DNATATA RNA polymerase tryptophan repressor repressor protein repressor tryptophan – repressor protein complex Synthesis pathway model When excess tryptophan is present, it binds to tryp repressor protein & triggers repressor to bind to DNA – blocks (represses) transcription gene1gene2gene3gene4 conformational change in repressor protein! 1234 repressor trp RNA polymerase trp

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

15. mRNA enzyme1enzyme2enzyme3enzyme4 operatorpromoter Inducible operon: lactose DNATATA RNA polymerase repressor repressor protein repressor lactose – repressor protein complex lactose lac repressor gene1gene2gene3gene4 Digestive pathway model When lactose is present, binds to lac repressor protein & triggers repressor to release DNA – induces transcription RNA polymerase 1234 lac conformational change in repressor protein! lac

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

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

18. 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

19. Positive gene control occurs when an activator molecule interacts 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.

20. 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

21. 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.

22. 2007-2008 Control of Eukaryotic Genes

23. 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?

24. 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

25. 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

26. 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

27. 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

28. DNA methylation Methylation of DNA blocks transcription factors – no transcription  genes turned off – attachment of methyl groups (–CH 3 ) to cytosine C = cytosine – nearly permanent inactivation of genes ex. inactivated mammalian X chromosome = Barr body

29. Histone acetylation Acetylation of histones unwinds DNA  loosely wrapped around histones enables transcription genes turned on  attachment of acetyl groups (–COCH 3 ) to histones conformational change in histone proteins transcription factors have easier access to genes

30. 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

31. 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

32. 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

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

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

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

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

37. 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

38. 6-7. Protein processing & degradation Protein processing – folding, cleaving, adding sugar groups, targeting for transport Protein degradation – ubiquitin tagging – proteasome degradation Protein processing movie

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

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

41. 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

42. 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 NEW! siRNA

43. Action of siRNA siRNA double-stranded miRNA + siRNA mRNA degraded functionally turns gene off Hot…Hot new topic in biology mRNA for translation breakdown enzyme (RISC) dicer enzyme

44. initiation of transcription 1 mRNA splicing 2 mRNA protection 3 initiation of translation 6 mRNA processing 5 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 7 protein processing & degradation 4 4 Gene Regulation

45. 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)

46. 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

47. 2007-2008 Turn your Question Genes on!