基因表达和调控

1. 基因表达和调控

3. The most direct control point • At the level oftranscription:Regulate by changing sets of genes’ expression level in response to environment alteration Efficiency and economics

4. Why is Regulation necessary • Not all genes are expressed continuously:the level of gene expression differ according to--cell types, stages of cell cycle • Organisms live inchanging environments • Regulation allows organisms to grow and reproduce optimally in different environments

5. Are all genes’ activity regulated? • The housekeeping genes (constitutive genes) • genes that are essential for normal cell function and are constitutionally expressed. • The regulated genes • Their activity is controlled in response to the needs of a cell or organism

6. Regulation of Gene Expression Operons: Fine Control of ProkaryoticTranscription

7. Operons: Fine Control ofProkaryoticTranscription • The genes, the operator, and the promoter constitute an operon • The genes are adjacent to each other and are transcribed into a polygenic (polycistronic) mRNA

8. The lac operon repression model • In the absence of lac

9. The repressor protein • The repressor protein is a tetramer, • repressor is an allosteric protein, with two domains: a DNA binding domain an inducer binding domain Inducer binding structural change at DNA binding domain lose its DNA binding ability transcription repression relieved.

10. In the presence of lactose The true inducer is allolactose

11. Regulation of Gene Expression in Eukaryote

12. Differences between Prokary. and Eukary. • Prokaryotes: unicellular, free living, gene organization--operons, control--short term T/C • Eukaryotes: unicellular or multicellular. No operon organization, control---short term and long term • goal: to coordinate the generation of new proteins in different cells at different times

13. Levels of gene expression control in eukaryotes mRNA Transcription mRNA processing mRNA transport mRNA translation mRNA degradation protein processing protein degradation

14. Transcriptional ControlShort Term Regulation • Transcriptional control regulates whether a gene is transcribed and the rate at which transcripts are produced

15. Transcriptional Control • Positive (negative) regulatory element (TATA, etc.) • Positive (negative) regulatory protein

16. Transcriptional Control Protein coding gene has 1. Core promoter elements: RNAPII, TFs 2. Regulatory elements: regulatory Proteins 3. Enhancers: regulatory Proteins

17. Core promoter elementsDNA sequences for RNAP II and TFIIs

18. A model of typical gene & regulatory elements

19. Transcription machinery binding and Chromosome remodeling Chromosome remodeling Complex ------ATP-dependent nucleosome remodeling (SWI/SNF) The effects of modification: a. Promoter accessibility: ------modification loosens up chromotin structure, increase nucleosome mobility. b. Attracts some specific DNA binding proteins:

20. Histone and T/C Regulation Transcription machinery binding and Chromosome remodeling

21. Histone and T/C Regulation Transcription machinery binding and Chromosome remodeling

22. Transcription Regulatory Proteins • DNA binding proteins: recognize specific DNA sequence or structure: • most of them have distinctive structural motifs: • Zn finger • leucine zipper • Helix-Turn-Helix. • Some have less distinctive structural motifs, therefore, have less restrictions for their DNA target. • Some regulatory proteins do not bind to DNA directly.

23. DNA binding domain: Zn finger • Zn binding involves two Cys and two His a.a. • the finger is a coiled coil • binds to the major groove of DNA double helix

24. DNA binding domain: leucine zipper • Leucine zipper proteins are dimers, the zipper helix is at the C-terminal of the protein • There are leucine at every 7th position of the helix, face to face in the dimer • The N-terminal helices has + charged amino acids: binding major groove

25. DNA binding domain: HTH/homeodomain • three short helices separated by turns • the helix proximal to C-terminal is needed for DNA binding • the other two helices needed for protein dimer formation • Drosophila: Homeodomain proteins controls the development of drosophila, all are DNA-binding proteins with HTH motif.

26. What’s different between specific and non-specific DNA bindings? • Specific DNA binding proteins interact with the specific bases in a given sequence • Non-specific DNA binding proteins mostly interact with the phosphate backbone instead of the base

27. Histone and Gene Regulation • Histone modification Phosphorylation Acetylation Methylation • Histone must be modified to loosen their grip on the DNA or be displaced from the DNA so that DNA strands can interact with transcription factors or regulatory proteins • In essence, the histones act as general repressors of transcription

28. Histone Acetylation and Transcription Regulation • Active chromatin: Hyper-acetylated • Inactive chromatin: Hypo-acetylated • Acetylation of the lysine at the out sphere of nucleosome: cause conformational change, destabilizes chromatin structure • Acetylation make nucleosomes becomes more accessible to transcription factors such as bromodomain bearing TFs • Protein (histone acetyl transferases,HATs; histone deacetylases, HDACs) are involved in this event

29. Histone Methylation and Transcription Regulation • Histone methylation and demethylation by histone methyltransferases and histone demethylases are also related to transcription regulation • Certain domains (chromo, tudor etc.) can recognize methylated histone

30. DNA Methylation and T/C Regulation

31. Cytosine (in DNA) methylation • There are many types of DNA methylase (DNMT)

32. DNA Methylation and T/C Regulation • Methylation could be a signal for DNA involved events: replication, transcription, repair et. al. (mCpG attract methylation sensitive DNA binding proteins (MeCP1), which in turn recruit Histone de-acetylation enzyme (Sin3 complex) histone de-acetylation gene inactivated • 5mC : Cytosines are methylated after replication • the percentage of 5mC varies from species to species.(3% for mammalian DNA, little or no in Drosophila and yeast) • The distribution of 5mC is non-random, Most 5mC was found in the sequence CG ( mCpG island)

33. Methylation and T/C regulation • HpaII/MspI RFLP study of genes with different activity----negatively correlated (30 genes examined) • Is this relationship a general picture? • Will all methylated C demethylated in active gene ? • Is methylation level change a necessity of T/C activity or a byproduct of it?

34. Over or Under methylation may have serious consequences • Mutation of methylase in mice is fatal • Fragile X syndrome: FMR-1(fragile X mental retardation)gene triplet repeat over expansion (CGG repeat #>200) and abnormal methylation ------T/C silenced • Abnormal methylation and cancer: promoter of tumor suppressor gene

35. Epigenetics • Chromosome Remodelling • Histone Acetylation and Methylation • DNA Methylation

36. RNA Processing Control

37. RNA Processing Control • Regulates the production of mature-RNA from precursor-RNA: • 1. Choice of alternative poly(A) site • to produce different pre-mRNA molecules • 2. Choice of alternative splicing site • to produce different functional mRNAs

38. RNA Processing Control • The product of alternative poly(A) or alternative splicing are proteins that are encoded by the same gene but differ structurally and functionally • Such proteins are called protein isoforms, and their synthesis may be tissue specific • Alternative poly(A) is independent of alternative splicing

39. Processing control model • A) control by polyA choice B) control by splices site choice

40. Control by PolyA and Splice site choice — human calcitonin gene (CALC) • CALC consists of five exons and four introns • This gene is transcribed in certain cells of the thyroid gland and in certain neurons of the brain • Alternative PolyA occurs with PolyA site next to exon 4, used in thyroid cells, and PolyA site next to exon 5, used in the neuronal cells

41. Alternative polyadenylation and alternative splicing resulting in tissue-specific products of the human calcitonin gene, CALC

42. RNA Transport Control • Perhaps there are up to 50% protein coding primary RNAs never leave nucleus, degraded.

43. RNA Transport Control • The spliceosome retention model: • spliceosome assembly competes with nuclear export • After splicing process, intron is associated with snRNPs before degradation • The methylated 5’cap is necessary for mRNA to be exported to the cytoplasm

44. mRNA Translation Control

45. Poly(A) tail can promote translation In general, stored inactive mRNA has shorter PolyA tails (15-90 As) than active mRNAs (100-300As) • Is the shorter tail synthesized as it is , or being truncated to what it is? 1.In oocytes of mouse/frog, the pre-mRNAs has long tails (300-400 As), 2. The mature stored mRNAs has short tail (40-60 As) 3. Actively translated mRNAs have 100-300 As

46. Same signal for deadenylation and polyadenylation in cytoplasm Deadenylation :in the 3’UTR of mRNA, upstream of AAUAAA sequence, there is an (AU)-rich element(ARE) as deadenylation signal (UUUUUAU). Polyadenylation :to activate a stored mRNA in this class, this signal (ARE element) is recognized by a polyadenylation enzyme and add ~150 As

47. mRNA degradation control

48. mRNA degradation control • Both rRNA and tRNA are very stable, but mRNA exhibits a diverse range of stability • Regulatory signals change mRNA stability • mRNA secondary structure & ARE sequence also affect mRNA half life

49. mRNA degradation control mRNA Tissue or Cell Regulatory Signal Half-Life of mRNA Vitellogenin Liver (frog) Estrogen 500h (16h) Vitellogenin Liver (hen) Estrogen 24h (<3h) Lipoprotein Liver (frog) Estrogen 20-24h (<3h)

50. Two mRNA decay pathways • Deadenylation -dependent decay pathway • poly(A) tail are deadenylated until the tail are too short to bind PAB (polyA binding protein) • Then 5’cap is removed (decaping) (DCP1) • 5’-to-3’ exonuclease degradation • Deadenylation-independent decay pathway • Yeast dcp1 mutant is capable to degrade mRNA • Decaping without being deadenylated