1 Genetics, gene and central dogma of molecular biology

2 生命怎麼變的如此複雜? 生物演化的三步曲：變異、遺傳與天擇 variation, inheritance and natural selection!

3 Darwin: Variation of Animals and Plants Under Domestication (1868)The power of selection, whether exercised by man or brought into play under nature through the struggle for existence and the consequent survival of the fittest, absolutely depends on the variability of organic beings. Without variability, nothing can be effected; slight individual differences, however, suffice for the work, and are probably the chief or sole means in the production of new species. Darwin: Variation of Animals and Plants Under Domestication (1868)

4 達爾文的遺傳學：Blending inheritanceIs blending inheritance compatible with natural selection？

5 Gregor Mendel (1823-1884) Theory of particulate inheritance

6 This seems to be the one correct way of finally reaching a solution to a question whose significance for the evolutionary history of organic forms cannot be underestimated Mendel, G., 1866 Versuche über Pflanzenhybriden. Ver. Naturforsch. Ver. Brünn 4: 3–47.

7 Lucky Mendel: he chose true-breeding plants!

8 Mendel cannot reproduce his results on peas in hawkweed!

9 Important terms in Mendelian genetics.Character: color of peas. Trait: yellow or white. Gene: unit of heredity. Allele: version of a gene produces a specific trait. Homozygous: having two copies of the same alleles for a given gene. Heterozygous: having two different alleles for a given gene.

10 Model and new technique provide insight!From gene to DNA Model and new technique provide insight!

11 無害的Ｒ細菌與有害的Ｓ細菌的抽取物培養，無害的Ｒ細菌會轉變成有害的Ｓ細菌！Ｓ細菌的抽取物中是什麼樣的成分會改變Ｒ細菌的遺傳程式(資訊)使Ｒ細菌轉變成Ｓ細菌？

12 奧斯卡．阿佛來(Oscar Avery) 1943 ＤＮＡ是攜帶遺傳資訊的分子！ How？

13 DNA is the molecule in life to store genetic information.May 21, Cambridge

14 Arthur Kornberg （亞瑟．孔伯）Set out an assay to purify an enzyme that could make DNA! Protein extract from E. coli + template DNA + substrates He guessed these would be: dATP; dTTP; dGTP and dCTP He guessed that Mg2+ would be required! 1956 找到複製DNA的酵素

15 Characteristic of DNA synthesis - IPrimers absolutely necessary Usually short stretches of RNA or RNA-DNA Some virus use proteins primers.

16 Characteristic of DNA synthesis - II5’ to 3’ directionality Leading strand vs. lagging strand End problems for linear DNA molecules when replication starts internally

17 Okazaki fragments: discontinue synthesis!

18 His son, Roger Kornberg received Nobel Prize in 2006 for his study of structure basis of gene transcription in eucaryotes

19 The bacterial chromosome and its manner of replication as seen by autoradiography. Cairns J. J. Mol. Biol. 6:208-13, 1963.

20 Is Kornberg’s enzyme responsible for DNA replication in vivo?DNA replication in E. coli proceeds at approximately 1,000 nucleotides/second, while the rate of synthesis by Kornberg’s polymerase averages only between 10 and 20 nucleotides/second. One cell contains approximately 400 molecules of Kornberg’s enzyme which did not correlate with the fact that there are only two replication forks in E. coli. Kornberg’s polymerase is insufficiently processive to copy an entire genome, as it falls off after incorporating only nucleotides.

21 Is Kornberg’s enzyme responsible for DNA replication in vivo?If your guess is no, how to prove your guess is right? 如果能找到一個 E. coli 沒有Kornberg’s enzyme activity!!! 怎麼樣去找這樣的E. coli

22 亂槍打鳥的實驗設計 Cairns' lab assistant Paula De Lucia created thousands of cell free extracts from E.coli colonies. Assayed them for DNA polymerase activity individually. After assay 3,478th clone, Paula isolated a viable mutant that lacked the polymerase activity. The mutant was named as polA by Cairns to credit "Paula" .

23 De Lucia P, Cairns J Nature 224: 1164–6; 1969."Isolation of an E. coli strain with a mutation affecting DNA polymerase". De Lucia P, Cairns J Nature 224: 1164–6; 1969. Popper否証論的最佳例子

24 Central dogma of molecular biology : a process of decodingGenetic code in DNA: A, T, G, C Genetic code in RNA: A, U, G, C 20 amino acids in protein

25 (Expression of information)DNA (genetic code) To make a unique protein with a specific amino acid sequence through transcription and translation Gene expression (Expression of information)

26 } } + (1) The discovery of initiation factors factor is required for bacterial RNA polymerase to initiate transcription on promoters  +  ' ' KD ~ 10-9 M } } ‘holoenzyme’ ‘core’ Can elongate but cannot begin transcription at promoters Can begin transcription on promoters and can elongate

27 How RNAP was discovered (Burgess, 1969)A. Assay for RNA polymerase: *ATP CTP GTP UTP E.coli lysate Calf thymus DNA buffer Look for incorporation of *ATP into RNA chains B. Initial purification Lysate various fractionation steps (DEAE column, glycerol gradient etc) Active fractions identified by assay

28 Peak 1 restored activityC. Improved purification of RNA polymerase: Improved fractionation lysate phosphocellulose column Labmate Jeff Roberts reported that the new, improved preparation of RNAP (peak 2) had no activity on  DNA salt 1 2 Activity (*ATP) CT DNA Fraction # OD 280 Peak 1 restored activity  increases rate of initiation g  Transcription  DNA Assay: incorporation P ATP SDS gel analysis Peak Peak 2  '   

29 (3) s undergoes a large conformational change upon bindingto RNA polymerase Free  doesn’t bind DNA  in holoenzyme positioned for DNA recognition Sorenson; 2006

30 (2) Bacterial promotersThere are several flavors of promoters  and  recruit RNAP to promoter DNA

31 Identifying eukaryotic “initiation factors”

32 Transcription Initiation by PolII requires many General Transcription FactorsRNA Pol II + NTPs + DNA containing a real promoter NO TRANSCRIPTION promoter Same experiment we discussed to identify sigma but in this case the result was very different RNA Pol II + NTPs + DNA with real promoter TRANSCRIPTION INITIATION and ELONGATION nuclear extract

33 Purification scheme for partially purified general transcription factors. Fractionation of HeLa nuclear extract (Panel A) and nuclear pellet (Panel B) by column chromatography and the molar concentrations of KCl used for elutions are indicated in the flow chart, except for the Phenyl Superose column where the molar concentrations of ammonium sulfate are shown. A thick horizontal (Panel A) or vertical (Panel B) line indicates that step elutions are used for protein fractionation, whereas a slant line represents a linear gradient used for fractionation. The purification scheme for pol II, starting from sonication of the nuclear pellet, followed by ammonium sulfate (AS) precipitation is shown in Panel B. (Figures are adapted from Flores et al., 1992 and from Ge et al., 1996) NAME # OF SUBUNITS FUNCTION TFIIA Antirepressor; stabilizes TBP-TATA complex; coactivator TFIIB Recognizes BRE;Start site selection; stabilize TBP-TATA; pol II/TFIIF recruitment TFIID TBP Binds TATA box; higher eukaryotes have multiple TBPs TAFs ~10 Recognizes additional DNA sequences; Regulates TBP binding; Coactivator; Ubiquitin-activating/conjugating activity; Histone acetyltransferase; multiple TAFs TFIIF Binds pol II; facilitates pol II promoter recruitment and escape; Recruits TFIIE and TFIIH; enhances efficiency of pol II elongation TFIIE Recruits TFIIH; Facilitates forming initiation-competent pol II; promoter clearance TFIIH ATPase/kinase activity. Helicase: unwinds DNA at transcription startsite; kinase phosphorylates ser5 of RNA polymerase CTD; helps release RNAP from promoter In vitro complementation; not a single factor—multiple, some of them almost as large as polII itself—TFIID, TFIIH;--work took many person years and extended into the 90’s—just to get facts straight. Which ones in common bet archae and euks? TPB and B; some archae groups have a proliferation of B subunits; TAFs a later addition; metazoans: some TBP related proteins and alternative tafs; differentiated muscles—trf3 and TAF3; low tbp check how tbp binds Go thru roles

34 Transcription Initiation by RNA Pol IIThe stepwise assembly of the Pol II preinitiation complex is shown here. Once assembled at the promoter, Pol II leaves the preinitiation complex upon addition of the nucleotide precursors required for RNA synthesis and after phosphorylation of serine resides within the enzyme’s “tail”. Initiation mostly examined on TATA promoters; actually a minority of promoters TBP binds tata box;minor groove recognition—depends on TA sequence, hbonds to edges of bases, and then 2 phenylala inserted widening narrow groove and introducing 80˚ bend in DNA also recognizes IIB; IIB also recog DNA, and has some of the roles of sigma during initiation process; enters active site cleft thru RNA exit channel; may block RNA TFIIA- prevents binding of certain regulators to TBP TFIIF binds to polymerase; enhances affinity of Polymerase for tbp and IIB; also has roles in elongation; ; cryo EM suggests that TFII opens clamp and orders promoter DNA; Note this important for mechanism of linitiation: if closed in PIC, melting must occur outside cleft and ss DNA enter active site; if open melting can occur in cleft IIE recrruits IIH—10 subunits : 3 enzymatic activities: CTD kinase(CDK7); 2ATPases—helicase (NER) and a translocase : Model, based on x-link, cryo EM and structurebinds downstream from unwound DNA; screws 15 bp DS DNA into active sit, leading to unwindingATP required for strand opening—helicase PO4 tail ;;we ill come back to the tailbut now I want you to notice that the CTD is very long (800å) 7X greater than the length of polymerase. And is located near the RNA exit channel Talk about ctd also; positioned near RNA exit channel; different forms of RNAP How did we discover all of this: functional identification; structures, crosslinking ( DNA-protein; protein-protein; cryo EM PIC = preinitiation complex

35 The Pol II promoter has many recognition regionsPositions of various DNA elements relative to the transcription start site (indicated by the arrow above the DNA). These elements are: BRE (TFIIB recognition element); there is also a second BRE site downstream of TATA TATA (TATA Box); Inr (initiator element); DPE (downstream promoter element); DCE (downstream core element). MTE (motif ten element; not shown) is located just upstream of the DPE. Go thru elements and then compare with proks TATA—superficial similarity with -10 region but fundamentallly differen mehanism, not at start site; doesn’t strand open; TBP binds and bends DNA serves as a platform for assembly of TAFs

36 Structure of RNAP in the three domainsUniversally conserved Archaeal/eukaryotic Archaea Eukarya Bacteria Features of enzyme: crab claw: DNA enters through the big channel in middle; 2 pincers; one is called the clamp and is moveable; open state accommodates ds DNA closed state ss DNA; closed during transcription; bridge helix; a series of moving parts connects from catalytic center to clamp; implications of closed vs open for initation: Note this important for mechanism of initiation: if closed in PIC, melting must occur outside cleft and ss DNA enter active site; if open melting can occur in cleft Discuss conservation; much greater at structural than sequence level. Extra subunits—away from center; discuss plasticity of enzyme—proliferation of enzymes in eukaryotes, insertions and deletions in proks Explain why discuss basics of system in prokaryotes Transcription Werner and Grohmann (2011), Nature Rev Micro 9:85-98 Extra RNAP subunits provide interaction sites for transcription factors, DNA and RNA, and modulate diverse RNAP activities

37 RNA polymerases in all living organisms are evolutionary relatedGre LUCA-Last universal common ancestor LUCA may have had elongating, not initiating RNA polymerase

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39 mRNA

40 Replication vs transcriptionSpeed nucs/sec: bacteria nucs/sec 50 nucs/sec: euks Error rate /109(including /104- 1/105 mismatch repair) Speed: 5-20fold slower--rep--once gets started keep going; trx; slower elongation regulation complex active site; NTP addition rate limiting for easier regulation Error rate--have discussed why rep needs to be so accurate; will see later in lecture the strategy used for accuracy in trx Job Transcribe segments of the genome at highly variable rates Copy every sequence in the genome once

41 How many polymerase? DNA dependent DNA polymeraseFor DNA replication and repair. 5 known Prokaryotic DNA polymerases. at least 15 Eukaryotic DNA polymerase DNA dependent RNA polymerase For gene transcription. RNA dependent RNA polymerase For RNA virus genome replication RNA dependent DNA polymerase Reverse transcriptase of retrovirus Telemerase to make telemere structure

42 In 1977, when viral mRNA was hybridized with its DNA, some loops were observed.

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47 How many genes do we have ?

48 Regulation of gene expression at different level!DNA (genetic code) what where when how much Regulation of gene expression at different level!

49 Classic paradigm of molecular biolotyAmino acid sequence of protein determines its secondary, tertiary and quaternary structure ! (Anfinsen, 1972)

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51 Paradigm Shift in Molecular Biology1, can protein carry inheritable information? 2, is one gene – one polypeptide still valid?

52 Kuru Discovered by Carleton Gajdusek in the 1950’s and 60’s among the South Fore people of New Guinea. Transmitted through ritual mortuary cannibalism where deceased individuals were consumed by their relatives to honor them. Between 1957 and 1968, over 1,100 South Fore succumbed to kuru. Early on it affected mostly women (80% vs men) but later also affected elderly and children at high rates as well.

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54 Prion Protein (PrP) The purification of the infective scrapie agentrevealed a protease-resistant fragment that copurifies with infectivity Cloning identifies the gene as 33-35kD glycoprotein PrP. PrP is insoluble and protease-resistant only in infected animals and accumulates in plaques in infected brain. Finally, knockout of PrP in mice renders them immune to the effects of infective prion.

55 Stanley Prusiner first coined the term prion in 1982 Prion: proteinaceous infectious particle

56 Sequence of prion protein

57 Models for the conversion of PrPc to PrPsc

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59 Paradigm Shift in Molecular Biology1, can protein carry inheritable information? 2, is one gene – one protein – one function valid?

60 One million dollars question: What is the nature of this factor?Changes of cellular markers during differentiation of HL-60 promyelocytes to macrophages as induced by T cell conditioned medium. Leuk Res 5:477, 1981 One million dollars question: What is the nature of this factor?

61 The Differentiation and Maturation Mediator for Human Myeloid Leukemia Cells Shares Homology With Neuroleukin or Phosphoglucose Isomerase Blood 87: ; 1996

62 The concept of “moonlighting” emerge!What does moonlighting mean?

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65 The multiple functions of a moonlighting protein may involve the same or different regions of the protein

66 A conformational change caused by dissociation of the 4Fe-4S cluster convertsaconitase from a cytoplasmic enzyme to a transcription factor (IRP-1) that binds to the iron response element (IRE) in the 5 or 3 untranslated regions of mRNAs involved in use of iron.

67 Insight of genetics Partial Dominance: dilute concentration.Codominance: human ABO blood group (AB) Overdominance: sickle-cell anemia Dominant: gain-of-function. Recessive: loss-of-function. Could a loss-of-function gene behave dominant? Negative dominant mutation. Genetic suppression: intra vs intergenic suppression!

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69 Scrapie Invariably fatal, chronic neurodegenerative disease.First reported in England, France and Germany in the 19th century. Long period of incubation (2-5 years). Affected animals rub their coats against trees, suffer ataxia, convulsions, blindness, anorexia, and eventually death. Death usually occurs within 1-6 months.

70 Early experiments Transmissibility was unintentially demonstrated by inoculation of a Scottish sheep a vaccine extract prepared from formalin treated brain of a scrapie-infected animal. Within 2 yrs, 10% of the flock contracted scrapie. Gajdusek notes similarity in brain pathology between Kuru and scrapie. He goes on to inject chimpanzees with Kuru brain extracts, after which they exhibit TSE pathology. Investigators follow up by showing transmissibility to animals of CJD, familial TSE, and GSS. Slow virus – isolation and characterization

71 Progression of BSE 1986: First case of BSE discovered in a cow that was fed livestock feed produced from a sheep that died of scrapie. Dr. Richard Lacey annouces that scrapie and BSE are the same disease and that “this beef was in the meat supply”. British government dismisses Lacey and cuts his research funding. They announce that scrapie renderings are still an acceptable form of livestock feed.

72 Progression of BSE (cont)1987: 700 BSE infected cows are reported in Great Britain. 1988: 7,000 infected cows. Law is passed declaring sheep rendering illegal. 1992: 36,000 infected livestock reported. 1994: 150,000 infected livestock reported and is identified in half of British cattle herds.

73 Animal Rendering In September 1995, reporter Van Smith of Baltimore’s Weekly City Paper visited Valley Proteins Inc, a Baltimore rendering plant: Smith observed these items listed: a horse, the grill grease and used frying oil from Camden yards, a baby elephant who died in Baltimore, Illinois, tons of waste meat and inedible animal parts from the local supermarkets and slaughterhouses, carcasses from the zoo, thousands of dogs, cats, raccoons, possums, deer, foxes, snakes, and the rest of the local animal shelters waste and road kill that must be disposed each month.

74 Crossing the line….. In 1996, a new form of CJD is discovered in the UK, termed variant CJD (vCJD). Linked with consumption of BSE-contaminated beef. Shares the symptoms of classic CJD, except the median age of death is 28 (contrasting with 68) and feature psychiatric and sensory symptoms with neurologic effects occuring later.

75 Molecular Mechanism Protein can convert between two conformations (a benign form and pathogenic state) at a certain frequency The second state can seed the formation of oligometric, insoluble aggregates that in turn form toxic amyloid plaques. During the oligomerization the prions corrupt the native form of the protein into a transmissible disease conformation.

76 Some proteins have more than one moonlighting functionThe super GAPDH!

77 6th reaction of glycolysisH-C=O H-C-OH CH2-O- PO3-2 1 Glyceraldehyde-3-phosphate (GAP) 2 3 Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) NAD+ + Pi NADH + H+ O C-O H-C-OH CH2-O- PO3-2 1 1,3-Bisphosphoglycerate (1,3-BPG) -PO3-2 2 3

78 What else GAPDH can do? a crystallin in the lens of diurnal geckos.It is involved in apoptosis. vesicular transport. nuclear tRNA export. a component of the OCT1 coactivator that controls transcription of histone 2B during S-phase. a low-affinity transferrin receptor in macrophages It is displayed on the surface and excreted into the medium by Bacillus anthracis Etc.

79 The same moonlighting function can be provided by different proteins