Genetics, gene and central dogma of molecular biology

1. Genetics, gene and central dogma of molecular biology 11-2-2015

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. 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 inheritance Is blending inheritancecompatible 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. 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！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, 1953 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 - I • Primers absolutely necessary • Usually short stretches of RNA or RNA-DNA • Some virus use proteins primers.

16. Characteristic of DNA synthesis - II • 5’ 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 25-50 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. "Isolation of an E. coli strain with a mutation affecting DNA polymerase". De Lucia P, Cairns J Nature224: 1164–6; 1969. Popper否証論的最佳例子

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

25. 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. B. Initial purification Lysate various fractionation steps (DEAE column, glycerol gradient etc) Active fractions identified by assay 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

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

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

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

31. Identifying eukaryotic “initiation factors”

32. RNA Pol II + NTPs + DNA containing a real promoter NO TRANSCRIPTION promoter RNA Pol II + NTPs + DNA with real promoter nuclear extract TRANSCRIPTION INITIATION and ELONGATION Transcription Initiation by PolII requires many General Transcription Factors

33. NAME # OF SUBUNITS FUNCTION TFIIA 3 Antirepressor; stabilizes TBP-TATA complex; coactivator TFIIB 1 Recognizes BRE;Start site selection; stabilize TBP-TATA; pol II/TFIIF recruitment TFIID TBP 1 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 2 Binds pol II; facilitates pol II promoter recruitment and escape; Recruits TFIIE and TFIIH; enhances efficiency of pol II elongation TFIIE 2 Recruits TFIIH; Facilitates forming initiation-competent pol II; promoter clearance TFIIH 9 ATPase/kinase activity. Helicase: unwinds DNA at transcription startsite; kinase phosphorylates ser5 of RNA polymerase CTD; helps release RNAP from promoter 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)

34. Transcription Initiation by RNA Pol II The 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”. PIC = preinitiation complex

35. The Pol II promoter has many recognition regions Positions 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.

36. Structure of RNAP in the three domains Universally conserved Archaeal/eukaryotic Bacteria Archaea Eukarya Transcription Extra RNAP subunits provide interaction sites for transcription factors, DNA and RNA, and modulate diverse RNAP activities Werner and Grohmann (2011), Nature Rev Micro 9:85-98

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

39. mRNA

40. Transcribe segments of the genome at highly variable rates Job Copy every sequence in the genome once Replication vs transcription Replication Transcription Speed 500 nucs/sec: bacteria 10-30 nucs/sec 50 nucs/sec: euks Error rate 1/109(including 1/104- 1/105 mismatch repair)

41. How many polymerase? • DNA dependent DNA polymerase • For 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.

47. How many genes do we have ?

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

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