Genetics gene and central dogma of molecular biology

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

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)

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

Gregor Mendel (1823 -1884) Theory of particulate inheritance

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.

Lucky Mendel: he chose true-breeding plants!

Mendel cannot reproduce his results on peas in hawkweed!

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. • • •

From gene to DNA Model and new technique provide insight!

DNA is the molecule in life to store genetic information. May 21, 1953 Cambridge

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: d. ATP; d. TTP; d. GTP and d. CTP He guessed that Mg 2+ would be required! 1956 找到複製DNA的酵素

Characteristic of DNA synthesis - I • Primers absolutely necessary – Usually short stretches of RNA or RNA-DNA – Some virus use proteins primers.

Characteristic of DNA synthesis - II • 5’ to 3’ directionality – Leading strand vs. lagging strand – End problems for linear DNA molecules when replication starts internally

Okazaki fragments: discontinue synthesis!

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

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

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.

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

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, 478 th clone, Paula isolated a viable mutant that lacked the polymerase activity. • The mutant was named as pol. A by Cairns to credit "Paula".

"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

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

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

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

How RNAP was discovered (Burgess, 1969) A. Assay for RNA polymerase: E. coli lysate *ATP CTP GTP UTP 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

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

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

(2) Bacterial promoters There are several flavors of promoters �and �recruit RNAP to promoter DNA

Identifying eukaryotic “initiation factors”

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

Purification scheme for partially purified general transcription factors. Fractionation of He. La 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 3 Antirepressor; stabilizes TBP-TATA complex; coactivator TFIIB 1 Recognizes BRE; Start site selection; stabilize TBP-TATA; pol II/TFIIF recruitment 1 ~10 Binds TATA box; higher eukaryotes have multiple TBPs Recognizes additional DNA sequences; Regulates TBP binding; Coactivator; Ubiquitin-activating/conjugating activity; Histone acetyltransferase; multiple TAFs TFIID TBP 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 ser 5 of RNA polymerase CTD; helps release RNAP from promoter

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

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 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.

Structure of RNAP in the three domains Universally conserved Archaeal/eukaryotic Bacteria Archaea Eukarya 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

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

m. RNA

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

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

In 1977, when viral m. RNA was hybridized with its DNA, some loops were observed.

How many genes do we have ?

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

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

Paradigm Shift in Molecular Biology 1, can protein carry inheritable information? 2, is one gene – one polypeptide still valid?

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.

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

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

Sequence of prion protein

c Models for the conversion of Pr. P to Pr. P sc

Paradigm Shift in Molecular Biology 1, can protein carry inheritable information? 2, is one gene – one protein – one function valid?

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?

The Differentiation and Maturation Mediator for Human Myeloid Leukemia Cells Shares Homology With Neuroleukin or Phosphoglucose Isomerase Blood 87: 4502 -4506; 1996

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

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

A conformational change caused by dissociation of the 4 Fe-4 S cluster converts aconitase 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 m. RNAs involved in use of iron.

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!

Scrapie • Invariably fatal, chronic neurodegenerative disease. • First reported in England, France and Germany in the 19 th 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.

Early experiments • Transmissibility was unintentially demonstrated by inoculation of a Scottish sheep a vaccine extract prepared from formalin treated brain of a scrapieinfected 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

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.

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.

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.

Crossing the line…. . • In 1996, a new form of CJD is discovered in the UK, termed variant CJD (v. CJD). • 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.

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.

Some proteins have more than one moonlighting function The super GAPDH!

6 th reaction of glycolysis 1 H-C-OH CH -O- Glyceraldehyde-3 -phosphate (GAP) H C=O 2 3 PO 3 -2 2 Glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) NAD+ + Pi NADH + H+ O 1, 3 -Bisphoglycerate (1, 3 -BPG) 1 - -PO H-C-OH PO CH -O-2 3 C O 2 3 2 -2 3

What else GAPDH can do? a crystallin in the lens of diurnal geckos. It is involved in apoptosis. vesicular transport. nuclear t. RNA export. a component of the OCT 1 coactivator that controls transcription of histone 2 B 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. • • •

The same moonlighting function can be provided by different proteins