Genetics, gene and central

dogma of molecular biology

11-2-2015
 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!

 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!

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？
奧斯卡．阿佛來 (Oscar Avery) 1943

ＤＮＡ是攜帶遺傳資訊的分子！ How ？
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: dATP; dTTP; dGTP and dCTP
• He guessed that Mg2+ 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 3,478th clone, Paula isolated a viable
mutant that lacked the polymerase activity.
• The mutant was named as polA 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

‘holoenzyme’ ‘core’

}
}

Can begin transcription Can elongate but cannot

on promoters and can begin transcription at
elongate promoters
 How RNAP was discovered (Burgess, 1969)
A. Assay for RNA polymerase: *ATP
CTP
E.coli lysate 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
Labmate Jeff Roberts
salt reported that the new,

Activity (*ATP)
improved preparation of

CT DNA
2
OD 280

RNAP (peak 2) had no

activity on  DNA
1
Peak 1 restored activity
Fraction #
 increases rate of initiation

SDS gel analysis

Transcription

Peak 1 Peak 2
 DNA
Assay:
' incorporationP ATP



 g 
(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
 (2) Bacterial promoters
There are several flavors of promoters

 and  recruit RNAP to promoter DNA

Identifying eukaryotic “initiation factors”
Transcription Initiation by PolII requires many General Transcription Factors

RNA Pol II
+ NTPs
+ DNA containing a real promoter

NO TRANSCRIPTION

promoter

RNA Pol II
+ NTPs
nuclear + DNA with real promoter
extract

TRANSCRIPTION INITIATION and ELONGATION

 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 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
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 (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

mRNA
 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)

Job Transcribe segments

Copy every sequence in
of the genome at
the genome once
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 mRNA 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 (PrP)

The purification of the

infective scrapie agent
revealed 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.
Stanley Prusiner first coined the term prion in 1982
Prion: proteinaceous infectious particle
Sequence of prion protein
 c sc
Models for the conversion of PrP to PrP
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 4Fe-4S 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 mRNAs 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 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.
 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
 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 (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.
150

100
CJDdeaths

50

0
1990 1995 2000 2005
year
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!
 6th reaction of glycolysis
1
Glyceraldehyde-3-phosphate -
H C=O
(GAP)
H-C-OH
2

3 CH -O- 2
PO3-2

Glyceraldehyde-3-phosphate dehydrogenase NAD+ + Pi

(GAPDH)

NADH + H+

O
1
1,3-Bisphosphoglycerate (1,3-BPG)
-C O -PO 3
-2

H-C-OH
2

CH -O- PO -2
3 3
2
 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.
The same moonlighting function can
be provided by different proteins