15

12

DNA
(genetic information) RNA
(gene) DNA
(genome)
DNA
3,000 (3x109 base pairs) haploid cell
70,000
10% DNA

diploid somatic cells (46 ) germ cells (23 )

central dogma
Transcription

Replication DNA
RNA
Reverse transcription

Translation

protein

DNA RNA

DNA

DNA replication

RNA
DNA

RNA


15
8

RNA transcription
RNA 3
mRNA, tRNA rRNA
RNA
translation
transcription translation
gene expression
DNA RNA

DNA
DNA 2
(copy) haploid genome
1. Nonrepetitive unique single-copy DNA
75% DNA

DNA structural genes

2. Repetitive DNA 25% DNA

DNA
2
2.1 Interspersed (dispersed) sequences DNA

DNA
2.1.1 Short interspersed nuclear elements (SINE)
100-500 Alu
single copy sequence 300 10
kb Alu family 300,000
500,000

15
9

2.1.2 Long interspersed nuclear elements (LINE)

500
LI family 5,000 7,000

2.1.3 Variable number of tandem repeats (VNTR)

(repeat unit) 15 80

2 kb
VNTR genetic marker

2.1.4 Inverted repeats (fold back DNA) linear

DNA 100-1000 stem loop
() 2 inverted repeats

2.2 Satellite DNA

tandem array

Satellite DNA centromere

telomere satellite DNA
minisatellite (
20 30 ) microsatellite short tandem repeat
(STR, 2 7 )
STR polymorphism
DNA

,


16
0

DNA

1.
12.1
- DNA

(Genetic (Roskoski R, Biochemistry, 1996.)
information)

nucleotide sequence
DNA

DNA replication

DNA

DNA

(mutation)
2.

DNA (DNA
repair)

16
1

DNA

DNA (DNA replication)

eukaryotic cells S-phase
(synthesis phase) cell cycle
DNA 2
DNA


(template)
-
(base
pairing) A T G C
DNA DNA 2

1 1
complementary DNA
semiconservative replication ( 12.1 )
(site of
origin)
(bi-directional) prokaryote
eukaryote
DNA (
12.1 )


16
2

12.1 DNA semiconservative

DNA
(Marks DB, Marks AD, Smith CM, Basic medical bioche
mistry, 1996.)

DNA
1. DNA (DNA template)
2.
DNA dATP, dGTP, dCTP dTTP
DNA

3. RNA primer
ATP, GTP, CTP UTP RNA primer


DNA
DNA

4. protein
12.2

16
3

5.
DNA
12.2
Protein

Gene(
s)

DnaA

dnaA

DnaC
DNA
polymerase
III
holoenzym
e

Pol I
Ligase
Gyrase

dnaC

DnaB
Primase
SSB

DNA (Zubay
GL, Biochemistry, 1998.)

dnaE
dnaQ
holE
dnaX
dnaX
holA
holB
holC
holD
dnaN
polA
lig
gyrA
gyrB
dnaB
dnaG
ssb

Function
Initiator protein: Binds oriC; promote doublehelix opening: DnaB loading
Complexes with DnaB; delivers DnaB to DNA

Polymerase
3 to 5 exonuclease
Unknown

Processivity factor: Sliding clamp

Prime removal and gap filling
Joins nascent DNA fragments
Type II topoisomerase; replication swivel, DNA
supercoiling, decatenation
3 to 5 helicase and activator of primase
Primer synthesis
Binds single-stranded DNA

DNA polymerase


16
4

DNA polymerase prokaryote

..1957 Arthur Kornberg
DNA DNA polymerase E.coli
12.2
I.

12.2 DNA polymerase (Marks DB,

Marks AD, Smith CM. Basic medical biochemistry, 1996.)
deoxyribonucleotide
free-OH 3
DNA 5 3
DNA
DNA
DNA


DNA polymerase I
DNA polymerase I activity
3
1. 35 exonuclease
(proofreading) DNA (
12.3 )

16
5

2. 53 exonuclease
(error-correcting) ( 12.3 )

12.3 exonuclease activity DNA polymerase (Stryer

L. Biochemistry, 1995.)
3. polymerase DNA
3
Small fragment

5 3
Exonuclease

Large fragment
(Klenow fragment)
3 5
polymerase
C
Exonuclease

12.4
enzymatic activities DNA polymerase (Strye
Biochemistry, 1995.)
2
DNA


16
6

DNA
polymerase II DNA polymerase III
DNA polymerase II
DNA polymerase III
DNA polymerase III
holoenzyme DNA
DNA
DNA polymerase
III 10 (
, , , , , , , , , ) subunit
polymerase activity, subunit 35 exonuclease
, , core active
subunit processivity
core enzyme
DNA
(processivity
DNA
polymerase template )
DNA
polymerase III holoenzyme
1000 DNA
polymerase I 100 ( 12.3)
12.3 Prokaryotic DNA polymerases (Mathews CK,
Van Holde KE. Biochemistry, 1996.)

II DNA polymerase eukaryote

eukaryote DNA polymerase
5 , , ,

16
7

( 12.4) DNA
polymerase
DNA DNA polymerase
DNA (fill gap)
DNA DNA polymerase
processitivity DNA polymerase
mitochondrial
DNA
DNA polymerase , 3 5
exonuclease activity (proofreading)
5 3 exonuclease activity DNA polymerase
lagging
leading
DNA
polymerase proliferating cell
nuclear antigen (PCNA)
12.4 DNA polymerases
(Roskoski R. Biochemistry, 1996.)

PCNA = proliferating cell nuclear antigen

DNA (DNA replication)

1. (initiation point)
E.coli Dna A ori
C
eukaryote


16
8

DNA ( ARS : autonomously

replicating sequence) DNA

DNA
replicon
2. (unwinding DNA)
helicase ( Dna B) eukaryote
DNA
DNA
ATP
replication
fork binding
protein single stranded DNA binding protein

( 12.5)

DNA

supercoil
supertwist
DNA
gyrase
DNA
topoisomerase II


supercoil

3.

RNA primer RNA
polymerase
primase

primosome
RNA

50-100 -

RNA primer
DNA

5 3
DNA

16
9


17
0

RNA free OH
group 3
deoxyribonucleotide
DNA polymerase
4.
DNA
DNA
polymerase

holoenzyme DNA
polymerase

RNA primer
53
DNA

complementary

nucleotide
sequence

DNA

12.5
DNA replication fork (Marks DB,
Marks AD, Smith CM. Basic medical
biochemistry, 1996.)

DNA

leading
strand
replication fork
replication fork Okazaki
fragment 40-200 eukaryote
prokaryote 1000-2000
Okazaki fragment
lagging strand

17
1

DNA polymerase RNA polymerase

53 ( 12.6)

12.6 DNA (DNA replication)

eukaryote (Marks DB, Marks AD, Smith CM. Basic medical
biochemistry, 1996.)
5. RNA primer
5
DNA polymerase ( E.coli) DNA polymerase (
eukaryote) RNA primer
deoxyribonucleotide
complementary DNA
DNA polymerase
DNA
3 -OH 5 -phosphate
DNA polymerase
DNA


17
2

6. DNA
DNA ligase DNA Okazaki
fragment deoxyribonucleotide 3 5
DNA ligase 3 OH 5 -phosphate ester bond
eukaryote
ATP
DNA
(leading strand)
DNA
ligase DNA


DNA 1

DNA DNA
1 1
(mitosis)
DNA (genetic information)

12.7 DNA
DNA 2 leading
lagging (Devlin TM. Textbook of biochemistry with
clinical correlations, 1997.)

DNA telomere

17
3

(telomere)

12.7 DNA RNA
primer RNA primer
DNA
- DNA
polymerase
DNA polymerase 5
3

germ cell
3
telomeric DNA telomerase
telomeric DNA tandem repeats
AGGGTT telomerase
tandem repeat
primer DNA

telomerase
primer telomerase RNA
oligonucleotide reverse
transcriptase ( DNA RNA )
telomerase
12.8 telomerase
DNA 3 DNA
complementary RNA template
RNA primer DNA
DNA

lagging DNA polymerase

DNA


17
4

12.8

telomere

telomerase
DNA
53
lagging
(Mathews
CK, van Holde KE.
Biochemistry, 1996.)

somatic cells
telomerase telomere

telomerase

telomerase

DNA (DNA Repair)

17
5

nucleotide sequence DNA

DNA

DNA ( 12.5)
1. nitrous acid,
hydroxylamine alkylating agent

cytosin
e
adenin
e
guanin
e

HNO2
HNO2
HNO2

uracil
Hypoxanthi
ne
Xanthine

ethidium bromide, acridines

(intercalation)
DNA DNA
DNA polymerase


LSD, aflatoxin

DNA
2. free radicals
phosphodiester bond, keto
amino form enol imino form

3. DNA polymerase


17
6

4.
Crossing over
meiosis

12.5
DNA (Lehninger AL, Nelson DL, Cox MM. Principles
of biochemistry, 1993.)

genetic
information DNA
replication

genetic
information

mutation

mutation

mutation
mutagen
mutation mutagenesis
mutation
DNA
1. (base
substitution) point mutation
DNA

17
7

1.1 transition


DNA A G C T
1.2 transversion

DNA A
G C T
2. deletion 1
1
3. insertion 1
1

mutation
microsatellite 2
1. Trinucleotide repeat expansions (Triplet repeats)
3
CGG, CAG CTG

mRNA
Fragile-X syndrome
FMR-1 CGG
50 copies CGG repeats
200 copies
triplet repeats spinobulbar muscular
atrophy, myotonic dystrophy, Huntingtons disease
spinocerebellar ataxia type I


17
8

12.9 trinucleotide repeats

(Morell V. Science 260: 1423, 1993.)
2. Microsatellite instability mutation
short
tandem repeats
mutation
DNA
DNA
DNA
DNA

DNA polymerase
DNA

DNA ligase ( 12.10)
1. Mismatch repair

(DNA replication)

(
Watson-Crick base
pair)

12.10
DNA (Marks DB,
Marks AD, Smith CM. Basic

17
9

parental DNA
methyl

DNA


methyl

mismatched base

mutation

mis-match repair
hereditary
nonpolyposis
colorectal
cancer
2. Base excision
repair

DNA
DNA glycosylase

12.11 mismatch repair

(Marks DB, Marks AD, Smith CM.
Basic medical biochemistry, 1996.)


18
0

N- glycosidic bond

deoxyribose

AP site
( apurinic
apyrimidinic site)
AP
endonuclease
sugarphosphate

3.
Nucleotide

excision repair

endonuclease
DNA
( mismatched
base base adducts)

pyrimidine dimer

DNA -

18
1

4 Uvr
ABC excinuclease (excision
nuclease)
DNA
12
,
Uvr D (helicase)


DNA polyme-rase I

deoxynucleotides
DNA

DNA ligase

excision
repair prokaryote
eukaryote

prokaryote
excinuclease
4
Uvr A, B, C
D

1617

4. Transcription
coupled repair

transcribed mRNA

12.12
DNA (Marks DB,
Marks AD, Smith CM. Basic
medical biochemistry, 1996.)
12.23 Excision repair
thymine dimer
Uvr ABC excinuclease
E. coli (Stryer L.


18
2

RNA
RNA
polymerase (

RNA transcription)

DNA template
excision repair

RNA polymerase

12.13 Excision repair

thymine dimer
Uvr ABC excinuclease
E.coli (Stryer L.
5.
Direct
repair

DNA
-

cyclo-butane

12.14
thymine dimer
DNA
(Zubay

12.15 Thymine dimer

photoreactivation
(Zubay GL.. Biochemistry, 1998.)

18
3

pyrimidine dimers
DNA photolyase
(photoreactivating
enzyme)
DNA
pyrimidine dimer
(visible light)
dimer

phosphodiester bonds
DNA

nucleotide-excision
repair
12.6 DNA
E.coli (L Matthews HR, Freedland R, Meisfeld RL.
Biochemistry. A short course, 1997.)
Repair
Type of
system
damage
Nucleoti Pyrimidine
de
dimers
of
excision other
structural
perturbations
Mismatc Nucleotide
h
mistatches

Base
Removal
of
excision uracil,
hypoxanthine,
xanthine, and
alkylated
bases
Direct
Chemical
reversal
of

E.coli
Human genes
proteins
ABC
XPA,
XPB/ERCC3,
exonuclease,
XPC, XPD/ ERCC2,
DNA
pol
I, XPE,
XPF/ERCC1
ligase
and
XPG/ERCC5,
ERCC4, ERCC6
MutH,
MutL, MLH1, PMS1, and
MutS,
DNA PMS2
(MutL
helicase, SSB, homologues) MSH2
DNA
pol
III, (MutS)
ligase,
exonuclease I
DNA
Yeast DNA pol
glycosylases,
(mammalian
DNA
AP
pol ), human DNA
endonuclease, glycosylase
DNA
pol
I,
ligase
O6O6-methylguanine
methylguanine DNA


18
4

methylation or DNA
methyltransfer
photoactivaases, DNA
tion
photolyases

methyltransferases,

Xeroderma
pigmentosum
DNA excision
repair

chromo-somal
breakage syndrome Blooms disease, Fanconis anemia
Progeria
DNA

DNA RNA
template

Rous sarcoma, Avian myeloblastosis, Raucher murine
leukemia (HIV, human
immunodeficiency virus) RNA
RNA

reverse
transcriptase
RNA-dependent DNA polymerase
DNA RNA
RNA-DNA hybrid
RNA DNA
DNA
DNA
DNA
12.16
reverse
transcriptase

18
5

clone in vitro
mRNA com-plementary DNA (cDNA)

12.16 retroviral genome (Mathews CK,

van Holde KE. Biochemistry, 1996.)

Transposable Elements (Transposons)

Transposable elements
jumping genes Barbara McClintock


18
6

1940

1983

Transposon DNA


transposase transposon

( 12.17)
Retroposon transposon
retroposon intermediate RNA reverse
transcriptase DNA RNA
DNA

12.17
transposons
(Marks DB,
Marks AD,
Smith CM.
Basic
medical
biochemistr
y, 1996.)

18
7

DNA Recombination
DNA

DNA
recombination
crossing over

meiosis,
recombination
genetic information


differentiate
plasma cells,
plasmid DNA DNA
,
DNA phage
DNA host

DNA host
DNA host

12.18
DNA recombination (Marks DB,
Marks AL, Smith CM. Basic
medical biochemistry, 1996.)


18
8

(genetic
enginee-ring)

DNA
DNA
genetic information
recombinant DNA

recombinant DNA
DNA

restriction endonuclease

DNA

DNA

(vector) DNA

(plasmid)

DNA
DNA
recom-binant DNA
DNA

(host cell)

DNA
DNA
cloning

DNA cloning

18
9

polymerase chain
reaction PCR
DNA 50-3000

PCR
DNA
2 primer
oligonucleolide
complementary
-
primer
complementary

primer 2
DNA

DNA polymerase Thermus aquaticus
E.
coli DNA
2

DNA

primers 4

4 DNA
polymerase
DNA 4 DNA
PCR 3, 4, 5,....n
DNA 2n
PCR 20-30
DNA


19
0

chorionic villi DNA

PCR



,
DNA
DNA PCR
DNA
DNA
vector (plasmid)

insulin, interferon, growth hormone, , , , globin

hemoglobin somatostatin

recombinant protein

RNA (Transcription)
RNA (transcription)

DNA RNA
RNA (copy)

RNA
rRNA 5s 1660 DNA
mRNA
diploid cell
-chain 2
11 1 -chain 4
16 2

19
1

(structural gene)

transcribed region 5 untranslated region,

exon,
intron

3
untranslated
region (regulatory
element)

promoter,
enhancer
silencer
eukaryotes

mRNA

DNA
intervening
sequence
introns

genetic
information


19
2

exon
DNA
genetic
information

12.19

a

prokaryote b
RNA


12.7
eukaryote
eukaryote (Devlin TM. Textbook of biochemistry
with clinical
(Kendrew J. The
correlations, 1997.)
ency-clopedia of

19
3

RNA
1.
2.
3.
4.

DNA (DNA template)

ribonucleoside triphosphate (ATP, GTP, CTP, UTP)
DNA-dependent RNA polymerase
transcription factor

DNA template
DNA 2
DNA
(template) RNA
(genetic locus) DNA template
antisense strand DNA
coding strand sense strand DNA
sense strand RNA
T RNA
U
DNA coding strand 5 ATGCCAGTAGGCCACT 3
(sense)
DNA template strand 3 TACGGTCATCCGGTGA 5
(antisense)
mRNA
5 AUGCCAGUAGGCCACU 3
DNA-dependent RNA polymerase
RNA
DNA RNA
polymerase
RNA RNA polymerase E.coli
6 2 ,
, , 1 (2 )

12.8
12.8 Subunits RNA polymerase E.coli
(Mathews CK, van Holde KE. Biochemistry, 1996.)


19
4

Subu

Mass
(Kd)

Functions

Number per
enzyme
molecule
2
1

36.5
151

155

70

11

Initiates synthesis
Forms
phosphodiester bonds
Binds DNA
template
Recognizes
promoter
Unknown

eukaryote RNA polymerase 3

I, II, III RNA
12.9
-amanitin
Amanita phalloides

RNA polymerase

RNA gene mt DNA
12.9 Eukaryotic RNA polymerases

19
5

Type

Localizati
on

Nucleolus

Cellular
transcript
s

Effect of
amanitin

18S, 5.8S, 28S,

Insensitive
rRNA
II
Nucleoplas mRNA precursors,
Strongly
m
hn RNA
inhibited
III
Nucleoplas tRNA, 5S rRNA,
Inhibited by high
m
snRNA, scRNA
concentrations
Mitochon Mitochondr products of all
Insensitive
d ia
mitochondrial genes
r
i
a
l
sn = small nuclear, sc = small cytoplasmic
transcription
RNA DNA
promoter
RNA polymerase
DNA promoter
conserved
consensus
sequences
RNA 5
1. prokaryote promoter
-10 sequence Pribnow box
sense strand TATAAT
-35 sequence
TTGACA +1
RNA
+2, +3, downstream
+1 5
1, 2 ,
upstream


19
6

upstream

downstream
-35
TTGACA

-10

+1
TATAAT

3
m RNA start site
2. eukaryote promoter
(TATAAA)
-25 TATA box (Hogness
Hogness-Goldberg box) TATA box promoters
CAAT box GC box
-40 -110
- 110
+1
5
3

CAAT

- 40
GC

box

- 25
TATA

box

mRNA start site

Two or more upstream activating sequences

Transcription factor
RNA polymerase eukaryote
promoter RNA
transcription factors
promoter RNA polymerase
transcription factor 12.10
transcription factor (TF) RNA polymerase
mRNA
2 TF

(housekeeping gene)
glycolysis, Krebs cycle,
organelles
transcribed

19
7

constitutive gene TF

transcribed

12.10 transcription factors RNA polymerase

(Roskoski R. Biochemistry, 1996.)
1. RNA polymerase transcription factors (TF)

2. Human RNA polymerase transcription factors (TF)

3. Human RNA polymerase transcription factors (TF)

promoter DNA eukaryote

transcription
transcription elements enhancers, silencers


19
8

response
elements upstream
downstream
transcribed DNA transcription elements
binding site transcription factors
RNA

enhancer immunoglobulin
B-lymphocytes
enhancer sequences transcription
transcriptional transactivators
silencers transcription response
elements
transcription factor signaling molecule
cyclic AMP response elements (CRE) DNA
CREB (cAMP response element
binding protein)
RNA
RNA polymerase DNA polymerase
RNA
primer RNA transcribed
RNA 5
3 RNA polymerase
3 5
RNA prokaryote

1. Initiation RNA polymerase

holoenzyme DNA promoter (sigma
factor)
closed promoter complex
DNA 17 open promoter complex
RNA

RNA adenine guanine
phosphodiester bond

19
9

5 RNA (PPP) (
12.20)
2. Elongation
phosphodiester bond
RNA 10
sigma factor core enzyme
( ) core enzyme
RNA
50 ( 12.20)
3. Termination
phosphodiester bond RNA polymerase DNA DNA
sigma factor
core enzyme RNA 2
rho ()
3.1 rho (rhoindependent termination)
3 RNA G C
(hairpin) U
4 - 8 ( 12.21)
3.2 rho (rhodependent termination) rho factor
RNA-dependent ATPase RNA
RNA polymerase
RNA ( 12.22)


20
0

12.20 initiation elongation

(Mathews CK, van Holde KE. Biochemistry, 1996.)
12.21
termination
transcription

RNA

hairpin loop
(Mathews
CK,
van
Holde
KE.
Biochemistry,
1996.)

20
1

12.22
termination transcription rho
protein. (Stryer L. Biochemistry, 1995.)
RNA (Posttranscriptional Processing)
RNA



,
(covalent modification)

mRNA
eukaryote,
RNA
mRNA


20
2

mRNA mRNA
mRNA
polygenic polycistronic mRNA mRNA

noncoding region (
)

mRNA eukaryote
precursor
heterogeneous nuclear
RNA (hnRNA) precursor mRNA
mRNA
mature mRNA

mature mRNA ( 12.23)

1. Capping 5' 7-methylguanosine

5'-5' triphosphate bridge
methyl
cap
cap 5'
phosphatases nucleases ribosome
mRNA
2. Polyadenylation polyadenylate (poly A)
tail 200 3'
poly A polymerase ATP
DNA template poly A mRNA
nucleases
3. Splicing intron
exon
introns (splice) mRNA precursor
intron
GU AG

20
3

5' Splice site

Branch site
AG GUAAGU
A
upstream exon
Intron
exon
(donor exon)
(acceptor exon)

3' Splice site

(Py)nNCAG G
downstream

12.23 mRNA (Devlin TM. Textbook of

biochemistry with clinical correlations, 1997.)
consensus sequence 5' introns
vertebrate AGGUAAGU 3' intron
(U C) 10
C AG


20
4

intron A
- 20 50 3' splice site
branch site mutation
(5' splice site, branch site 3' splice site)
(aberrant splicing)

aberrant splicing 19
3' splice site intron G A
3' acceptor site ( 12.24)
mRNA codon

12.24 mutation (G A)
intron -thalassemia (Stryer L. Biochemistry, 1995.)
mRNA
phosphodiester bond upstream exon (exon 1)
5' intron 2'-OH adenosine branch
site 5' intron 2'5'phosphodiester bond
(lariat) ( 12.25) exon 1
3'OH 5'
exon 2 3',5'-phosphodiester bond exon 1 2
intron lariat
exon
RNA 300
snRNA (small nuclear RNA)
scRNA (small cytoplasmic RNA) RNA
snRNPs (small nuclear ribonucleoprotein
particles; snurps) scRNPs (small cytoplasmic ribonucleoprotein
particles; scurps)
mRNA precursors ( 12.11)
spliceosome
60S snRNP splicing
antibody

20
5

(systemic lupus
erythematosus, SLE) splicing
12.11 Small nuclear ribonucleoprotein particles
mRNA precursors (
Stryer L. Biochemistry, 1995.)
SnRNP

U1
U2
U5
U4
U6

Size
of
Function
snRNA
( nucleotides
)
165
Binds the 5' splice site and then the 3
splice site
185
Binds the branch site
116
145
Assembles the spliceosome
106

12.25

exon
small
nuclear
ribonucleoprote
in
particles
(Marks
DB,
Marks
AB,
Smith
CM.
Basic medical
biochemistry,
1996.)


20
6

Alternative splicing
Pre-mRNA hnRNA alternative
splicing

alternative splicing
donor exon acceptor exon
hnRNA
tyrosine hydroxylase (
12.26) tyrosine hydroxylase 14 exons
4 isoforms
alternative splicing isoforms 4
adrenal medulla isoform 1
2 85%
isoform

20
7

12.26 alternative splicing hnRNA

tyrosine hydroxylase (Roskoski R. Biochemistry, 1996.)
4. mRNA editing RNA
edited
transcription gene
mRNA
transcribed gene

RNA

RNA
editing apoprotein B
lipoproteins
gene
apoprotein B gene
apo B100 4,563

2,152
codon 2153 CAA
glutamine
cytosine condon 2153 RNA transcript
deaminated uracil UAA
stop codon translation
2152 48% Apo B-100


20
8

apo B-48 ( 12.27)
12.27
RNA editing
(Stryer

L.

Biochemistry,
1995.)

rRNA
rRNA eukaryote
2 precursor rRNA 45S
nucleolus 5S rRNA
precursor
nucleoplasm pre-ribosomal RNA
5S rRNA 45S rRNA
5' rRNA 18S, 5.8S 28S
spacer sequences methylation
hydrolysis rRNA 12.28
rRNA
prokaryote
3
23S, 16S 5S

polycistronic primary
transcripts


12.28

20
9

12.28

rRNA 45S prerRNA eukaryotes
proka-ryotes

primary trans-cripts
23S,16S
5S (Devlin TM.
Textbook
of
Biochemistry with
clinical correlations,
1997.)
tRNA
tRNA nucleoplasm RNA
polymerase III (
12.29)
1. 5 ribonuclease P
(ribozyme) 3 exonuclease
2. intron exon

3. CCA-sequence 3

4.
tRNA
modified base methyl

12.29


21
0

tRNA (Devlin
TM.
Textbook of
biochemistry
with clinical
correla-tions,
1997.)

tRNA

1.
tRNA

tRNAphe
phenylalanine tRNA
50
20
tRNA 1
tRNA
isoaccepting tRNA
tRNA
aminoacyl-tRNA synthetase

21
1

isoaccepting tRNA
tRNA

tRNA
2.
tRNA TC loop

5S rRNA (
prokaryote) 5.8S rRNA (
eukaryote)
3. codon mRNA
anticodon loop

1. DNA

DNA (replication)
S phase cell cycle
DNA

DNA
RNA
DNA
polyamines
putrescine spermidine
RNA
DNA
H3N-CH2- CH2- CH2-NH3

putrescine

H3N- CH2- CH2- CH2-NH2 CH2 CH2- CH2- CH2-NH3

spermidine
2. RNA


21
2

RNA

RNA
mRNA
,
(infection)

21
3

1. DNA

1.1 DNA
(intercalation) DNA
DNA RNA
alkylating agent, proflavin, chloroquine, actinomycin D

1.2 DNA
DNA bleomycin, streptonigrin, mitomycin

-radiation
1.3
1.1 DNA polymerase
aphidicolin, dideoxy NTPs, arabinosyl NTP
N-ethylmaleimide (NEM)
1.4
1.2 DNA gyrase
(topoisomerase II) novobiocin, oxalinic acid
nalidixic acid, etoposide, teniposide
1.5
1.3 DNA
reverse transcriptase
azidothymidine (AZT),
dideoxyinosine (ddI),
dideoxycytidine (ddC) AIDS

RNA
2.1 DNA
DNA RNA
actinomycin D, adriamycin, mithramycin
2.2
RNA
polymerase
2.


21
4

2.2.1 rifampicin,

rifamycin
initiation
RNA
2.2.2 -amanitin
RNA polymerase II
RNA polymerase III
elongation
2.2.2 streptolydigin RNA elongation
2.3 poly A tail
mRNA cordycepin
(3-deoxyadenosine)

mRNA

(turnover rate) mRNA

endonuclease

exonuclease
nucleotide nucleoside
DNA (DNase)
RNA (RNase)
nucleotidase, nucleoside phosphorylase,
phosphatase

21
5

-1-
-1-

salvage pathway modified

base

(Translation)

RNA

3 - 4 /

(0.5 - 1.1 /)

globin chain

- prokaryote
eukaryote
-
RER
- 20


21
6

-
mRNA
-

-
NH2-terminal
COOH-terminal

1.
20 L-isomer side
chain genetic code (
12.13)
tRNA
3
activation
aminoacyl-tRNA synthetase, Mg2+
ATP aminoacyl-tRNA
2. tRNA
50 tRNA
1 clover leaf
L-shape 3 ( tRNA
3) tRNA

aminoacyl-tRNA synthetase tRNA

complementary codon 3
mRNA anticodon
3 anticodon codon
antiparallel
3 codon 5 anticodon

wobble position (
12.12) wobble
A G C U inosine (I) C, U
A ( G)

21
7

A U G C
wobble base pairing
tRNA 1 codon 1 codon
tRNA 50
61 codons codon
( codon
3 wobble base tRNA)
3. Genetic code

(nucleotide sequence) DNA (

RNA ) DNA transcribed
mRNA ribosome
(amino
acid sequence) DNA

mRNA
mRNA DNA

..1960
3
triplet code codon
1 mRNA
4 A, U, G C
64 (
12.13)
genetic code degenerate
2
AUG ( methionine


21
8

12.30

(Roskoski R. Biochemistry, 1996.)
12.12
codon-anticodon
wobble base pairing (Zubay GL.
Biochemistry, 1998.)

initiation
codon) UGG ( tryptophan) (
12.13) 61
3 UAA, UAG UGA
stop nonsense codon
coding region mRNA

21
9

genetic code
universal
prokaryote eukaryote genetic code
(<10
) UGA
= tryptophan, AUA = methionine, CUA = threonine
genetic code mRNA codon
(commaless)
(non-overlapping)
genetic code

12.13 The universal genetic code (Kendrew J. The
encyclopedia of molecular biology, 1994.)

gene mutation


22
0

RNA
2
1. regulatory element
transcrip-tion
RNA

2. structural gene (transcription unit)

2.1 missense mutation

DNA (base substitution)
mRNA codon

CAU (His) CAG (Gln)
2.2
nonsense mutation
codon code
stop codon

truncated polypeptide chain CGA (Arg) UGA (stop)
2.3
chain termination mutation sense
mutation stop codon
codon code

2.4 splicing mutation ()

mRNA

2.5
silent mutation
DNA mRNA
code
AAA
(Lys) AAG (Lys)
2.6 frameshift mutation
( 3 )

22
1

codon mutation
( 12.31)

12.31 frameshift mutation

A (base insertion) (Marks DB, Marks AD,
Smith CM. Basic medical biochemistry, 1996.)

interaction

4. Aminoacyl-tRNA synthetase

tRNA (cognate tRNA)
20

amino acid + ATP + tRNA aminoacyl-tRNA + AMP +PPi

PPi 2Pi
pyrophosphate (PPi)

aa-tRNA 1 2 ATP

aminoacyltRNA synthetase (aa-tRNA synthetase)


22
2

genetic code mRNA aminoacyltRNA
tRNA
tRNA
aa-tRNA synthetase
(proofreading)
aa-tRNA
aa-tRNA
tRNA
tRNA high energy ester bond COOH group 3 -OH group tRNA
peptide
bond
5. Ribosome
ribosome
ribonucleoprotein ribosome prokaryote
70S 2 50S 30S
eukaryote 80S
60S 40S ( 12.32) ribosome
P site
A site aminoacyl tRNA

22
3

12.32 70S ribosome prokaryotic

ribosome (A) 80S ribosome eukaryotic ribosome (B)
(Roskoski R. Biochemistry, 1996.)

3 initiation,
elongation termination
12.14
12.15
eukaryotes
12.14 factors
(Roskoski R. Biochemistry, 1996.)


22
4

12.15 factors
eukaryote (Roskoski R. Biochemistry, 1996.)

Initiation
coding sequence
mRNA AUG
5 mRNA
mRNA

ribosome mRNA 3

mRNA
3 synthetic mRNA
genetic code
poly(AAC)n 3

poly(AAC)n AAC AAC AAC AAC AAC (Asn)n

AA CAA CAA CAA CAA C
(Gln)n

22
5

A ACA ACA ACA ACA AC

(Thr)n

mRNA

AUG 5 mRNA
AUG methionine

tRNAi initiation
AUG tRNA
methionine tRNAi


1. mRNA
2. Met-tRNAi ( prokaryote formyl methionyltRNA)
3. ribosome
4. initiation factor (IF)
8.4
5. GTP
6. ATP eIF
cap mRNA stemloop
(initiation)
12.33
1. eIF2a GTP Met-tRNAi ternary
complex
2. ribosome 2 40S
eIF3 ternary complex eIF4c
eIF2a.Met-tRNAi. GTP, eIF3.40S, eIF4c complex
3. complex mRNA 5
preinitiation complex ATP IF
5 3
AUG


22
6

4. anticodon tRNAi base

pairing AUG mRNA GTP
GDP
5. IF GDP
ribosome 60S initiation
complex
prokaryote IF eukaryote
initiator codon AUG mRNA AUG

Shine-Dalgarno sequence 10
upstream AUG
initiation complex ribosome 80S (2
40S 60S) Met-tRNAi P
site ribosome anticodon tRNAi AUG
codon mRNA
initiation complex ribosome

translation

22
7

12.33 initiation complex

(Devlin TM. Textbook
of biochemistry with clinical correlations, 1997.)
(Elongation)
initiation complex
ribosome mRNA ribosome
mRNA
termina- tion codon

mRNA
12.34
1. initiation complex
2. aminoacyl-tRNA complex
eEF1 GTP
3. elongation factors (EF)
4. GTP GDP

1.
eEF-1 GTP aa-tRNA
initiation complex A site
2. base pairing base codon mRNA
anticodon aminoacyl-tRNA complementary
eEF-1 GTP GDP.eEF-1 GDP
ribosome aminoacyl-tRNA A site
ribosome


22
8

3. peptide bond peptidyl transferase

ribosome ester bond COOH tRNA P site COOH group -NH2 group A
site peptidyl-tRNA A site
4. translocation peptidyl-tRNA A site
P site eEF-2 translocase
GTP
A site aminoacyl-tRNA
2
termination codon
5.
uncharged tRNA (deacylated tRNA)
P site E site eEF-1.GTP
eEF-1.GDP
eEF-1 ( 12.34)

Termination
ribosome
termination codon UAA, UAG
UGA release
factor (eRF) GTP A site
deacylated tRNA E site peptide
tRNA
ribosome peptidyl transferase
hydrolase ester bond
tRNA ( 12.35)

22
9

12.34

(elongation) (Devlin TM,
Textbook of biochemistry with clinical correlations, 1997.)


23
0

12.34

()

23
1

12.35 (termination)
(Devlin TM, Textbook of biochemistry
with clinical correlations, 1997.)


23
2

12.36 eukaryote
(1) initiation complex (2-6)elongation
termination ribosome,
aa = amino acid (Roskoski R. Biochemistry, 1996.)

Polyribosome (polysome) ( 12.37)

mRNA ribosome
ribosome

mRNA ribosome

ribosome

23
3

12.37 (a) polyribosome E.coli

(b) polyribosome ribosome
(Mathews CK, van Holde
KE. Biochemistry, 1996.)

2 -

DNA (mtDNA) 16,569

base pairs (bp) rRNA 2 tRNA
22 13


ATP
mtDNA

mtDNA mutation mtDNA
mitochondrial DNA
diseases

- DNA
transcribed mRNA
ribosome tRNA

1.
ribosome


23
4

2. genetic code UGA =

trp, AGA AGG = stop

prokaryote eukaryote

prokaryote

plus strand RNA

minus strand RNA DNA reverse
transcriptase mRNA

host eukaryote prokaryote

GUG
overlap
RNA 2
( coat protein L protein bacteriophage MS2)

ribosome

(amino acid sequence)

-
(one dimension)

(self-assembly)
denature

native form (folding)

23
5

molecular chaperone


prolyl isomerase cis-trans
isomer proline

prokaryote
eukaryote
12.16
12.16
(Garrett RH, Grisham CM. Biochemistry, 1995.)


23
6

1.
prokaryote
antibiotic

2. eukaryote


Diphtheria toxin eEF2 ADP-ribosylation translocation
peptidyl-tRNA A P site
eEF-2 + NAD+
(active)
diphtheria toxin
ADP-ribosyl-eEF-2 + nicotinamide + H+
(inactive)
3. eukaryote
prokaryote puromycin
tRNA peptidyl-puromycin peptidyl
transferase peptidyl
puromycin
premature termination

2
1.
2.

23
7

mRNA

transcription
( germ cell) DNA

1
transcribed mRNA
mRNA

mRNA

mRNA -

transcription
steroid, thyroxin, calcitriol retinoic acid
receptor binding protein
transcription factor

1.

3 mRNA,
ribosome
protein factor

1.1 mRNA
mRNA
mRNA

1.1.1 mRNA
mRNA
transcription


23
8

(genetic dosage)
Down
syndrome

1.1.2 mRNA
translation
cap nucleotide sequence stemloop mRNA mRNA
ribosome
1.1.3 mRNA
repressor protein anti-sense RNA ( RNA
complementary DNA sense strand)
ribosome
1.1.4 poly A tail
(stability) mRNA
1.2

(short half life)

albumin,
transthyretin

1.3 ribosome
(protein-synthesizing machinery)
activity phosphorylation dephosphorylation
allosteric regulation
globin chain
heme
allosteric inhibitor
(heme-controlled
inhibitor) eIF-2

23
9

stable complex eIF-2 eIF-2B

( 12.38)

12.38 initiation globin chain

(Mathews CK, van Holde KE, Biochemistry, 1996.)
2.

cofactor

eukaryote
(half-life)
( 12.17 )

N-terminal (N-end rule)

( 12.17 )

PEST


24
0

sequence
(proline-glutamate-serine-threonine)

24
1

12.17 half-life ()
N-terminal () (Zubay GL. Biochemistry, 1998.)

2

1. Ubiquitin-ATP dependent pathway

(regulatory protein), nuclear
protein (myc, myb), tumor suppressor protein (p53)
(cyclins)
marked
ubiquitin
protease


24
2

Ubiquitin

marker

-COOH
ubiquitin -NH3
lysine
isopeptide bond
3
E1 E2 E3 (
12.39)
protease complex

ubiquitin

exopeptidase

MHC (major histocompatibility
complex) class I, II
cell surface

immune MHC


antigen presentation
2.
Lysosome
degradation

protease lysosome
cathepsins

24
3

2.1

(
12.40 A)
2.2 organelle
(autophagic
lysosome) ( 12.40
B)
2.3

endocytosis ( 12.40
C)
2.4 (
12.40 D)

12.39
ubiquitin- ATP
pathway
(Zubay
GL.
Biochemistry, 1998.)


24
4

12.40 lysosome
(Mathews CK, van Holde KE. Biochemistry, 1996.)
Protein translocation (Protein targetting)

signal sequence
(receptor)

2
1. Post-translational translocation
ribosome

free polysome

peroxisome
2. Cotranslational translocation

ER (endoplasmic reticulum)
Golgi apparatus lysosome
(secretory protein)
ribosome
ribosome signal sequence
signal recognition
particle (SRP) SRP G-protein (guanine nucleotide binding
protein) GTP SRP

24
5

ribosome
ER
SRP docking protein ( 12.41)
ribosome ribosome receptor
protein conduction channel
GTP
SRP GDP Pi SRP
ribosome
GTP SRP
ribosome
RER
membrane protein sequence
hydrophobic -helix

signal sequence
Golgi apparatus

12.41 endoplasmic reticulum

(Roskoski R. Biochemistry, 1996.)
Secretion

secretory protein


24
6

ribosome ER signal peptide

N-terminal
lumen ER Golgi apparatus
exocytosis

exocrine trypsinogen,
chymotrypsinogen, amylase
endocrine insulin, glucagon
fibroblast
collagen, proteoglycans

plasma proteins
mammary gland milk proteins
plasma cell
immunoglobulins
2 ( 12.42)
1. constitutive secretion

plasma protein
fibroblast
2. regulated secretion

secretory vesicle
secretory vesicle exocytosis

24
7

12.42 protein translocation secretion (Roskoski R.

Biochemistry, 1996.)


24
8

(Post-translational Processing)
ribosome

post-translational processing
proteolytic cleavage covalent
modification


1.
active form
1.1 zymogen

trypsinogen ()
trypsin (
)
pepsinogen
pepsin ()
Factor XII () Factor
XIIa
(injury
surface)
plasminogen () plasmin (fibrin clot)

activa-tion

1.2 prohormone

proinsulin
insulin
proglucagon
glucagon
pro-opiomelanocortin (POMC) ACTH + MSH +
-endorphin
2. side chain
( 12.18)
12.18
(Devlin TM. Textbook of biochemistry with clinical correlations,
1997.)

24
9

2.1 -carboxylation glutamate

calcium binding protein carboxylglutamate Ca++


25
0

12.43 -carboxylglutamate
vitamin K-dependent oxidation (Roskoski R. Biochemistry, 1996.)
2.2 Hydroxylation side chain
proline lysine proline hydroxylase lysyl
hydroxylase hydroxyproline hydroxylysine
H-bond ,
(glycosylation) cross link
tropocollagen
hydroxylation

25
1

12.44

collagen
(Mathews CK,
van Holde KE.
Biochemistry,
1996.)

12.45


procollagen (Gal =
galactose, Glc =
glucose,
GlcNAc = Nacetylglucosami
ne,
Man
=
mannose) (Voet
D, Voet JG.
Biochemsitry,
1995.)

2.3

N-methylation


25
2

2.3.1 lysine
cytochrome C, calmodulin, histone
2.3.2 histidine

trimethyllysine

carnitine

3. Phosphorylation
ATP protein kinase

protein kinase 1-3%

phosphorylation
activity
ATP + Protein
phosphoprotein + ADP
serine
threonine tyrosine

casein
casein

4. Acylation prenylation
lipid
lipid anchor
4.1 Acylation acetyl group
meristoyl group (C-14) N-terminal
Acetyl
CoA + H2N-protein
acetyl-NH-protein + CoA
4.2 Prenylation farnesyl group (C-15)
geranylgeranyl group (C-20) cysteine Cterminal Ras (cell-transforming GTP binding protein),
transducin retina -subunit G-protein
( 12.46)

25
3

5. Glycosylation ER Golgi apparatus

glycosyltransferase side chain
glycosylation 2
5.1 N-linked glycosylation
amide asparagine (Asn-X-Ser)
14
lipid dolichol phosphate
membrane ER oligosaccharide N-linked
3 high mannose complex
hybrid ( 12.47) N-linked glycosylation
cotranslational process
antibiotic bacitracin
tunicamycin
5.2 O-linked glycosylation
OH serine threonine
Golgi apparatus Olinked glycosylation post translational process
nucleotide
1
blood group antigen ABO


25
4

12.46
Protein prenylation
proteolysis (3) methylation
(Roskoski R. Biochemistry, 1996.)

(1) farnesylation, (2)

receptor
lysosome
mannose-6-phosphate
receptor lysosome
mannose-6-phosphate
lysosome lysosome

(inclusion body)
I-cell disease mucolipidosis II
lysosomal storage disease

25
5

12.47 N-linked glycosylation () GlcNAc-Asn linkage

( ) oligosaccharide N-linked
(Roskoski R. Biochemistry, 1996.)

12.48 O-linked glycosylation () GlcNAc-Ser linkage

() Blood group glycoproteins (Roskoski R. Biochemistry, 1996.)
(Protein abnormalities)
2
1.
2.

osmolality


25
6

1. (extrinsic factor, exogenous cause)

1.1

1.2
1.3

1.4 (Infection)
2. (Intrinsic factor)

25
7

1. ,

, . 1
:
, 2536.
2. Champe PC, Harvey RA. Lippincott s illustrated reviews:
Biochemistry. 2nd ed. Philadelphia: J.B. Lippincott Company,
1994.
3. Devlin TM. Textbook of biochemistry with clinical correlations.
4th ed. New York: Wiley Liss, Inc., 1997.
4. Horton HR, Moran LA, Ochs RS, Rawn JD, Scrimgeour KG.
Principles of biochemistry. 2nd ed. New Jersey: Prentice-Hall
International Inc., 1996.
5. Kendrew J. The encyclopedia of molecular biology. Oxford:
Blackwell Science Ltd., 1994.
6. Lehninger AL, Nelson DL, Cox MM. Principles of biochemistry.
2nd ed. New York: Worth Publishers, 1993.
7. Mark DB, Marks AD, Smith CM. Basic medical biochemistry.
A clinical approach. Baltimore: Williams & Wilkins, 1996.
8. Mathews CK, van Holde KE. Biochemistry. 2nd ed. Menlo Park:
The Benjamin/Cummings Publishing Company Inc., 1996.
9. Matthews HR, Freedland R, Miesfeld RL. Biochemistry: A short
course. New York: Wiley-Liss, Inc., 1997.
10. Morell V. The puzzle of the triplet repeats. Science 1993; 260:
1422-3.
11. Roskoski R. Biochemistry. Philadelphia: W.B Saunders Co.,
1996.
12. Stryer L. Biochemistry. 4th ed. New York: W.H. Freeman and
Company, 1995.
13. Voet D, Voet JG. Biochemistry. 2nd ed. New York: John
Wiley&Sons, 1995.
14. Zubay GL. Biochemistry. 4th ed. Dubuque: Wm.C. Brown
Publisher, 1998.


25
8