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Text Book of Molecular Biology
Department of molecular biology and biochemistry
Contents
Chapter 1
Nucleotide Metabolism
Chapter 2
Polynucleotides and Nucleic Acids
Chapter 3
DNA Biosynthesis and Recombination
Chapter 4
RNA Biosynthesis
Chapter 5
Protein Synthesis and Processing
Chapter 6
Integration of Expression and Transmission of Genetic Information
Chapter 7
Regulation of Prokaryotic Gene Expression
Chapter 8
Regulation of Eukaryotic Gene Expression
Chapter 9
Coupling of Gene Expression and Cell Signaling
Chapter 10
Basic Methodology in DNA Manipulation
Chapter 11
Recombinant DNA Technology
Chapter 12
Strategies for Analyzing Gene Structure
Chapter 13
Strategies for Analyzing Gene Expression and Function
Chapter 14
Gene and Disease
Chapter 15
Gene Diagnosis and Gene Therapy
Chapter 1
Nucleotide Metabolism
Section 1
Structure and function of nucleic acids (PP.9-13)
I. The building blocks (or monomeric units) of nucleic acids are nucleotides.
II. The nucleotide is composed of base, pentose and phosphate
Bases:
There are four bases adenine (A), Guanine (G),cytosine(C) and thymine (T) in DNA
There are also four bases A, G, C and U (uracil) in RNA.
A and G are purines, C, T, and U are pyrimidines.
P9 fig
Tantomers of Purines and Pyrimidines:
There are two forms (or tantomers) of purines, the amino form and the imino form.
There are also two forms of pyrimidines, the keto form and the enol form.
A tantomer is an isomer that differs from another in the location of hydrogen and a
double bond.
The interconversion of the tantomers, such as keto and enol forms, is referred to as
tantomerization.
P10 fig
Rare Bases:
A,T,G,U and C are the most common bases in nucleic acids. Minute amounts of
modified forms of the above bases are also found in nucleic acids. They are rare bases
and of a large variety. Most of them are methylated five common bases.
P10 tab1-7
Pentoses:
In RNA the pentose is ribose whereas in DNA it is 2’-deoxyribose (β-d-2’)
P11 the top fig
Nucleosides:
A nucleoside consists of a base covalently bonded by the glycosidic bond to the
1’-position of a ribose or 2’-deoxyribose. There are ribonucleosides and
deoxynucleosides. All the naturally occurred nucleosides are of anti-conformation.
P11 the middle fig
Nucleotides:
A nucleotide is a nucleoside with one or more phosphate groups bound covalently to
the 3’-，5’- or (in ribonucleoside only，the 2’-position) by phosphoester bond.
There are ribonucleotides and deoxynucleotides.
In the case of the 5’-position, up to three phosphates may be attached, to form, for
example, adenosine 5’-triphosphate (ATP) or deoxyadenosine 5-monophoshate
(dAMP) etc.
P11 fig1 -6A
The most nucleotides in the organisms are 5’ –phosphate nucleotides.
Cyclic nucleotides:
cAMP and cGMP are formed from ATP and GTP respectively. They act as
intracellular second messengers.
P11 fig 1-6B
III. Nucleotides have a lot of physiological and biochemical functions.
1. Nucleotides are building blocks of DNA and RNA.
2. Nucleotide derivatives, such as U-DPG, CDP-DG etc, are metabolic intermediates
which participate in macromolecule biosynthesis.
3. ATP is the direct energy-supplier.
4. AMP and ADP are components of many co-enzymes.
5. GTP participate in the synthesis of the cap of the eukaryotic mRNA and
tetrahydrpterodine.
6. cAMP and cGMP are second messengers.
7. ppGpp, 2’5’oligo A participate in physiological and biochemical regulation.
8. GDP and GTP are important components of G-protein.
IV. The physico-chemical properties of the nucleotide are utilized for its isolation and
identification.
1. Because of the conjugate double bond in the base, nucleotides have the
UV-absorption in the range of 220-290nm, with the peak at 260nm. Each kind of
nucleotide has the specific UV-absorption spectrum. It is not only utilized for
identification but also for quantitative determination of the nucleotide.
P14 fig1-7
2. The dissociation properties of nucleotide are utilized for the separation and
purification of nucleotides.
Section 2
Nucleotide Metabolism (PP.259-274)
I. Outline for Nucleotide Metabolism
1. Nucleotides have a lot of biological functions.
2. There are two pathways for the formation of nucleotides, the de novo synthesis and
the salvage pathway. The biosynthetic origins of purine and pyramiding ring
atoms are as follows:
P260 fig 13-1
3. Degradation of nucleotides and the salvage pathway are also of important
biological significance.
P261 fig 13-2
4. PRPP is at the crossing of the de novo synthesis and salvage pathway.
P261 fig 13-3
II. Anabolism and catabolism of purine nucleotides
1. Three features of synthesis of purine nucleotides
a. Liver, small intestine and thymus are the three organs in which purine nucleotides
are mainly de novo synthesized.
b. Synthesis of purine nucleotides begins with R-5-P and ATP, and the initially
synthesized purine nucleotide is IMP.
c. AMP and GMP are synthesized from IMP.
IMP is synthesized in a pathway comprised of 11 reactions
R5P ─ PRPP ─ PRA ─ ─-------─IMP
P262 fig 13-4
from IMP to AMP
IMP+Aspartate ─ AS ─ AMP+fumarate
from IMP to GMP
IMP ─ XMP
XMP+glutamine ─ GMP+glutamate
P263 fig 13-5
2. Regulaion of Purine Nucleotide Synthesis
(1)The IMP pathway is regulated at its first two reactions: those catalyzing the
synthesis of PRPP and PRA.
a. Feedback inhibition:
Synthesis of PRPP is inhibited by both ADP and GDP.
Synthesis of PRA is inhibited by IMP, AMP and GMP.
b. Feed forward activation
R-5-P and ATP activate PRPP synthesis.
R-5-P and ATP activate PRA synthesis.
(2)Regulation of the IMP to AMP or GMP pathway
a. Feedback inhibition
AMP and GMP are competitive inhibitors of IMP and inhibit AMP and GMP
pathway respectively.
b. Reciprocal activation
GTP activates the synthesis of AMP, whereas ATP activates the synthesis of GMP.
This reciprocity serves to balance the production of AMP and GMP.
P266 fig 13-7
3. Salvage Pathway of Purine Nucleotides
(1) Using purine and PRPP as precursors to synthesize AMP, GMP and IMP.
APRT
PRPP+A ───────AMP+PPi
HGPRT
PRPP+G───────GMP+PPi
HGPRT
PRPP+I───────IMP+PPi
(2) AK catalyzes the AR phosphorylation
AK
AR+ATP ─────── ADP+AMP
4. Purine nucleotide is degraded to the end product uric acid, in the human body.
P265 fig 13-6
III. Anabolism and Catabolism of Pyrimidine Nucleotides
1. (1)denovo synthesis of pyrimidine nucleotides begins with CO2 and glutamine
CPSⅡ
CO2 + Gln ─────── CP
There are two different types of CPS: CPSⅠ and CPSⅡ
CPSⅠ is located in the mitochondria of the hepatocyte, which participates in the urea
synthesis.
CPSⅡ is a part of a multifunctional enzyme located in the cytosol. The product CP is
utilized to synthesize pyrimidine nucleotides.
(2) The initially synthesized pyrimidine ring containing product is OA.
OA and PRPP form OMP. Decarboylation of OMP yields UMP
P267 fig 13-8
(3) UTP is synthesized form UMP and CTP is formed by amination of UTP.
(4) UDP is formed from UMP. dUDP is formed by reduction of UDP by
ribonucleotide reductase. dUDP is hydrolyzed to dUMP. Methylation of dUMP
yields dTMP(TMP).
p268 fig 13-9
2. Regulation of synthesis of pyrimidine nucleotides
(1) Feed forward activation
a. ATP activates PRPPK and CPSⅡ.
b. PRPP activates OPRT.
(2) Feedback inhibition
a. UMP feedback inhibits CPSⅡ
b. UMP and CTP feedback inhibit ATPase
c. ADP and GDP feedback inhibit PRPPK
d. CTP feedback inhibits CTPS
3. Salvage pathway of pyrimidine nucleotides
(1) Using U or OA as precursors to synthesize UMP or OMP.
PRPP+U (OA) ───────UMP (OMP) + PPi
(2) Using UR, CR, or TdR to synthesize UMP, CMP or TMP
UR (CR) + ATP───────UMP (CMP) + ADP
TdR + ATP ---------------TMP + ADP
4. Catabolism of pyrimidine nucleotides
Products of U and C catabolism are NH3, CO2 and B-alanine, while products of T
catabolism are NH3, CO2 and β-aminoisobutyric acid.
P269 fig 13-10
IV. The Conversion of Nucleotides in Vivo
1.Reduction of NDPs (N=A, G, U, C) forms dNDPs.
Reduction of the 2’-hydroxyl of NDPs catalyzed by ribonucleotide reductase, forms
the corresponding dNDPs.
Reduction requires thioredoxin, thiredoxin reductase NADPH and FAD besides
ribonucleotide reductase.
P270 fig 13-11
2. NDPs and NTPs are interconvertible
(1) from UMP to UDP, AMP to ADP
UMP+ATP─────UDP+ADP
AMP+ATP─────ADP+ADP
(2) Interconversion of NDP and NTP
XDP+YTP ───────XTP+YDP
P270 fig 13-12
V. Nucleotide Metabolism and Medicine
1. Metabolic disorders of purine nucleotides may cause diseases.
(1) A defect in HGPRT causes Lesch-Nyhan Syndrome.
(2) Gout is a metabolic disorder of purine catabolism.
(3) ADA deficiency is associated with an immunodeficiency disease.
(4) Metabolic disorder of pyrimidine nucleotides is rarely associated with clinically
significant abnormalities.
2.The Mechanism of antimetabolites is blocking the synthesis of nucleotides
(1) Antimetabolites are analogues of metabolites or coenzymes of the nucleotide
synthesis pathways.
P273 fig 13-13
(2) There are key reactions in the nucleotide synthesis pathways, which are
antimetabolites’ targets.
6MP, the adenine analogue, is the competitive inhibitor of HGPRT.
6MP nucleotide, the analogue of IMP, inhibits the conversion of IMP to AMP or GMP.
Azaserine, the analogue of glutamine, inhibits de novo synthesis of purine nucleotides.
MTX and aminopterin are the analogues of folate and inhibit DHFR.
Arabinosylcytosine inhibits the conversion of dCDP from CDP and the biosynthesis of DNA.
5FU is an irreversible inhibitor of thymidylate synthase.
Chapter 2
Polynucleotides and Nucleic Acids
Section 1 Polynucleotides(PP.44-64)
Deoxynucleotides or ribonucleotides respectively are joined into a polymer by the
covalent linkage of a phosphate group between the 5’-hydroxyl of one deoxyribose
(or ribose) and the 3’-hydroxyl of the next.
This kind of bond or linkage is called a phosphodiester bond, since the phosphate is
chemically in the form of a diester.
Polydeoxynucleotides and polyribonucleotides are nucleic acids. The former are
known as DNA, and the latter RNA.
A nucleic acid chain can be seen to have a direction. Any nucleic acid chain, unless it
is circular, has a free 5’-end and a free 3’-end.
The 5’-end to 3’-end arrangement of the nucleotide (or base) in a nucleic acid chain is
called the sequence of the nucleic acid chain.
The sequence of a nucleic acid chain is the primary structure of the nucleic acid chain.
There is a convention to write the sequence with the 5’-end at the left.
A stretch of DNA sequence might be written 5’-GTCA-3’ or even just GTCA
P45 fig 3-1
Section 2
Structure and Function of DNA
Ⅰ.DNA double-helix structure is the secondary structure of DNA
DNA double-helix structure model was put forth in 1953 by Watson and Crick.
DNA double-helix model:
1. Two separate and anti-parallel chains of DNA are wound around each other in a
right-handed helical path, with the sugar-phosphate backbones on the outside and the
bases, paired by hydrogen bonding and stacked on each other, on the inside.
Adenine does pair with thymine by two hydrogen bonds; guanine pairs with cytosine
by three hydrogen bonds.
The two chains are complementary; one specifies the sequence of the other.
P47 fig 3-2
2. Between the backbone strands run the major and minor grooves, which also follow
a helical path.
A single turn of the DNA double-helix contains ten base pairs. The distance spanned
by one turn of the helix (pitch) is 3.4nm .the diameter of the helix is 2nm.
3. The stability of a DNA double-helix is maintained by the hydrophobic base
stacking and the hydrogen bonding.
Multi-forms of double-helix DNA
The above-mentioned DNA double-helix is the B–form of DNA double-helix
(B-DNA). the other well-known forms of double-stranded DNA are A-DNA and
Z-DNA.
P48 fig 3-3
Triplex DNA, tetraplex DNA and DNA hairpin:
1. Hoogsteen base pair
It is a nucleic acid base pair that differs from the Waston-Crick base pair.
In the Hoogsteen adenine-thymine base pair the 6-NH2 and N-7 of the adenine are
hydrogen bonded respectively to the 4-O and H-3 of the thymine. That forms T*A=T.
The Hoogsteen guanine-cytosine pair requires that N-3 of the cytosine is
protonated .In this base pair the 6-O and N-7 of the guanine are hydrogen bonded
respectively to the 4-NH2 and the protonated N-3 of cytosine.(C*G≡C)
P50 fig 3-5
2. triplex DNA
It is a three-stranded structure formed by duplex DNA in association with an
oligonucleotide, when purine or pyrimidine bases occupy the major groove of the
DNA double-helix adopting Hoogsteen base pairing with the Waston-Crick
base-pairs.
P50 fig 3-5
3. mirror repeat
5’------- TC TC TC TC CT CT CT CT------------3’
3’------- AG AGAG A G GAGA GAGA----------5’
4. conditions of triplex DNA formation
(1) double stranded DNA ,one strand is polypurine, the other strand polypyrimidine
(2)mirror repeat
(3)acidic pH
If the Hoogsteen pairing occurs , triplex DNA is formed.
For example, the above mirror repeat, one strand is polypyrimidine the other strand
polypuine. Under acidic pH condition it is denatured. The polypyrimidine strand folds
back and participates in the formation of triplex DNA.
5. tetraplex DNA or quadruplex DNA
Tetraplex DNA is a four-stranded DNA structure adopted by sequences rich in
guanine bases.
There are two major classes of tetraplex DNA. The first involves the folding back of a
repetitive sequence of guanines resulting in anti-parallel strands. The other is
characterized by the association of four independent parallel strands
5-GGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGG-3
5
3
One strand folds back three times:
5-GGGGGGGG-3, 5-GGGGGGGG-3, 5-GGGGGGGG-3, 5-GGGGGGGG-3
Four strands associate
5---------------------3
5---------------------3
5---------------------3
5---------------------3
P50 fig 3-6
6. DNA hairpin
Palindrome structure or inverted repeat for example
----------------------------------
------
-----
5-TTAGCAC∣GTGCTAA-3
5-TGCGATACTCATCGCA-3
3-AATCGTG∣CACGATT-5
3-ACGCTATGAGTAGCGT-5
-----------------------------------------Perfect IR
partial IR
If the inverted repeat DNA is denatured the single stranded DNA may form DNA
hairpin after renaturation.
P49 fig3-4
Ⅱ.The supercoil structure is the DNA tertiary structure
Closed-circular DNA and supercoil
1. The E coli chromosome is a closed-circular DNA of length 4639Kb, which resides
in a region of the cell called the nucleoid.
2. The eukaryotic mtDNA and ctDNA are also closed-circular DNAs.
3. Closed-circular DNA means that the two complementary single strands in the DNA
molecule are each joined 5’ to 3’ and form a double stranded circle.
4. Supercoiling is the coiling of the DNA axis upon itself. That results in supercoil
structure of DNA.
P50 fig 3-7
5. E coli DNA, mt DNA,ct DNA are closed-circular supercoil DNA.
III. The highly ordered DNA-protein complex makes up the eukaryotic chromosomes.
1. Nucleosome is the basic unit of the chromatin. The major protein components of
chromatin are histones. They are small basic proteins which bind tightly to DNA.
There are five families of histones:H1 ,H2A,H2B,H3 and H4. H2A,H2B,H3 and H4
are known as core histones.
Two molecules of each families of core histones : (H2A)2 (H2B)2(H3)2(H4)2 , make up
the octameric histone core.
P52 fig 3-9A
The histone core with 146 bp of DNA wrapped 1.8 times in a left-handed fashion
around it is the nucleosome core (core particle).
P52 fig 3-9B
A single molecule of H1 stabilizes the DNA at the point at which it enters and leaves
the nucleosome core.
There is the linker DNA of 60 bp or so between neighbouring nucleosome cores.
P53 fig 3-9C
Nucleosomes are arranged in a ‘beads on a string’ structure.
P53 fig 3-9D
2. The ‘beads on a string’ structure is organized into chromatin/chromosome
Ⅳ. DNA is the vehicle of inheritance.
1. DNA is the carrier of genetic information, which is demonstrated by Avery’s
experiment of pneumococcus transformation.
2.The whole genetic information of a virus or a cell is the genome of the virus or the cell.
3.Chromatin in interphase can be seen under microscope in two types. The less
densely packed variety is termed euchromatin, which is genetically expressed, and the
more densely packed variety is termed heterochromatin, which is transcriptionally
inactive.
Section 3
Structure and Function of RNA
Ⅰ. There are four major types of RNA in the animal cell.
P53 table 3-4
Ⅱ. Most eukaryotic mRNA contains five parts: the cap, the 5’UTR,the coding region,
the 3’UTR and the poly A tail.
P54 fig 3-11
Ⅲ. structural features of tRNA
1. The tRNA molecule is rich in rare bases.
P55 fig 3-12
2. The secondary structure of a tRNA molecule is like the cloveleaf.
P55 fig 3-13
3. The tertiary structure of a tRNA molecule is L-shaped.
P56 fig 3-14
Ⅳ.The structure of rRNAs is more complicate than that of mRNA or tRNA.
There are three types of prokaryotic rRNAs:5SrRNA,16SrRNA and 23SrRNA, while
there are four types of eukaryotic rRNAs:5SrRNA,5.8SrRNA,18SrRNA and
28SrRNA.
P57 table 3-5
rRNAs are important components of the ribosome. The ribosome is the location
where the protein is synthesized.
Ⅴ.There are a lot of snmRNAs in the cell
P57 table 3-6
RNomics studies the type, the structure, the function ,and the expression profile of
snmRNAs in the cell.
1.SnRNAs participate in pre-mRNA splicing
P58 table 3-7
2. Ribozymes or catalytic RNAs participate in splicing of particular RNAs.
3. snoRNAs participate in modification of rRNAs and tRNAs
4. siRNAs participate in post-translational regulation
Section 4
Physico-chemical properties of nucleic acids
Ⅰ.Nucleic acids are susceptible to hydrolysis under acidic or basic conditions, They are
also hydrolyzed by nucleases
Ⅱ. Nucleic acids are amphipathic compounds
1. Bases are tantomeric
There are the keto form and the enolate form of G, U and T, and the amino form and
the imino form of A and C.
2.Bases are basic under the physiologic condition
3.Nucleic acids are acidic compounds
Ⅲ. Nucleic acids solution esp. DNA solution has a high viscosity
Ⅳ. Nucleic acids absorb UV light due to the conjugated aromatic nature of the bases.
The wavelength of maximum absorption of light by nucleic acids is 260nm,(lambda
max=260) which is conveniently distinct from the lambda max of protein(280nm).
The absorption properties of nucleic acids can be used for detection, quantitation and
assessment of purity of nucleic acids.
Ⅴ. Denaturation, Renaturation and Molecular Hybridization of Nucleic Acids
1.When a solution of dsDNA is heated above a characteristic temperature, its native
structure collapses and its two complementary strands separate and assume the
random coil conformation.
P60 fig 3-17
The DNA denaturation process is accompanied by a qualitative change in the DNA’s
physical properties, such as decreasing viscosity and increasing UV260 absorption
(hyperchromic effect) of the DNA solution and loss of the DNA biological activity.
2. Tm (melting temperature)
DNA denaturation is a cooperative process. The collapse of one part of the structure
(at the ends of the dsDNA molecule or AT-rich internal regions) destabilizes the
remainder. The temperature corresponding to the mid-point at the DNA thermal
melting curve is known as Tm. It is a function of the G+C content of the DNA
sample.
P60 fig 3-18
3. Denatured DNA can be renatured. Slow cooling the denatured DNA allows time for
the wholly complementary DNA strands to find each other and the sample can
become fully double-stranded by base pairing (annealing).This process is called DNA
renaturation. The renaturation of regions of complementary between different nucleic
acid strands is known as hybridization.
P61 fig 3-19
Southern blotting is used to examine DNA samples.
The Southern blotting procedure is as follows:
a. DNA molecules are separated by agarose gel electrophoresis.
b. They are denatured with alkali.
c. Transferred to a nylon or NC membrane.
d. Hybridized with labeled probe
e. Detection
Northern blotting is used to examine RNA sample.
Section 5
Nucleases
Ⅰ. grouping nucleases
1. according to the substrate specificity: DNases and RNases
2. according to the hydrolytic style: exonucleases and endonucleases
3. according to the substrate secondary structure specificity ssDNA specific nucleases
and dsDNA specific nucleases.
Ⅱ. RNases are RNA endonucleases
Ⅲ. DNases hydrolyze DNA
Ⅳ. N-glycosidases remove the base from the nucleotide
Chapter 3
DNA Biosynthesis and Recombination
Section 1
The General Features of genome Replication
1. Genome is the total genetic information possessed by an organism.
2. The Common Features of DNA replication:
(1) DNA replication is semiconservative.
In 1958 Meselson and Stahl demonstrated experimentally that DNA replication is
semiconservative.
P319 fig 16-1
(2) The point at which separation of the DNA strands and synthesis of new DNA takes
place is known as the replication fork.
P319 fig 16-2
(3) DNA replication is bidirectional.
a. replicon : Any piece of DNA which replicates as a single unit, is called a replicon.
b. origin : The initiation within a replicon always occurs at a fixed point known as
the origin.
c. bidirectional replication : Usually two replication forks proceed bidirectionally
away from the origin and the strands are copied as they separate until the terminus
is reached.
(4) DNA replication is semi-discontinuous.
Because DNA can only be synthesized in a 5’3’ direction, at each replication
fork, one strand ( the leading strand ) is synthesized as one continuous piece, while
the other strand ( the lagging strand ) is made discontinuously as short fragments in
the reverse direction.
These short DNA fragments are called Okazaki fragments. They are joined by DNA
ligase and form the lagging strand.
(5) Origin contains short AT-rich repeat sequences. E coli’s origin is called oriC.
It is 245bp long and contains three 13bp-direct repeat and four 9bp-inverted repeat
sequences.
(6) DNA replication needs priming.
a. Most DNA replications are primed by RNA
A RNA primer is synthesized and DNA polymerase elongates with DNA at the
3’-end of the RNA primer.
b. There are also DNA priming or nucleotide priming.
(7) Multi-enzymes and proteins participate in DNA replication.
(8) DNA replication is of high fidelity.
3. There are several DNA replication mechanisms.
(1) Reverse transcription
(2) Double stranded circular DNA replication
θ replication, rolling circle replication, D-loop replication
(3) Single stranded DNA virus genome RF intermediate
(4) Integrated retrovirus such as HIV RNA intermediate
4. There are several RNA replication mechanisms.
(1) ds RNA virus genome
two strands replication
(2) (+)ss RNA virus genome
A negative sense RNA as the intermediate
(3) (-)ss RNA virus genome
A positive sense RNA as the intermediate
(4) retrovirus RNA genome
DNA intermediate
Section 2
DNA Replication
I. Features of DNA Polymerases:
1. The substrates of DNA polymerases are dNTPs and the primer-template junction.
3’_____________________________________________________5’
---------⊙ here + dNTP
( dNMP )n + dNTP ─────── ( dNMP )n+1 + PPi
The template directs the DNA polymerase to synthesize a daughter strand
following the base-pairing rules.
2. The active center of DNA polymerases catalyzes DNA synthesis.
3. The semi-closed right-handed structure of the DNA polymerase is composed of
three domains.
4. The protein of “ sliding clamp “ encircles the DNA template and accounts for the
processivity of the DNA polymerase.
5. The 3’  5’exonuclease active center of the DNA polymerase is responsible for the
proofreading.
P322 tab16-1
II. DNA replication in E coli
1. E coli DNA replication is initiated at oriC in a process mediated by a multiprotein complex including DnaA, DnaB, DnaC etc.
It involves wrapping of the DNA around Dna- ATP complex and separation of the
strands at AT-rich sequences with the help of DnaC. The helicase DnaB then binds
and extends the single stranded region for copying.
P321 fig 16-3
2. Primase synthesizes RNA primer.
DnaG, the primase, a specific RNA polymerase, attaches to the DNA and synthesized
a short RNA primer.
3. DNA polymerase Ⅲ synthesizes DNA and DNA polymerase Ⅰ removes the primer.
4. The holoenzyme of DNA polymerase Ⅲ:
P323 fig 16-4
(1) dsDNA unwinding:
As the parental DNA is unwound by DNA helicases and single-stranded binding
protein( SSB ), the resulting positive supercoiling has to be relieved by the
topoisomerase DNA gyrase .
(2) elongation:
A mobile primosome ( DnaB-DnaG complex ) synthesizes multiple primers on
the lagging strand’s template. DNA polymerase Ⅲ holoenzyme elongates both
leading and lagging strands ( Okazaki fragments ).
P324 fig 16-5
(3) formation of lagging strand:
The 5’  3’exonuclease activity of DNA polymerase Ⅰ removes the RNA primer
of the Okazaki fragment and the polymerase function simultaneously fills the gap
with DNA. DNA ligase joins Okazaki fragments to form lagging strand.
(4) Tus recognizes and binds to the TER sites. That terminates replication.
(5) Only at the full methylated oriC can initiate replication.
(6) Two types of topoisomerases are required in DNA replication.
III. DNA Replication in Eukaryotes
1. There are five types of common eukaryotic DNA polymerases: pol.α, pol. β, pol. γ ,
pol. δ, and pol. ε.
2. Eukaryotic and prokaryotic enzymes and protein factors that participate in
DNA replication at the replication fork are comparable.
3. After the pol. α/primase complex initiates replication pol. δ starts elongation.
4. There are two mechanisms of removing primers:
(1) RNase HI and FEN1-dependent mechanism
(2) helicase Dna2 and FEN1-dependent mechanism
P330 fig 16-10
5. The eukaryotic chromosome replicates only once in a cell cycle.
(1) pre-RC forms in the G1-phase and is activated in the S-phase.
(2) Cdk controls the formation and activation of pre-RC.
P330 fig 16-11, 12
6. Telomerase participates in the replication of telomere DNA.
(1)The problem of replicating the ends of linear chromosomes :
The ends of linear chromosomes cannot be fully replicated by semi-discontinuous
replication as there is no DNA to elongate to replace
the RNA removed from the 5’end of the lagging strand. Thus genetic information
could be lost from the DNA.
P331 fig 16-13
(2) Telomere and telomerase solve the problem :
To overcome this, the end of eukaryotic chromosomes ( telomeres ) consist of
hundreds of copies of a simple, non-informational repeat sequence ( TTAGGG in
humans ) with the 3’end overhanging the 5’end. The enzyme telomerase contains a
short RNA molecule, part of whose sequence is complimentary to this repeat. This
RNA acts as a template for the addition of these repeats to the 3’overhang by repeated
cycles of elongation. The complimentary strand is then synthesized by normal lagging
strand synthesis leaving a 3’overhang.
P332 fig 16-14
IV. DNA Replication in Mitochondria and Phages
1. mtDNA is replicated in D-loops.
2. Phage’s circular DNA is replicated in rolling circle.
Section 3
Reverse Transcription
Ⅰ. The discovery of reverse transcriptase develops the central dogma put forth by
Crick.
Ⅱ. Reverse transcriptase has three enzymatic activities :
1. RDDP activity : It transcribes the RNA template onto a complimentary DNA
strand to form an RNA-DNA hybrid.
2. RNase H activity : The enzyme then degrades the RNA template.
3. DDDP activity : It replicates the resulting single-stranded DNA to form the
duplex DNA.
P335 fig 16-17
Ⅲ. Retroviruses have diploid, positive sense RNA ( mRNA ) genomes, and replicate
via a dsDNA intermediate, the provirus. The provirus is inserted into the host cell’s
genome. The provirus genome is expressible. The gene products are mRNA, the
retrovirus genome, and various proteins, including reverse transcriptase. The
retrovirus particle is packaged in the host cell and the mature virion buds from the
host cell surface.
P335 fig16-15,16
Section 4
The Repair of DNA Damage
Ⅰ. Physical or chemical agents may cause DNA damage. There are also replication
errors. DNA damages must be repaired. There are several DNA repair systems in both
prokaryotic and eukaryotic cells.
P337 tab16-3
Ⅱ. Mismatch repair system repairs the replication errors.
Replication errors which escape proofreading have a mismatch in the daughter
strand. Hemi-methylation of the DNA after replication allows the daughter strand to
be distinguished from the parental strand. The mismatched base is recognized and
bound by MutS, MutL and MutH participate in cleavage the daughter strand. A piece
of the error containing daughter strand is removed by exonucleases. The gap is filled
by DNA polymerase Ⅲ and the nick is ligated by DNA ligase.
Ⅲ. There are different kinds of DNA lesions caused by physical or chemical agents.
Ⅳ. There are at least six DNA damage repair systems in E coli.
1. Photoreactivation
2. BER
P337 fig16-18
P338 fig16-19
3. NER
P339 fig16-20
4. Recombinational repair
5. Translesion DNA synthesis,TLS
P340 fig16-21
6. Mismatch repair
Section 5
DNA Recombination
I. Homologous recombination is the most general recombination of DNA.
II. Site specific recombination is the integration of genetic information between the
specific sites.
1. The integration of λ phage DNA.
2. The site specific recombination of bacteria.
3. The rearrangement of immunoglobulin.
III. Transpositional recombination is DNA shift mediated by insertion sequences or
transposon.
Chapter 4
RNA Biosynthesis and Posttranscriptional Processing
Section 1
RNA Synthesis Overview
Ⅰ. There are two forms of RNA biosynthesis: DNA dependent RNA synthesis and
RNA dependent RNA synthesis.
II. DNA dependent RNA synthesis is asymmetric transcription. That is in a gene, one
strand is the template strand and the other is the coding strand. Only the template
strand is transcribed. In a piece of DNA which harbours several genes, the template
strand of these genes may not always on the same DNA chain.
P349 fig 17-1,17-2
Section 2
DNA dependent RNA synthesis
I.RNA polymerases catalyze RNA synthesis.
1. DDRPs initiates RNA synthesis directly.
RNA biosynthesis is also called transcription. It is the synthesis of a single-stranded
RNA from a double-stranded DNA. RNA synthesis occurs in the 5’→ 3’direction
and its sequence is complimentary to that of the DNA template strand and
corresponds to that of the DNA coding strand.
RNA polymerase moves along the DNA and sequentially synthesizes the RNA chain.
DNA is unwound ahead of the moving polymerase, and the helix is reformed behind
it.
P355 fig 17-10
The reaction is as follows :
( NMP)n＋NTP ────── ( NMP )n+1 + PPi
P350 fig 17-3
2.There is only one kind of prokaryotic RNA pol. while there are three kinds of
eukaryotic RNA pols: RNA pol I, RNA pol II and RNA pol III. RNA pol I is located
in the nucleoli. It is responsible for the synthesis of the precursors of 18SrRNA,
28SrRNA and 5.8SrRNA. RNA pol II is located in the nucleoplasm and is
responsible for the synthesis of mRNA precursors and some snRNAs. RNA pol III is
located in the nucleoplasm. It is responsible for the synthesis of 5SrRNA, tRNAs,
snRNAs and scRNAs.
3.The holoenzyme of E coli RNA pol. is composed of core enzyme ( α2ββ’ ω )and σ
subunit.
Each eukaryotic RNA pol. has 12 or more different subunits. The largest two
subunits are similar to each other and to the β and β’ subunits of E coli RNA pol.
Other subunits in each enzyme have homology to the α subunit of the E coli
enzyme .Five additional subunits are common to all three pols , and others are
polymerase specific.
The largest subunit of RNA pol II has a seven amino acid repeat at the C
terminus called carboxyl-terminal domain (CTD).This sequence YSPTSPS is
repeated 52 times in the mouse RNA pol II . CTD is subject to
phosphorylation.
Ⅱ.RNA pol recognizes and binds to the promoter thus inhibits transcription.
1. subunit is responsible for recognizing the promoter.
Promoters contain conserved sequences which are required for specific binding of
RNA pol and transcription initiation.
The σ70 promoter of E coli consists of a sequence of between 40 and 60 bp. With
this sequence, there are short regions of extensive conservation which are critical for
promoter function. The -10 sequence is a 6 bp region present in almost all
promoters .The consensus -10 sequence is TATAAT. It is generally 10 bp upstream
from the start site. The -35 sequence is also a 6bp region recognizable in most
promoters . The consensus -35 sequence is TTGACA. The base at the start site of
transcription is almost always a purine and is assigned as position +1 .There is another
consensus sequences, the AT rich UP element, locates between -40 to -60 bp in some
genes of high expression.
P 352 fig 17-4
Many prokaryotes, including E coli , have multiple sigma subunits .The most
common sigma subunit in E coli is σ70 . Alternative classes of consensus promoter
sequences (e.g. heat shock promoters ) are recognized only by an RNA pol containing
an alternative σ subunit .
The σ subunit enhances the specificity of the core enzyme α2ββ’ω, for promoter
binding . The polymerase finds the promoter -35 and -10 sequences by sliding along
the DNA and forming a closed complex with the promoter DNA. Around 17bp of the
DNA is unwound by the polymerase, forming an open complex.
The first several bases are added without the enzyme movement along the DNA.
When initiation succeeds the enzyme releases the σ subunit and forms a ternary
complex of polymerase (the core enzyme), DNA and nascent RNA and elongation
begins.
P355 fig 17-11
2. Eukaryotic RNA pol Ⅱ alone can not initiate transcription. It has a lot of helpers
called transcription factors such as TFⅡA, TFⅡB…etc.
P352 table 17-1
P353 fig 17-6
3.
Transcription initiation of eukaryotic RNA pol Ⅰ or pol Ⅲ is similar to that of
RNA pol Ⅱ.
Ⅲ. There are two types of prokaryotic transcriptional termination.
1. ρ(rho)–independent termination
Self-complementary sequence at the 3’-end of genes causes formation of hairpin
structure in the RNA which act as the terminator. The stem of the hairpin often has
a high content of G.C base pairs giving high stability, causing the polymerase to
pause. The hairpin is often followed by several Us which result in weak RNA
template DNA strand binding. This favor dissociation of the RNA strand, causing
transcription termination.
P355 fig 17-12
2. ρ(rho)–dependent terminator sequence which requires an additional protein factor
rho, for efficient transcription termination. rho binds to specific sites in
single-stranded RNA. It hydrolytes ATP and moves along the RNA towards the
transcription complex, where it enables the polymerase to terminate transcription.
Section 3
RNA Processing
Ⅰ.Eukaryotic mRNA processing includes 5’capping, 3’ polyadenylation and splicing.
1.5’-capping of pre mRNA (hnRNA)
This is the addition of a 7-methylguanosine nucleotide (m7G) to the 5’-end of an
RNA pol Ⅱ transcription when it is about 25-30 nt long .The m7G or cap, is added in
reverse polarity (5’to 5’) , thus acting a barrier to 5’-exonuclease attack, but it also
promotes splicing , transport and translation.
P356 fig 17-13,17-14
2. 3’ cleavage and polyadenylation
Most eukaryotic pre-mRNAs are cleaved at a polyadeylation site and poly(A)
polymerase (PAP) then adds a poly A tail around 250nt to generate the mature 3’-end.
3. Splicing
In eukaryotic pre-mRNA processing, intervening sequences (introns) that interrupt
the coding regions (exons) are removed (spiced out) and the two flanking exons are
joined .This spicing reaction occurs in the nucleus and requires the intron to have a
5’-GU and an AG-3’ and a branch point sequence. In a two-step reaction, the intron is
removed as a tailed circular molecule, or lariat, and is degraded.
Splicing involves the building of snRNPs to the conserved sequences to form a
spliceosome in which cleavage and ligation reactions take place.
P357 fig17-15,
P358 fig 17-16
P360 fig 17-17, fig 17-18
4. Alterative mRNA processing is the conversion of pre-mRNA species into more
then one type of mature mRNA . This can result from the use of different poly(A)
sites (alternative polyadenylation )or different patterns of splicing (alternative
splicing).
P.360 fig 17-19, fig 17-20
Ⅱ.Nucleases participation in tRNA and rRNA processing
1. An initial 30S transcript is made in E coli by RNA pol transcribing one of the
seven rRNA operons. Each contains one copy of the 5S,16S and 23S rRNA coding
regions , together with some tRNA sequences. This 6500 nt transcription folds and
complexes with some proteins, becomes methylated and is then cleaved by
specific nucleases to release the mature rRNAs.
2. In eukaryotes RNA pol transcribes the rRNA genes , which exist in tandem repeats,
to yield a 45S pre-RNA which contains one copy each of the 18S ,5.8S and 28S
sequences . Various spacer sequences are removed from the long pre-rRNA
molecule by a series of specific cleavages. Many specific ribose methylation take
place directed by snRNAs, and the maturing rRNA molecules fold and complex
with ribosome proteins.
P361 fig 17-21, fig 17-22
3. Prokaryotic and eukaryotic pre-tRNA processing are similar. It includes:
a. 5’end and 3’end cleavages by specific RNases.
b. base modifications, such as methylation, reduction, deamination etc.
c. splicing: many eukaryotic pre-tRNA are synthesized with an intron. It is removed
by enzymatic cutting and joining mechanism.
d. 3’ terminal CCA is added by the enzyme tRNA nucleotidyl transferase if there is
no CCA sequence at the 3’terminus.
P362 fig 17-23, P363 fig 17-24
Ⅲ.Catalytic RNAs (ribozymes )can perform self-splicing reactions
Several biomedical reactions can be carried out by RNA enzymes or ribozymes. They
can cleave themselves or other RNA molecules, or perform self-splicing reactions.
There is an intron in the large subunit rRNA of Tetrahymena that can remove itself
from the transcription in vitro in the absence of protein. The process is called
self-splicing and requires guanosine , or a phosphrylated derivative , as cofactor.
P364 fig 17-25
Ⅳ. Some m RNA undergo editing
An unusual form of mRNA processing in which the sequence of the mRNA is altered
is called RNA editing. In man, editing cause a single base change from C to U in the
apolipoprotein B mRNA, creating a stop codon in the mRNA in intestinal cells at
position 6666 in the 14500nt molecule. The unedited RNA in the liver makes
apolipoprotein B100, a 512 KD protein , but in the intestine editing causes the
truncated apolipoprotein B45(241 KD) to be made.
P364 fig 17-26, fig 17-27
Section 4
RNA Dependent RNA Synthesis
Most viruses have RNA as the genome . They are called RNA viruses .RNA viruses
require virus-encoded RNA dependent RNA polymerases (RDRPs) for their
replication. Retroviruses also belong to RNA viruses . They use an RDDP (reverse
transcriptase) to replicate via a DNA intermediate.
Chapter 5
Protein Biosynthesis and Processing
Section 1
Components Required for Protein Biosynthesis
Ⅰ.mRNA is the template of protein biosynthesis.
1. The genetic code contains 64 codons. 61 codons are sense codons, whereas 3
codons UAG, UGA and UAA are stop codons
2. Five features of the genetic code:
a. Universality or almost universality
All organisms, from bacteria to human being use the universal genetic code.
However, since 1980, it has been discovered that mitochondria, which have their own
small genome, utilize a genetic code that differs slightly from the standard universal
code.
b. Direction: Codons in the coding sequence of mRNA are arranged from 5’ to
3’direction.
c. Commaless: The triplet codons arranged in the coding sequence of mRNA are
commaless, one after the other . Deletion or insertion one or two nucleotides of
the triplet will cause frame shift mutation.
d. Degeneracy: There are 61sense codons, while there are only 20amino acids. 18
out of 20 amino acids have more than one codon to specify them.
e. Wobble: The codon in mRNA base pairing with the anticodon in tRNA may not
observe the standard base pairing rules (A pairs with T , U pairs with A and C
pairs with G).G-U, I-A ,I-C and I-U are wobble base pairs.
P369 tab 18-1
Ⅱ.tRNAs transfer amino acids.
When charged by attachment of a specific amino acid to their 3’-end , to become
aminoacyl-tRNAs, tRNA molecules act as adapter molecules in protein synthesis.
Ⅲ. Ribosomes are the locations where protein synthesis takes place.
Ribosomes are complexes of rRNA molecules and specific ribosomal proteins ,and
these large RNPs are the machines the cell uses to carry out translation.
The E coli 70S ribosome is formed from a large 50S and a small 30S subunit. The
large subunit contains one each of the 23S and 5SrRNA and more than 30 different
proteins. The small subunit contains a 16SrRNA molecule and more than 20 different
proteins. The mammalian 80S ribosome is composed of one large 60S subunit and
one small 40S subunit. The small subunit contains one 18SrRNA molecule and the
large subunit contains one 5SrRNA , one 5.8S rRNA, one 28S rRNA and more than
forty proteins.
P371 fig 18-1
IV. Protein biosynthesis system also includes substrates and cofactors
1. amino acids
2. aminoacyl-tRNA synthetases
3. mRNA
4. initiation factors
5. elongation factors
6. release factors
7. ATP
8. GTP
9. Mg2+
10. ribosome
P372 tab 18-2
Section 2
Proteins synthesis Takes Place in Five stages
The five stages are formation of aminoacyl tRNAs, initiations, elongation, termination
and peptide folding and post-translational processing .
Ⅰ.Aminoacyl tRNAs are synthesized from amino acids and tRNAs by aminoacyl tRNA
synthetases. The reaction takes place in two steps:
P374 fig18-2
Proofreading occurs when a synthetase carries out step 1. of the aminoacylation
reaction with the wrong , but chemically similar amino acid. It will not carry out step
2, but will hydrolyze the aminoacyl adenylate (E-AA-AMP) instead.
Ⅱ. Initiation
A special tRNA(initiator tRNA), recognizing the AUG start codons, is used to initiate
protein synthesis in both prokaryotes and eukaryotes. In prokaryotes, the initiator
tRNA is first charged with Met by methionyl- tRNA synthetase. The methionine
residue is then converted to N-formyl-methionine by transformylase. In eukaryotes,
the methionine on the initiator tRNA is not modified . There are structural differences
between E coli initiator tRNA and the tRNA that inserts internal Met residues.
In prokaryotes, initiation requires the large and small ribosome subunits, the m RNA,
the initiator tRNA, three initiation factors(IFs) and GTP. IF1 and IF3 bind to the 30S
subunit and prevent the large subunit binding. IF2 + GTP can then bind and will help
the charged initiator tRNA to bind later. This small subunit complex can now attach to
a mRNA molecule via its ribosome-binding site. The initiator tRNA can then
base-pair with the AUG initiation codon which releases IF3 thus creating the 30S
initiation complex. The large subunit then binds, displacing IF1 and IF2+GDP , giving
the 70S initiation complex which is fully assembled ribosome at the correct position
on the mRNA (the initiator tRNA with the initiation start codon AUG occupies the P
site )
P375 fig18-3
Ⅲ.Elongation
Elongation involves the three elongation factors, (EF-Tu, EF-Ts and EF-G),GTP,
charged tRNA and the 70S initiation complex (or its equivalent ).It takes place in
three steps.
1.entrance (registration)
A charged tRNA is delivered to the A-site as a complex with EF.Tu and GTP. The
GTP is hydrolyzed and EF-Tu-GDP is released which can be re-used with the help of
EF-Ts and GTP(via the EF-Tu-EF-Ts exchange cycle)
P376 fig18-4
2. peptide bond formation
23SrRNA has the peptidyl transferase activity . It makes a peptide bond by joining the
two adjacent amino acids without the input of more energy.
P377 fig 18-5
3.translocation
Translocase (EF-G) with energy from GTP, moves the ribosome one codon along the
mRNA, ejecting the uncharged tRNA and transferring the growing peptide chain from
the A-site to the P-site. Now ,the A-site is vacant and ready for another round of the
three –step ribosomal cycle.
P377 fig18-6
Ⅳ.Termination
When any one of the stop codons enters into the A-site, RF1 recognizes stop codons
UAA and GAG, RF2 recognizes UAA and UGA. They are helped by RF3 which
hydrolyzes GTP and make peptidyl transferase join the peptide chain to a water
molecule, thus releasing it.
P378 fig 18-7
Ⅴ. Folding and Posttranslational Processing See next section.
Section 3
Folding and Posttranslational Processing
Ⅰ. The nascent peptide undergoes posttranslational modification.
1. trim or modification of N or C terminal
2. restricted hydrolysis; polyprotein processing and protein splicing
3. various modification types of amino acid residues
Ⅱ.Peptide folding results in formation of higher orders of polypeptides’ structure.
1. Peptide folding is a complex process
2. Molecular chaperones participate in peptide folding
Molecular chaperones are proteins that assist in protein folding. They have following
functions:
a. binding to short hydrophobic segments in nascent polypeptides, thereby
preventing aggregation.
b. establishing an isolated environment for protein folding without interference.
c. promoting protein folding and disaggregation.
d. unfolding folded proteins in case of stress.
There are two classes of molecular chaperones: ribosome-binding molecular
chaperones and non-ribosome-binding ones.
Ⅲ.Assembly of protomers (subunits) forms oligomers or polymers (multi-subunit
proteins).
Section 4
Clinical Relatives in Protein Synthesis
Ⅰ. Many viruses co-opt the host cell protein synthesis machinery.
Ⅱ. Many antibiotics work because they selectively inhibit protein synthesis in bacteria.
Ⅲ. Certain toxins or cytokines inhibit the host cell or virus protein synthesis
1. Diphtheria toxin is an enzyme. It transfers the ADP-ribosyl group from NAD+ to a
certain histidine residue in eEF-2. That results in inactivation of eEF-2 and
inhibition of protein synthesis.
P384 fig 18-14
2.The interferons are a group of glycoproteins produces by a variety of cells in
response to several stimuli such as viral infections, antigens, mitogens etc.
Type Ⅰ interferons protect cells from viral infection by two mechanisms.
a. stimulating the phosphorylation and inactivation of eIF2 which is required to
initiate protein synthesis.
b. inducing synthesis of 2’-5’ oligo A which activates RNase L and causes
degradation of viral RNA.
P385 fig 18-15
Chapter 6
Integration of Expression and Transmission of
Genetic Information
Section 1 Genomics
I. Genome is the total genetic information possessed by an organism.
The eukaryotic genome includes the complete set of the haploid chromosomal DNA
and mtDNA and ctDNA.
The prokaryotic genome includes the nucleoid DNA and plasmid DNA.
II. Genomics consists of structural genomics, functional genomics and comparative
genomics.
III. The main task of the structural genomics is mapping the genes and DNA sequence
on a large scale.
1. Mapping the genes is the important strategy of HGP. It consists of genetic
mapping and physical mapping.
2. DNA sequencing on a large scale is based on construction of BAC cloning system
and shotgun sequencing.
3. Bioinformatics is the important subject for predicting gene structure and function.
IV. Functional genomics investigates the laws of gene activities systematically.
1. Identifying genes in the DNA sequence by whole genome scanning.
2. Searching homologous genes by BLAST.
3. Verifying gene function by experiments.
4. Describing the profile of gene expression by transcriptomics and proteomics.
Section 2
Transcriptomics
I. Transcriptomics studies the expression and functions of the total RNA.
II. The core of transcriptomics is to analyze the profile of gene expression.
III. The main techniques of transcriptomics are DNA microarray,SAGE and MPSS.
IV. RNomics studies the total SnmRNAs.
Section3 Proteomics
I. The central task of proteomics is to study the total protein composition and the law
of protein activities in the cell.
II. 2-DE and MS are the important techniques of proteomic studies.
III. Studies on protein- protein interaction are the important contents of recognizes
protein function.
Section 4
Omics application in medicine
I. Genomics of diseases investigates the mechanism of diseases.
II. Pharmacogenomics studies the influence of genetic mutation on pharmacentical
effect and doxicity.
III. Cancer genomics elucidates tumor genes.
IV. Metabonomics determines the composition of small molecules in an organism /
cell and tueir metabolism and interaction.
Chapter 7
Regulation of Prokaryotic Gene Expression
Section 1
Principles of Gene Regulation
Gene expression is the process by which gene products are made.
Ⅰ.The common rules of Gene expression in both prokaryotes and eukaryotes.
1. The temporal and spatial specificity of gene expression
2. The cis-acting elements and trans-acting factors in gene regulation
3. The positive and negative regulation of gene expression
4. DNA-protein interaction is the molecular basis of gene regulation.
Ⅱ. Regulation of transcriptional initiation is the key step of gene regulation.
1. Structural gene is a gene that codes for the synthesis of a protein or a RNA
molecule with a non-regulatory function (e.g. rRNA, tRNA)
2. Regulation of gene is the result of a complex control hierarchy which include
chromatin remodeling, translational control, RNA processing translational control,
post translational processing, protein or enzyme activation, protein or enzyme
inactivation or degradation.
Ⅲ. Induction and repression are the basic rules of regulation of prokaryotic
transcriptional initiation.
1. An operon is a unit of prokaryotic gene expression which includes co-ordinately
regulated structural genes and control elements which are recognized by
regulatory gene products.
P402 fig 20-1
2. Induction: an inducer (usually a substrate for a catabolic pathway) turns on gene
expression.
3. Repression: a repressor (usually a product of a biosynthetic pathway) turns off
gene expression.
4. Features of prokaryotic gene expression.
a. Regulation of transcription is the key step of gene regulation.
b. Operon regulation.
c. Negative controls are predominant.
d. Genes in an operon are all transcribed together on the mRNA, called a polycistronic
mRNA.
e. Many genes are constitutive genes (or house keeping genes ). They code for gene
products required for almost all cells and are routinely transcribed and expressed.
Section 2
The Operon of Bactria
Ⅰ. The lactose operon
The lacZ, lacY and lacA gene are transcribed from a lacZYA transcription unit under
the control of a single promote. They encode enzymes required for the use of lactose
as a unique carbon source. The lac I product, the lac repressor is expressed from a
separate gene upstream from lac operon.
P406 fig 20-5
Ⅱ.The lac operon is turned off by the lac repressor
The lac repressor is made up of four identical protein subunits, it binds to operator
DNA sequence that overlaps lac promoter, and blocks transcription from lac promoter.
The turned off lac operon can be turned on by lac inducer.
When lac repressor binds to the inducer (such as allolactose or IPTG), it changes
conformation and can not bind to the operator. The lac operon is turned on.
cAMP-CAP acts as the positive regulator of lac operon.
The lac promoter is a weak promoter.
RNA polymerase binding to the promoter and transcription initiation need activation
by cAMP-CAP complex.
CAP is a transcriptional activator which is activated by binding to cAMP.
cAMP levels rise when glucose is lacking.
cAMP-CAP complex binds to a site just upstream from lac PO region and activates
RNA polymerase binding to the promoter and transcription initiation.
P407 fig 20-7
Ⅲ. Other types of operon regulation.
Section 3
Regulation of Translational Gene Expression
I. Two mechanisms of trp operon regulation.
1.Repression
The trp repressor binds L-tryptophan, the end product of trp synthesis
pathway, to
form a complex that specifically binds to trp operator , so as to negatively regulate
operon transcription.
The trp repressor-operator system dose not fully account for the trp synthesis in E
coli.
2.Attenuation
There is a 161-nt-leader sequence (trp L) between the operator and the structure gene
trp E.
P411 fig 20-11
The trp leader RNA transcribed from trp L contains four regions of complementary
sequence which are capable of forming alternative hairpin structures.
One of these is a kind of p-independent terminator, called attenuator.
The leader RNA contains an efficient ribosome-binding site and encodes a 14-amino
acid peptide (lead peptide) which contains two trp residues.
When trp is low the ribosome will pause at the codon , while transcription of trp
operon by RNA polymerase goes on because of no attenuator formation.
When trp is high the leader peptide is synthesized, while transcription of trp operon
terminates because of attenuator formation.
Attenuation depends on the fact that transcription and translation are tightly coupled
in prokaryotes.
Translation can occur as a mRNA is being transcribed.
It depends on the conditions of leader peptide synthesis whether or not the full-length
mRNA is transcribed.
Ⅱ. SOS response
Agents that damage DNA induce a complex system of cellular changes in E coli
known as SOS response.
SOS response results from the expression of a network of operons.
These operons are coordinately controlled and form a regulon.
Lex A protein represses all the operons for SOS response.
RecA gene is induced by heavy DNA damages and RecA protein is synthesized.
RecA protein is a kind of protease.
It hydrolyzes the Lex A protein, thus SOS regulon is turned on.
That results in SOS response. SOS repair is error-prone.
Ⅲ. The synthesis rate of ribosomal proteins is coordinated with that of r RNAs
Ⅳ. Regulation of mRNA stability provides another control mechanism of gene
expression.
Section 4
Regulation of Gene Expression in Phage
The life cycle of phage lambda.
Lysogenic and lytic pathways.
P417 fig20-17
Bacteriophage lambda infects bacteria.
In lytic infection, phage particles are released from the cell by lysis. However in
lysogenic infection phage particles integrate their genome into that of the host cell.
Such a phage is called the prophage and such a process, the lysogeny, and such a host
cell, lysogen.
The prophage may be stably inherited through several generation before returning to
lytic infection by inductions.
Chapter 8
Regulation of Gene Expression is Eukaryotic
Section 1
Control of Transcription of Initiation
I. Eukaryotic cis-acting elements are composed of promoter and regulatory DNA
sequences.
1.TATA box /initiator or CpG island is the eukaryotic core promoter.
Some RNA Pol Ⅱ promoters contain a sequence celled a TATA Box which is situated
25-35 bp upstream from the start site. Other genes contain an initiator element which
overlaps the start site .These elements are required for basal transcription complex
formation and transcription initiation. Many RNA pol Ⅱpromoters consist of both
TATA box and initiator.
Some RNA pol Ⅱpromoters contains several transcription start sites distributed in a 20
to 200 bp region.
These promoters have no TATA box and initiator .They have clusters of CG sequences
called CpG islands upstream from the start sites.
2.Elements reside 100-200 bp upstream from the promoter are generally required for
efficient transcription. Examples include CAAT and GC boxes.
3. Enhancers and silencers are sequence elements which can regulate transcription
from thousands of base pairs upstream or downstream. They may contain a variety
of sequence motif which are recognized and bound by regulatory proteins. Enhancers
activate transcription while silencers inhibit transcription.
P 421 fig 21-1
Ⅱ.Transcription factors are composed of different functional domains. DNA binding
domains, and transcription activation domains are found both in prokaryotes and
eukaryotes.
DNA binding domains.
a. HTH: a recognition alpha-helix interacts with the DNA and is separated from
another alpha-helix by a characteristic right angle beta-turn.
b. Zinc finger domains include the C2H2 Zinc fingers which bind Zn2+ through two
Cys and two His residues and also the C4 fingers which bind Zn2+ through four Cys
residues.
P423 fig21-3
c. Basic domains are associated with leucine zipper at helix-loop-helix dimerization
domains. Dimerization is generally necessary for basic domain binding to DNA.
1. Transcription activation domains
a. Acidic activation domains contain a high proportion of acidic amino aids and are
present in many transcription activators.
b. Glutamine-ride domains and proline-rich domains contain a high proportion of
glutamine residues and a continuous run of proline residues respectively.
They can activate transcription.
Ⅲ.The “turn-on” mechanism of eukaryotic genes is more complex than that of
prokaryotic genes.
1. Chromatin remodeling includes two important steps, nucleosome remodeling and
modification of histones.
2. Assembly of transcription inhibition complex on the core promoter needs not only
the RNA pol, but also various general transcription factors.
P428
fig21-8
Section 2
Posttranscriptional Control
I. 5’ end capping and 3’end polyadenylation of eukaryotic mRNAs.
Ⅱ.Splicing and alterative splicing.
Ⅲ. mRNA editing.
Ⅳ. mRNA transporting from the nucleus to the cytoplasm.
Ⅴ. mRNA compartmentation in the cytoplasm.
Ⅵ. mRNA stability.
Ⅶ. mRNA polyadenylation in the cytoplasm.
Ⅷ. nonsense-mediated mRNA decay.
Ⅸ. RNA interference can make posttranscriptional gene silence.
Section 3
Translational Control
I. Translational initiation is controlled by phosphorylation /dephosphorylation of
eIF2.
Ⅱ.5’ UTR and 3’ UTR binding proteins may negatively regulate translation.
Ⅲ. Some eukaryotic mRNA contains more than one start codons
Chapter 9
Coupling of Gene Expression and Cell Signaling
Section 1
General mechanism of cell signal transduction
I. There is a complex signal transduction network in the cell.
II. The cell receives the first messenger via the receptor.
III. The concentration and distribution of the second messenger are important factors for the signal
transduction
IV.
Chapter eight
Recombinant DNA Technology
Section one
Overview of Recombinant DNA Technology
Ⅰ. Recombinant DNA technology is also called DNA cloning or molecular cloning.
1. Cloning is a lab procedure that produce clones.
2. Clones are individuals formed by an asexual process so that they are genetically
identical to the original individual, for example, bacterial colonies, multiple
identical copies of a gene, etc.
Ⅱ.DNA cloning facilitates the isolation and manipulation of fragments of an
organism’s genome by replicating them independently as part of an autonomous
vector.
The procedure:
1. Select the cloning vector such as the plasmid and isolate the DNA fragment of
interest.
2.
3.
4.
5.
6.
make the recombinant DNA molecule
transform the host cell such as E coli
recombinant DNA molecule replicates in the bacteria
select the transformed bacteria
isolate the recombinant DNA molecules and analyze them
P423 fig 20-1
Section Two
Molecular Tools in Recombinant DNA Technology
Ⅰ.The useful tool enzymes
1. Restriction endonuclease:
Restriction endonucleases are bacterial enzymes which can cleave DNA
symmetrically in both strands at specific recognition sequences to leave a
5’-phosphate and a 3’-OH.They leave blunt ends, or protruding 5’ or 3’ terminal
(cohesive or sticky ends)
REs form part of the restriction-modification defense mechanism against foreign
DNA.
They are the basic tool enzymes of molecular cloning.
P424 table 20-1
P425 fig 20-3
2. DNA ligase joins breaks in a dsDNA backbone with a 5’-phosphate and a 3’-OH in
a ATP+ or NAD+ dependent reaction. Requires that the ends of the DNA be
compatible, i.e. blut with blut, or complementary sticky ends.
3. CIP and Klenow flagment of DNA pol. I are also required in molecular cloning.
Ⅱ.Cloning vectors must be capable of being replicated and isolated independently of
the host’s genome and have enough capacity to clone foreign DNA fragments.
1. Three essential elements of plasmid DNA
a. There is at least one origin of replication.
b. It must contain a selectable marker such as resistance to antibiotics.
c. It must contain cloning sites.
P427 fig 20-8
2. Phage DNA is used as the vector to construct libraries.
3. Cosmid, BAC, and YAC can be used to clone large or very large genomic
fragments from humans and other species.
4. In cultured animal cells, vectors have often been based on viruses which naturally
infect the required species ,either by maintaining their DNA extra chromosomally
(such as adenovirus ) or by integration into the host genome (such as retrovirus).
5. Most of the vectors for use in eukaryotic cells are constructed as shuttle vectors.
They incorporate the sequences required for replication and selection in E coli (ori,
ampr) as well as in the desired host cells.
Ⅲ.Host cells provide the place for replication and expression.
Section Three
DNA cloning
Ⅰ.The core process of DNA cloning is construction of recombinant DNA molecules.
1. Construction of recombinant DNA molecules.
a. Select the appropriate DNA cloning strategy.
b. Use restriction endonucleases to cut the vector at a unique site for the cloning of
foreign DNA.
c. Make the recombinant DNA molecule by joining the vector DNA and the foreign
DNA fragment.
2. Amplification, screening and isolation of recombinant DNA molecules.
a. It is the process of take up of foreign DNA ,usually plasmids, by bacteria
( transformation ).
b. The introduction of foreign DNA into animal cells is called transfection.
c. The introduction of foreign DNA packaged in the virus particle into host cells is
called infection.
d. The process of identify particular clones containing the gene of interest from
among the very large numbers of others is called screening.
3. Marker screening , probe hybridization, and DNA sequencing are used to isolate
and identify recombinants.
a. Marker screening includes antibiotic resistance screening, alpha- complementation
and marker rescue.
b. Colony , plaque and molecular hybridization screening require the labeled probe.
c. Expression screening is based on the fusion protein with the vector encoded
beta-galactosidase or the detection of gene product by specific antibody.
Ⅱ. DNA cloning can be utilized to isolate and clone a single gene
1. Candidate genes may be derived from genomic or cDNA library.
A gene library is a collection of different DNA sequences from an organism each of
which has been cloned into a vector for ease of purification, storage and analysis.
There are two types of gene library that can be made depending on the source of the
DNA used . If the DNA is genomic DNA, the library is called a genomic library.
If the DNA is cDNA ,the library is called a cDNA library.
P432 fig 20-10
2. Screening to isolate one particular clone from a gene library routinely involves
using a nucleic acid probe for hybridization. The probe will bind to its complementary
sequence allowing the required clone to be identified.
3. Yeast one-hybrid system is used to isolate coding sequences of transcription factors.
P433 fig20-11
4. Yeast two-hybrid system is used to isolate coding sequences of proteins that interact
with the “bait” protein.
P433 fig 20-12
Section four
Analysis of Cloned DNA
Ⅰ. Digesting recombinant DNA molecules with restriction enzymes, alone and in
combinations, allows the construction of a diagram (restriction map) of the molecule
indicating the cleavage positions and fragment sites.
Ⅱ. DNA sequencing tells the exact sequence of a DNA molecule . Sanger’s enzymic
method uses dideoxynucleotides as chain terminators to produce a ladder of
molecules generated by polymerase extension of a primer.
Ⅲ.Genomics and Proteomics require methods of Bioinformatics. Bioinformatics
describes the development and use of softwares to analyze biological data.
1. detecting ORFs of a genome
2. determing intron-exon boundaries
3. screening of promoters
4. determing motifs and domains of proteins
5. sequence alignment
Ⅳ. Molecular hybridization and blotting technique can detect specific genes or their
expression.
1. Southern blotting detects specific genes on nylon membrane hybridize to a
particular probe.
P436 fig 20-13
2. Northern blotting detects RNA.
3. There are other methods to detect target genes or gene expression.
a. RNase protection experiment can show which parts of the RNA are protected by
bound probe.
P436 fig 20-14
b. RT-PCR is a method of rapid detection of mRNA.
P437 fig 20-15
c.Real time PCR detects gene expression quantitatively
d.In situ hybridization and FISH can locate the position of gene expression in the cell
and on the chromosome respectively.
e.DNA microarray can detect gene expression profile in a cell.
P437 fig 20-1
Section Five
Application of Recombinant DNA Technology in Biology and
Medicine
Ⅰ.Bioactive proteins may be expressed by cloned genes.
1. E coli gene expression system is easy to manipulate and very efficient and hence
economy.
2. Modified proteins may be expressed in eukaryotic expression systems.
3. There are cell free translation systems such as wheat germ extract or rabbit
reticulocyte lysate etc. They are commonly used to check the activity of an
mRNA.
Ⅱ. It is very useful to change just one or a few specific nucleotides in a sequence
(site-directed mutagenesis) to test the effect, such as the importance of each residue in
a transcription factor binding site, suspected critical amino acids in a protein ( by
altering codons in the cDNA molecule ).
Originally, site-directed mutagenesis used phage M13 mediated method. Now much
site-directed mutagenesis is carried out using PCR.
Ⅲ .RNAi can be used to study gene function.
RNA interference is a mechanism of gene regulation in organisms. When a dsRNA
molecule which is homologous to an endogenous mRNA coding sequence, is
introduced in to a cell, it will cause degradation of the mRNA and result in
posttranscriptional gene silence. The dsRNA used is the siRNA.
P440 fig 20-17
Ⅳ.Transgene and gene targeting are useful techniques of studying gene function.
1. Transgenic mice are made by using microinjection.
2. Gene targeting includes gene knock-out and gene knock-in.
3. Transgene technique is used to establish animal models of human diseases.
P441 fig 20-18
Ⅴ. Molecular cloning accelerates development of pharmacological and medical
recombinant proteins and vaccines.
Ⅵ. Applications of recombinant DNA technology in medical diagnosis and gene
therapy.
1. DNA cloning is a useful tool in molecular medical diagnosis such as diagnosis of
hereditary diseases, identification of microbe species, diagnosis and analysis of
tumors etc.
2. Attempts to correct a genetic disorder by delivering a gene to a patient is
described as gene therapy. Although gene therapy is still in its infancy, it has great
potential.
HOMEWORK
Translate the summary into English.
Chapter Nine
Signal Transducer and Cellular Signal Transduction
Section One
The General of Signal Transduction
Ⅰ. Extracellular chemical signal molecules are divided into two types: soluble and
membrane binding signal molecules.
1. Chemical signal communication undergoes evolution from simplicity to
complexity.
2. Soluble chemical signal molecules consist of three classes : hormones ( endocrine
signals ), cytokines ( paracrine or autocrine signals ), and neurotransmitters.
P522 table 25-2
3. Cell-surface molecules are also important extracellular signal molecules.
Ⅱ. Specific receptors receive extracellular signals.
1.Chemical signals are transduced into the cell through ligand-receiptor interaction.
2. Receptors may be cell-surface molecules or intracellular molecules.
Ⅲ. Structural, quantitative and distribution changes of signal molecules are the
working basis of the signal transduction network.
1. Signal transduction pathways are composed of second messengers and signal
transducers. Signal transduction pathways form signal transduction network
through crosstalking.
2. Signal transduction is based on protein conformational changes.
P524 fig 25-1
3. Quantitative changes and translocation of intracellular transducers are the
important working style of regulation of signal transduction.
Section Two
Small Chemicals as Transducing Messengers
Ⅰ. The necessary features of the second messenger
Ⅱ. Cyclic nucleotides ( cAMP and cGMP ) are important second messengers.
P525 fig 25-2
Cyclic nucleotides are allosteric effectors and can regulate enzyme or protein activity.
P526 fig 25-4 table 25-3
Ⅲ. Certain lipids can act as second messengers
1. PLC and PI3K can catalyze the synthesis of DG and PIP3 respectively. DG and
PIP3 are lipid second messengers.
P527 fig 25-5
2. Lipid messengers may be also allosteric effecters.
3. The target molecule of PIP3 is PKB.
Ⅳ. The second messenger Ca2+ has a lot of target molecules.
Ⅴ. Effects of nitric oxide are mediated by cGMP.
1. Nitric oxide synthases catalyze NO formation.
2. NO has many physical and patho-physical effects.
Section Three
Proteins Function as Signal Transducers
Ⅰ. Protein kinases and protein phosphatases regulate the activity of receptors and
protein transducers.
1. Phosphorylation and dephosphorylation may regulate protein activity.
2. Most protein kinases are serine/threonine kinases and tyrosine kinases.
3. MAPK cascade is the core of many signal transduction pathways :
a. Growth factor signaling : Ras, Raf and MAPK pathway that regulates cell
proliferation and differentiation.
b. JNK/SAPK pathway is activated by stresses caused by radiation, osmotic
pressure, temperature etc.
c. P38MAPK pathway responds to inflammation and apoptosis signals.
4. PTK transduces signals of cell proliferation and differentiation
a. Growth factor receptors belong to PTKs
P533 fig 25-9
b. There are also non-receptor PTKs
P533 fig 25-10
5. Phosphatidases ( phosphatases ) attenuate signals stimulated by protein kinases.
Ⅱ. G protein and small G protein are activated by GTP and performs a function, in the
meanwhile GTP is hydrolyzed to GDP.
1. Hetero-trimeric G proteins mediate signal transduction directly from G-protein
coupled receptors to downstream effecter molecules.
2. Members of Ras superfamily are important signal transducers.
Section Four
The Structural Basis of Signal Transduction Network
Ⅰ. Signal transduction complexes are characteristic.
1. Signal transduction complexes ensure that signal transduction is highly efficient,
precise and diverse.
2. Signal transduction complexes are located on membrane or cytoskeleton.
3. The composition of a signal transduction complex is changeable dynamically.
Ⅱ. Protein-protein interaction domains are the basis of complex formation.
1. Protein interaction domains mediate protein-protein interaction.
P536 fig 25-12
2. SH2 domain is a Src homology 2 domain, that interacts with the specific
phosphotyrosine residue in target proteins.
3. SH3 domain is a Src homology 3 domain, that interacts with the specific
proline-rich motif in target proteins.
4. PH domain binds to membrane bound phospholipids.
Ⅲ. Adapter proteins and scaffolding proteins
1. Adapter proteins link transducers of signal transduction pathways.
P537 fig 25-13
2. Scaffolding proteins ensure that signal transduction is specific and highly
efficient.
Section Five
General Signal Pathway Mediated by Membrane Receptors
Ⅰ. Ion channel coupled membrane receptors receive signals that activate the flow of
ions across the membrane.
Ⅱ. GPCRs activate a G protein that performs a function via a second messenger.
1. Activation of a G protein initiates signal transduction
P539 fig 25-15
2. Activated G protein regulates downstream enzyme activity.
3. Glucagon receptor mediates AC-cAMP-PKA pathway.
P540 fig 25-16
4. Angiotensin II receptor mediates PLC-IP3/DG-PKC pathway.
P541 fig 25-17
Ⅲ. Protein kinase coupled receptors perform functions via a protein kinase cascade.
1. Protein tyrosine kinase coupled receptors have similar signal transduction
pathways.
2. Ras-MAPK pathway is the main pathway of EGFR.
P542 fig 25-18
3. There are other enzyme coupled receptors in the cell.
HOMEWORK
Explain the following terms in English :
1.second messenger
2. receptor
3. signal molecule
4. signal transduction
5.G protein
6. adapter protein
7. scaffolding protein
8. MAPK cascade
Text Book of Molecular Biology
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