Nucleic acids, Gene expression and

Recombinant DNA technology

• Describe chemical structure of DNAs
• derive the concept that DNA is the carrier of genetic
information
• general concepts of how genes are expressed
• general concept of how DNA is replicated
• gene manipulation and expression

The chemical components in DNA

(1) Nucleotides and nucleic acids
The importance of nucleotides
(a) Monomeric unit of DNAsÆthe storage and expression of genetic information
(b) Nucleoside triphosphate (ATP) as energy source.
(c) ATP and ADT regulate the activities of numerous metabolic processes.
(d) Nucleotide derivatives, nicotinamide adenine dinucleotide, flavin adenine
dinucleotide, and coenzyme A, are required in many enzymatice reactions.
(e) Nucleotides themselves have catalytic activities, such as ribozymes.

(2) Nucleotides, nucleosides, and bases

Nucleotides=phosphate + five-carbon sugar (+ nitrogenous base)
Ribonucleotide: The monomeric units of RNA
Deoxyribonucleotides: The monomeric units of DNA
C5’ of pentose-the phosphate- C3’
of phosphate
Nucleoside-absence of phosphate
group
 NDPs and NTPs are Polyprotic Acids

The D-ribose is is an important sugar used in genetic materials. It is not used as

energy source but is a part of the backbone of RNA and DNA. The D-ribose
undergoes cyclization, forming a five-membered ring.

(1) The base linked to to C1’ of the ribose through glycosidic bond. The linkage of
the base is on the same side of the ribose as does the phosphate group (called E
configuration)
(2) Protons from the phosphoric acid groups are dissociable; Resulting phosphate
anions form tight complexes with Mg++ and Ca ++

The nitrogeous bases

Planar, aromatic and
heterocyclic compounds
originally derived from
purine or pyrimidine.
Pyrimidines connect to
ribose at N1 postions;
Purines connect to ribose
at N9 positions. The
other important feature of
the base is the Keto-enol
tautomeric shifts

Tautomerism refers to an equilibrium

Strong UV absorbance (max. 260 nm) for all bases
except for cytosine.
between two different structure of the
same compound. Usually the tautomers
differ in the point of attachment of a
hydrogen atom. One of the most common
examples of a tautomeric system is the
equilibrium between a ketone and its enol
form,
 Structure and Nomenclature of
Nucleic Acids
(1) Nucleic acids are polynucleotides;
(2) Nucleotides are linked by phosphodiester bridges
from 3’ to 5’;
(3) Polymers of ribonucleotides are ribonucleic acids, or
RNA;
(4) Polymers of deoxyribonucleotides are
deoxyribonucleic acids, or DNA;
(1) DNA are found in different cells and viruses. They
code for genetic information;
(2) DNA nearly always forms double helix;
(3) The two strands of DNA are complementary through
base-pairing interactions;
(4) Chargaff’s rules:
G=C and A=T
G and C and A and T are complementary bases
DNA base composition varied from 25% to 70% among
different organisms.

Double helical DNA structure

The DNA structure was determined by Watson and Crick in
1953. This finding of the helical structure of the DNA also ties
together the results of several diverse studies.
(1) Chargaff’s rule: A=T, G=C
(2) The bases are dominantly in keto tautomeric forms.
(3) DNA is in helical structure. The planar aromatic bases forms
a parallel rings which is parallel to the helical axis.
The Watson-Crick B-DNA structure
B-DNA is regarded as the biologically functional native form
of DNA. The features of B-DNA are
(1) Two polynucleotides strands wind about a common axis.
(2) The helix is in right-handed twist and the diameter is ~20Å.
(3) The bases are in the core of the helix and sugar-phosphate
backbones are coiled about the helix’s periphery.
(4) The two DNA strands are antiparallel.
 Double helical DNA structure
(5) The planes of the bases are nearly
perpendicular to the helix axis.
(6) The complementary bases pairing of
the bases result in the hydrogen bonding
interactions which associate the two
DNA strands in the helix.
(7) The B-DNA has 10 base pairs per
turn, the rise is 3.4 Å, and the pitch is 34
Å.
(8) Unique Watson-Crick base pairs-A.T
or C.G. These base pairs are
interchangeable without disturbing
helical structure.
(9) The helical structure has two exterior
grooves, major and minor grooves, which
show the asymmetric properties of the
bases.

DNAÅÆ heredity
(1) Transforming principle is DNA
In 1928, Frederick Griffith٬Ҕషӝ‫ޑ‬ςύ‫ޑک‬
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౜ࢲS form(virulent)‫ݹޤ‬๵.
In 1994, Oswald Avery, Colin Macleod, and
Maclyn McCartyว౜transforming principleࣁ
DNAǶ
(2) Bacteriophage T2གࢉჴᡍ
In 1952, Alfred Hershy and Martha Chase٬Ҕࢲ
ᡏ32PǴ32S radio labeled T2Ꮨ๵ᡏǴགࢉεဉఎ
๵ࡕǴϩ‫ࡕ݋‬жᏘ๵ᡏ‫ܫ‬৔‫܄‬኱‫ނۓ‬፦ࣁ30%
32P, 1% 32S, ຓჴѝԖᏘ๵ᡏDNAࣁᒪ໺ࡕж‫܌‬

ሡ‫ނޑ‬፦Ƕ
 Denaturation and renaturation of DNA

When solution of DNA is heated, the native DNA

structure collapsed.
(1) Each strand is in flexible and fluctuating
conformation state known as random coil.
(2) Physical properties change, such as viscosity,
and UV absorbance.

Hyperchromic effect:
The UV absorbance of the denatured DNA
increases by 40% in all wavelength, whereas the
absorbance curve does not change.
ѓკຓჴDNA denaturation is a cooperative
processӢࣁ֎Ӏ‫܄‬፦‫ׯ‬ᡂჹᔈ‫ࡋྕޑ‬ጄൎࡐઞ.
ஒRenatured DNAჴᡍచҹှନࡕ, DNA཮ӣᙟ
ᚈިconformation.ӕ౛, RNA-DNA hybridization
Ψёа‫׎‬ԋᚈި่ᄬǶ

Gene expression and replication

The central dogma of Molecular Biology
DNAÆ transcription (m-RNA) Æ
(modification) Æ translation (protein)
Other routes are possible
RNAÆ RNA
RNAÆ DNA (reverse transcription)
DNAÆ protein (?)
ՠࢂproteinÆXRNA or DNA
Transcription: Synthesis of RNA
The enzyme, RNA polymerase, synthesizes
RNA in the 5’Æ 3’ direction and appends
NTPs (ATP, GTP, CTP, UTP) to the free 3’-
OH group of the grouping RNA.
 The process of transcriptional regulation

The characteristics of the sequence of the DNA template

(1) Initiation site
(2) Control sites: regulates transcription on or off by proteins known in prokaryotes as
activator or repressor and in eukaryotes as transcription factor.
(3) termination
E. Coli lac operon genes transcriptional control

Translation: synthesis of protein

Proteins are synthesized under the direction of the
corresponding mRNA. The ribosome machinery for making
proteins comprises two-thirds RNA and on-third protein
(~2500 kD in prokaryotes and ~4200 kD in eukaryotes)
mRNAύ‫؂ޑ‬Οঁ‫ׇ‬ӈᆀϐࣁcodon(ӵፐҁύ‫)߄ޑ‬Ǵ཮ჹ
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Aminoacyl-tRNA೏஥ԿribosomeՏ࿼Ǵҗ‫ܭ‬tRNA‫ڀ‬Ԗanti-
codon‫ׇ‬ӈǴӢԜ཮ပԿribosome‫ޑ‬A-siteǶribosome໽ϯ
ϸᔈஒ

peptidyl-t-RNA౽Կ
Aminoacyl-tRNAǴԶҔၸ‫ޑ‬
tRNAҗP-site ౽рǶ
 Genetic codes
Ӣࣁ3ঁDNA‫ׇ‬ӈჹᔈ΋ঁữ୷ለǴ‫܌‬
аᕴӅԖ64 ঁcodonsǴ‫ځ‬ύ61ঁჹᔈ
‫ډ‬ữ୷ለǴԶSTOP codon ࣁUAA,
UAG, ϷUGAǶMet ǴTrpѝԖ΋ঁჹ
ᔈcodon ;Leu, Ser, ArgԖϤঁჹcodon,
ӢԜgenetic codeᆀϐࣁdegenerateǴՠ
ࢂჹᔈӕ΋ঁữ୷ለ‫ޑ‬codonৡ౦ӧಃ
ΟঁਡለǶ
The first synthesized a.a. is Methionine
Even though the initiation codon is AUG, the tRNA that recognizes this initiation
codon is different from the tRNA that delivers a polypeptide’s internal Met
residue to the ribosome.
How did the ribosome select the initiation codon from among the many AUGs?
(1) In prokaryote, ӧinitiation codon 5’ᆄ΢ෞ‫ׇ‬ӈᡣribosomeᒣᇡଆ‫ۈ‬codonǶ
(2) In eukaryote, ӧmRNA‫ޑ‬5‘ᆄcapଆ‫ࡕۈ‬ಃ΋ঁAUGջࣁଆ‫ۈ‬.
ߕຏ: tRNAҗamino-acyl-tRNA synthetasesஒa.a.к༤ԿtRNAǶ

DNA replication
DNA replication requires
(1) Deoxynucleoside triphosphate (dNTP)
(2) DNA polymerase: can only extends(5’Æ3’)
an existing polynucleotide (primer) that is
base paired to the DNA’s template strand.
(3) Primers are RNA
In E. Coli, these primers are made by both
RNA polymerase and primase.
(4) Both of the DAN strands are simultaneously
replicated at the replication fork where
(i) Leading strand is synthesized in 5’Æ3’
direction.
(ii) The complementary strand (the lagging
strand) is synthesized discontinuously.
 DNA replication
DNA polymeraseIII in E. Coli is a DNA
replicase and synthesizes the leading
strand and most of the lagging DNA
strand.

DNA polymerase I in E. Coli has both

the DNA polymerase activity and
5’Æ3’exonucleonase activity.
Pol I’s 5’Æ3’ exonuclease and DNA
polymerase activity work in concert. It removes
the RNA primers and replacing them with DNA.

The synthesis of the lagging strand is completed by

sealing the nicks between multiple discontinuously
synthesized segments.
DNA ligase covalently linked adjacent 3’-OH abd
5’-phosphate group and sealed the gap.

DNA is semi-conservatively replicated

The semiconservative nature of DNA
replication was demonstrated in 1958
by Mathew Meselson.
The DNA replication results in two
molecules of duplex DNA, each
consisting of one strand from the
parent molecule and a newly
synthesized complementary strand.
Experiment:
(1) To grow E. Coli using 15NH4CL as
a sole nitrogen source for 14
generations.
(2) Transfer cell to 14N-containing
medium.
(3) Equilibrium density gradient
ultracentrifugation method to monitor
DNA as a function as cell growth.
 Error correction in DNA replication
In E.Coli
RNA polymerase error: 1/104
DNA polymerase error: 1/108~1010
In DNA replication, the error was guarded by
(1) Using RNA primers to increase the DAN
replication fidelity of the lagging strand.

(2) Both pol I and pol III have 3’Æ5’ exonuclease activities, which degrade the newly
synthesized 3’-end of a daughter strand. This reaction is activated by non-Watson-Crick
base pairing.
(3) A series of enzymes were used to detect and correct residual erros in replication and
DNA damages caused by UV radiation, mutagens, and spotaneously hydrolysis. Then,
in E. Coli, Pol I replaces the damage DNA segments excised by these enzymes.

The polymerase chain reaction

In 1985, Kerry Mullis devised the PCR method, a basis of “cell-free molecular cloning”.
 The applications of PCR
The principle of PCR
A heated denatured DNA sample is incubated with heat stable DNA polymerase,
dNTP, and two oligonucleotide primers. The primer sequences flanked the DNA
sequence of interest to direct the DNA polymerase to synthesize new complementary
strands. Multiple cycles of heat denaturation at 95 ºC and elongation at lower
temperature amplify the gene of interest to multiple copies.
*** beware of the sample contamination
Applications:
(1) Diagnosis of infectious diseases and gene mutations
(2) Amplify RNA
RNAÆ reverse transcription to generate DNA ÆPCR
(3) Asymmetric PCR
Single strand DNA PCR with one primer
(4) Nested PCR
PCR with one pair of primersÆ product goes through second round of PCR with a
second pair of primers annealed to the target fene within its ampified region.
(5) Direct sequencing

Restriction endonuclease
To use restriction endonucleases to
manipulate genes with precisely
defined sequence.
Type I, III r.e.: have both the
endonuclease and the methylase
activity. Type I r.e. cleaves DNA at a
possibly 1000 bp from the recognition
site. Type III cleaves 24 to 26 bp
distant from the recognition site.
Type II r.e.: cleave DNAs at specific
sites within the recognition sequence.
The resulting cut DNA sequence may
be with cohesive or sticky ends or
blunt ends.
The recognition sequence may well
be in palindromic sequence.
 Cloning vectors
(1)Plasmid-based vectors
Plasmids are circular DNA about ~5000 bp in size.
• Plasmid copy number may be subjected to stringent
control of one to few copies per cell, or to relaxed
control of 10 to 700 copies per cell.
• Antibiotic selection
• Polylinker site for gene insertion

(2) Virus based vectors

Bacteriophages O vector is used to clone DNAs of up to 16 kb. The gene of
interest is inserted into the central third of the 48.5-kb genome.
Filamentous bacteriophage M13
baculovirus
(3) YAC and BAC vectors
Yeast artificial chromosomes (YACs) and Bacterial artificial chromosomes (BACs)
vectors were used to amplified long DNA segments. The former are linear DNA
segments required for replication in yeast, whereas the latter is used in E. Coli.

Gene manipulation
 Southern blot: identify seqcific DNA sequence

• Gel electrophoresis of double stranded DNA sample

• the gel is soaked in 0.5 M NaOH to convert the DNA to the single-stranded form.
• blot on nitrocellulase membrane
• soaked in solution containing 32P-label single stranded DNA or RNA probe
• autoradiogram

Construct of the genomic library

Shotgun cloning to construct genomic library:
Chromosom DNA is cleaved to fragments of clonable size, and
inserted in cloning vector to construct plasmid-, phage-, or
cosmid-based genmic libraries.
Selection of the correct clones was done by in-situ
hybridization. The correct clones were subject to a process
called “chromosome walking” to complete the DNA
sequencing.

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 RNA molecules
RNA occurs in multiple forms (can be double helix but not necessary) and copies;
Messenger RNA codes template for protein synthesis;
Ribosomal RNA constitute the catalytic core of the ribosome;
Transfer RNA is the adaptor between nucleic acids and proteins;
Small nuclear RNA are essential component of splicesome;
microRNA regulates gene expression.
RNA but not DNA is susceptible to base-catalyzed hydrolysis