NUCLEIC ACIDS

JULIUS P. MARIO, RMT,

MS
 While transfer (t) RNA molecules have
many features in common, the primary feature
that sets them apart is their specificity for
different amino acids and the
corresponding specific differences of
their anticodons.

Each tRNA is an L-shaped, single chain

composed of up to 93 ribonucleotides.
Each contains up to 15 methylated bases
and about half of the nucleotides are
base-paired into double helices.

The 5’ end is often guanosine and is

always phosphorylated. The 3’ end is
CCA. Activated amino acids attach to the
terminal 3’-hydroxyl group of the
Before DNA replication can actually begin, unwinding
protein must open segments along the DNA
double helix.

DNA-directed RNA polymerase (primase) catalyzes

the synthesis of a complementary RNA primer
of approximately 50 to 100 bases on each DNA
strand.

Then DNA directed DNA polymerase III adds

deoxyribonucleotides to the 3’ end of the
primer RNA, replicating a segment of DNA, the
Okazaki fragment.

DNA polymerase I then removes the primer RNA

and adds deoxyribonucleotides to fill the gaps
between adjacent Okazaki fragments. The
fragments are finally joined together by DNA
 S-adenosylmethionine donates methyl
groups in the biosynthesis of
creatinine phosphate, epinephrine,
melatonin, and phosphatidylcholine.

Thymine, however, receives a methyl group

from N5, N10-methylenetetrahydrofolate.

S- adenosylmethionine donates the methyl

group of methionine in many biochemical
reactions, leaving S-adenosylhomocysteine.
These reactions include methylation of
guanidoacetic acid to yield creatine and of
phosphatidylethanolamine to yield
phosphatidylcholine.
The final step of purine catabolism in primates is
the oxidation of xanthine to uric acid.
Overproduction or undersecretion of uric acid causes
the urate crystal formation of gout.

Urea, arising from allantoic acid, and allantoic acid

itself are the excreted products of purine
catabolism in fish, but in mammals urea arises
from the catabolism of amino acids.

Xanthine and orotic acid, which are not normally

excreted, are intermediates in purine and
pyrimidine metabolism, respectively.

Purine synthesis is a metabolic pathway that is

common to bacteria and humans. Nitrogen
fixation, photophosphorylation, mucopeptide
synthesis, and fermentation occur in plants or
bacteria but not in humans. Thus,
photophosphorylation, which is equivalent to
Carbons 4, 5 and nitrogen 7 of purines are
derived from glycine. The glycine
molecule is incorporated intact into the
purine ring. Carbons 2 and 8 arise from
activated derivatives of
tetrahydrofolate; carbon 6 arises from
carbon dioxide.

In early step in pyrimidine synthesis is the

reaction catalyzed by aspartate
transcarbamoylase:
carbamoyl phosphate + aspartate 
carbamoylaspartate.

Carbamoyl phosphate is not an intermediate in

In a general sense, the mechanism of protein synthesis
in eukaryotic cells is similar to that found in
prokaryoties; however, there are significant
differences.

Cycloheximide inhibits elongation of proteins in

eukaryotes, while erythromycin causes the same
effect in prokaryotes. Thus, one is an antibiotic
beneficial to humans, while the other is a poison.

Cytoplasmic ribosomes of eukaryotes are larger,

sedimenting at 80S instead of 70S. While
eukaryotic cells utilizes a specific tRNA for
initiation, it is not formylated as in bacteria.
Finally, eukaryotic mRNA always specifies only
one polypeptide as opposed to prokaryotic mRNA,
which may specify the synthesis of more than one
gene product per mRNA (polycistronic).
The pyrimidine ring is formed from
carbamoyl phosphate and aspartic acid.

Carbamoyl phosphate arises from the

reaction of glutamine and HCO3- with C4
derived from bicarbonate and N3 from the
amide N of glutamine. N1 and carbons 4
and 6 are derived from aspartic acid, with
carbon 4 being the side-chain carboxyl
carbon.

Folic acid derivatives are not required in the

synthesis of the pyrimidine ring, but 5,10-
methylenetetrahydrofolic acid is required
for the synthesis of thymine from uracil.
In bacteria, cytidine triphosphate leads to feedback
inhibition of aspartate transcarbamoylase.
Aspartate transcarbamoylase has been shown to
consist of 12 subunits, six each of catalytic and
regulatory subunits.

In mammals, uridine triphosphate inhibits

pyrimidine biosynthesis at the level of the
cytoplasmic carbamoyl phosphate synthetase rather
than at the level of aspartate transcarbamoylase.

Bovine pancreatic ribonuclease cleaves RNA at a

point distal to the 3’-phosphate of a pyrimidine
nucleotide. The products, therefore, are a
pyrimidine nucleoside 3’-phosphate and
oligonucleotides with a terminal pyrimidine
3’-phosphate.
Amanita phalloides, the mushroom known
as the destroying angel, leads to a
considerable number of deaths each year.
It produces the cyclic octapeptide
amantin, which is composed of several
unusual amino acids. The major site
of the toxin’s action is RNA
polymerase II, to which it binds very
tightly.

RNA polymerase II is responsible for the

synthesis of mRNA.
The melting temperature (Tm) of duplex
DNA is the temperature at which half the
base pairs are denatured.

Because cytosine- guanine )C-G) base pairs

have three rather than two hydrogen bonds,
a high content of C + G increases Tm by
virtue of the positive straight-line
relationship. According to Chargaff’s rules,
in duplex DNA the content of A + G = C + T.

One way of assaying the denaturation is to

measure the optical density of the DNA
(Usually at 260 nm). Denatured DNA has
an optical density approximately 40
percent greater than native DNA.
The Watson-Crick model of DNA structure predicts
a double-stranded helix with hydrogen bonds
between A-T and G-C bases on the inside of the
helix. The chains run in an antiparallel, or
opposite, direction. Covalent phosphodiester
bonds exist only between sugar moieties within
each chain and not between their attached bases.

Histones are a family of basic protein (rich in lysine

and arginine) that are bound noncovalently but
stoichiometrically to DNA. They are of relatively
low molecular weight (10,000-20,000) and
separable into five classes, largely on the basis of
their lysine and arginine content.

Histone IV from calf thymus and pea buds

discloses a remarkable degree of homology:
these diverse organisms exhibit a difference in
only 2 out of 102 amino acids that constitute
DNA replication entails pairing of thymine with
adenine and guanine with cytosine. The chains
of the double helix are thus bonded in part by a
hydrogen linkage between amino and keto
groups. The strands themselves are synthesized
in an antiparallel direction, i.e. the 5’ 3’
sequence f phosphodiester bridges mentioned in the
question specifies its complement in a 3’  5’
direction.

AUG is the codon for both methionine and N-

formylmethionine.

In E. coli, AUG is the chain-initiating codon and N-

formylmethionine is the first amino acid
incorporated into the nascent polypeptide.

In mammals, AUG is also thought to the codon for

chain initiation, but methionine (rather than N-
formylmethionine) is the N-terminal amino acid.
Insertion of one extra nucleotide causes a
frameshift mutation and mistranslation
of all the mRNA transcribed from
beyond that point in the DNA.

Transition, transversion and deletion of a

triplet cause mutations usually involving an
error in the identity of only one amino acid
(missense), or removal of the amino acid
from the sequence, or no error at all in the
amino acid sequence. There is a chance also
that transition and transversion will give a
“nonsense,” or chain-terminator,
mutation, and this is about as likely to be
lethal as is a frame shift.
ATP is required for the esterification of
amino acids to their corresponding
tRNAs. This reaction is catalyzed by the
class of enzymes known as aminoacyl-tRNA
synthetases. Each one of these enzymes
is specific for one tRNA and its
corresponding amino acid.

Amino Acid + tRNA + ATP  Aminoacyl-tRNA + AMP

PPi

As with most ATP hydrolytic reactions that

release pyrophosphate,
pyrophosphatase quickly hydrolyzes the
product to Pi making the reaction
essentially irreversible.

Since ATP is hydrolyzed to AMP and PPi

Dimerization between thymines (thymine dimers)
on the same polynucleotide strand is caused by
ultraviolet radiation (260 nm) and stops
replication at that point. The dimers can be
excised and repaired by enzymes that include
ligase or can be dissociated by further exposure
to longer (330 to 450 nm) or shorter (230 nm)
wavelength in the process of
photoreactivation.

In HbS, a valine residue replaces a glutamic acid

on the B chain as the result of a point mutation
in one nucleotide base. This single nucleotide
alteration at the second position of the triplet
consists in a change of thymine to adenine.

Formylmethionine is thought to be always the first

amino acid at the N-terminal of polypeptide
synthesized in E. coli.

Two discrete tRNAs for methionine exist in E. coli

During the course of protein synthesis in a ribosome,
peptidyl transferase catalyzes the formation of
peptide bonds. However, when a stop codon is
reached, such as UAA, UGA, or UAG, aminoacyl-
tRNA does not bind to the A site of a ribosome.
One of the proteins known as a release factor binds
to the A site of a ribosome. One of the proteins
known as a release factor binds to the specific
trinucleotide sequence present. This binding of the
release factor activates peptidyl transferase to
hydrolyze the bond between the polypeptide and
the tRNA occupying the P site. Thus, instead of
forming a peptide bond, peptidyl transferase
catalyzes the hydrolytic step leading to the
release of newly synthesized proteins. Following
release of the polypeptide, the ribosome
dissociations into its major subunits.

 The chain-terminating (nonsense) codons are UAA,

UAG, and UGA. Termination of a polypeptide chain
RNA polymerase, which requires a DNA
template in order to synthesize a
ribonucleic acid polymer, usually
transcribes one strand of double –helical
DNA. The direction of growth of the RNA chain
is from the 5’ to the 3’ end, and the
product is never a circular molecule.
Unlike DNA polymerase, RNA pol does not
require a primer.

Sigma (σ) factor is the subunit of RNA

polymerase that confers specificity of
initiation on the core enzyme. In the
presence of sigma factor, RNA polymerase
will choose the correct strand of duplex
DNA for transcription and
The first step in the incorporation of amino acid
into polypeptide chains is known as the
activation step, which is catalyzed by aminoacyl-
tRNA synthetases.

The reaction involves the esterification of amino

acids to their specific tRNA at the expense of
ATP. Pyrophosphate formed in the reaction is
hydrolyzed rapidly by pyrophosphatase, which
ensures the rapid completion of the reaction.

Other steps include initiation, involving the binding

of mRNA and the first aminoacyl-tRNA to the
30S subunit of the ribosome,

elongation, in which the polypeptide chain is

lengthened by addition of another aminoacyl-
tRNA;

and termination, which releases the polypeptide

The differences between prokaryotic and eukaryotic
mRNA are profound.

In bacteria, primary transcripts of mRNA can be used

immediately and directly for translation, while in
eukaryotes, primary mRNA must beprocessed,
modified, and transported from the nucleus to
the cytoplasm.

Prokaryotic mRNA can either code for a single

polypeptide (monocistronic) or for several
polypeptide chains (polycistronic). Eukaryotic
mRNA is always monocistronic. Primary transcripts
of eukaryotic mRNA are synthesized with introns
that must be removed. In addition, eukaryotic mRNA
contain 5’ caps of 7-methylguanosine and often
have 3’ poly A tails.

The bases found in DNA are adenine, guanine, cytosine,

and thymine, whereas RNA contains uracil in place of
thymine. Thus an animal fed tritiated thymine
Tetracycline blocks the binding of
aminoacyl-tRNA with initiator sites on
30S subunits of ribosomes, causing an
irreversible inhibition of protein
synthesis. The transcription of DNA, the
binding of mRNA to ribosomes, and the
formation of aminoacyl-tRNA are not affected.
Release of peptide from mRNA-tRNA
complexes does not occur inasmuch as
peptide bonds are not formed.

Chloramphenicol interacts with the 50S

subunits of prokaryotic ribosomes to
inhibit the process of polypeptide chain
elongation.

Streptomycin, on the other hand, binds to

the 30S subunit of ribosomes, resulting in
In E. coli, the RNA polymerase holoenzyme is
composed of four subunits. The sigma subunit of
the intact enzyme is essential for specific
initiation in that it allows RNA polymerase to
recognize promoter sites.

Following the initiation of RNA synthesis, the sigma

subunit dissociates from the other subunits of the
polymerase, leaving the core enzyme .

Only eukaryotic mRNAs are capped at their 5’ end.

Prokaryotic RNAs and eukaryotic tRNA and rRNA
are not capped. The cap is composed of 7-
methylguanylate attached by a pyrophosphate
linkage to the 5’end. The cap protects the 5’ end
of mRNAs from nucleases and phosphatase and is
essential for the recognition of eukaryotic mRNAs
in the protein-synthesizing system.

For the termination of RNA synthesis, rho protein is

necessary.
During translation, messenger RNA (mRNA) codons
dictate the amino acids to be synthesized into
proteins. This is in contrast to transcription, which
yields mRNA complementary to a sequence of
DNA, along with ribosomal RNA (rRNA) and
transfer RNA (tRNA).

The two subunits of ribosomes are composed of

proteins and rRNA. Ribosomes are found in the
cytoplasm, in mitochondria, and bound to the
endoplasmic reticulum.

Guanine-cytosine (G-C) base pair bonds are stronger

than adenine-thymine (A-T) bonds. DNA with a high
G-C content is more stable and melts at a higher
temperature than DNA that is rich in A-T. This
stability difference is a consequence of the existence
of only two hydrogen bonds between A and T,
compared with three between G and C.
The initiation but not the elongation of polypeptide
chain synthesis (in E. coli) involves N-terminal N-
formylmethionine incorporation as specified by
codons AUG and GUG.

Peptide chain elongation on the ribosomal mRNA

employs peptidyl transferase after codon-specific
binding of aminoacyl-tRNA, using energy from
GTP and the factors G, Tu, and Ts.

Four high-energy phosphate bonds are utilized

during the overall process of linking together two
amino acids via peptide bond formation. The
equivalents of two bonds from ATP are used
during activation of amino acids, since ATP is
hydrolyzed to AMP and inorganic pyrophosphate (PPi).
One bond from GTP is used during binding of
aminoacyl-tRNA to the active ribosomal site, and
another bond from GTP is hydrolyzed during the
attachment of N-formylmethionyl-tRNA to mRNA
and the association of ribosomal subunits. The
step where actual peptide bond formation is catalyzed
The cell walls of gram-positive bacteria
exhibit a 200- to 800-A-think, electron-
dense outer layer consisting of N-
acetylmuramic acid, N-acetylgocosamine,
and D- and L-amino acid peptides. The
enzyme lysozyme partially hydrolyzes cell
walls. Penicillin inhibits cell wall protein
synthase.

Transformation is characterized by the

bacterial uptake of soluble DNA released
by a donor cell. Transformation takes place
only in bacteria that can utilized the high-
molecule-weight DNA of the medium.
Although originally discovered in the
pneumococcus, transformation also occurs in
The adenylic acid moiety of ATP is the precursor of
nicotinamide adenine dinucleotide (NAD+), flavin
adenine dinucleotide (FAD), and coenzyme A
(CoA).

In addition to ATP, NAD+ synthesis requires

phosphoribosylpyrophosphate (PRPP), niacin and
glutamine. Pellagra is a disease caused by the
dietary insufficiency of niacin or tryptophan, the
precursor of niacin.

Biosynthesis of FAD is accomplished by the transfer

of AMP from ATP to riboflavin 5’-phosphate.

Pantothenate, cysteine, and ATP contribute to the

formation of CoA.

Nucleoside triphosphates are the substrates for

both RNA and DNA polymerases, and both
enzymes add bases at the free 3’ end of the
growing to DNA. The enzyme that synthesizes
Interferon is a cell-specific protein
produced by cells infected by a virus. It
elicits production of an antiviral protein
both in cells that produce it and in those
that are exposed to it. While all animal
cells are capable of yielding interferon,
cells of the reticuloendothelial system are
the major source in an infected animal.
Interferon production is dependent on
transcription and translation.

Spontaneous point mutations are most

likely to arise from tautomeric shift of
hydrogen atoms in a purine or
pyrimidine base. This shift causes an
altered base pairing during DNA
replication. Several mutagenic agents such
as 5-bromouracil and 2-aminopurine
The primary transcripts of all eukaryotic
mRNA are capped at their 5’ end. The cap
composed of 7-methylguanylate
attached by a pyrophosphate linkage to
the 5’end is known as cap 0. One of the
adjacent riboses is methylated in cap 1,
and both of the adjacent riboses are
methylated in cap 2.

When prokaryotic monocistronic mRNAs

are artificially capped, translation will occur
in a eukaryotic, in vitro translation system.

Humans cannot synthesize phenylalanine;

therefore it is always an essential amino acid.
Because phenylketonuric patients cannot
hydroxylate phenylalanine to form
tyrosine, in these individuals tyrosine
Transcription of the lactose operon “o” is
initiated by binding of RNA polymerase
(including sigma factor) to the promotor
region, ‘p.” The region designated “i” governs
repressor synthesis, and repressor protein from
“i” is the negative control exerted n the operon
at the site designated “o” . The regions “z” “y,” and
“a” specify β-galactosidase, galactoside
permease, and acetylase enzymes, respectively.

The smallest unit of DNA capable of coding for

the synthesis of a polypeptide is the cistron.

A gene sequence under the coordinated control

of a single operator is called an operon.

The promoter is the site of binding of RNA

polymerase and initiation of transcription.

A replicon is a unit of DNA that contains a signal

Amino acids are activated by specific aminoacyl-tRNA synthetases
that join them to their unique tRNA with the consumption of two
high-energy phosphate bonds.

Next, the 30s rRNA subunit binds initiation factor 3, mRNA, and
initiation factor 1. Aminoacyl-tRNA, initiation factor 2, and GTP join
the 30S subunit complex, which unites with the 50S subunits.

Aminoacyl-tRNA is bound to the initiation codon of mRNA, a process

driven by the hydrolysis of GTP to guanosine diphosphate (GDP)
and inorganic orthophosphate (Pi). The three initiation proteins
are then released from the complex.

In ensuring steps of elongation, aminoacyl-tRNA binds to elongation-

factor protein together with GTP. GTP hydrolysis energizes the
addition of the new aminoacyl-tRNA to the active (A) site of the
ribosome.

Next, peptidyl transferase catalyzes formation of a peptide bond

between the carboxyl group of the preceding amino acid and the
amino group of the newly added amino acid. The ribosome
advances one codon forward on the mRNA, moving the peptidyl-
tRNA from the A site to the peptidyl (P) site, thereby clearing the A
site for the next incoming aminoacyl-tRNA. This final step, which is
called translocation, requires a second elongation factor
and consumes another mole of GTP.
Mitochondria of eukaryotic cells contain
four to six molecules of circular DNA
The nuclear DNA of eukaryotes and
the E.coli DNA genome are much
larger.

Initiation of protein synthesis in E. coli

mitochondria, and chloroplasts
requires N-formylmethionyl-tRNA
association with ribosomes. In general,
initiation of translation on cytoplasmic
ribosomes of eukaryotes is similar.
However, the special tRNA used in
eukaryotes for initiation is methionyl-
tRNA, which is not formylated.
Condensation of carbamoyl phosphate and
aspartate to yield N-carbamoylaspartate
provides the carbon and nitrogen atoms
that are then formed into a pyrimidine
ring structure called dihydro-orotate.

The carbamoyl phosphate utilized for

pyrimidine ring synthesis is, itself, formed
in the cytosol from glutamine, ATP and
HCO3-. This may be contrasted to carbamoyl
phosphate consumed in the synthesis of
urea. The latter is formed in mitochondria
from NH3, ATP and CO2.
The final step in DNA replication is the closure of a
single DNA chain in prokaryotes or the joining
of the breaks in DNA chains in eukaryotes. This
is accomplished by an enzyme known as DNA
ligase. This enzyme catalyzes the formation of a
phosphodiester bond between the free
3’-hydroxyl of the end of one DNA chain and
the 5’ end of the other. DNA ligase in mammals
and bacteriophages, ATP drives the reaction,
while in bacteria, NAD+ is the energy source.
DNA ligase is specific only to double helical DNA;
that is, single-stranded DNA will not be joined
by ligase. DNA ligase closes nicks in double-
stranded DNA during repair as well as during
normal synthesis. It is also used as a tool during
genetic recombination.

Primase synthesizes RNA primers at the start of

DNA synthesis. Rep protein unwinds the DNA
helix. DNA gyrase introduces superhelical
twists during replication. DNA pol I erases
primer and fills in gaps at the end of DNA
DNA polymerase III catalyzes the step-by-
step polymerization of
deoxyribonucleoside 5’-triphosphate RNA
or DNA primer strand. During the course of
this magnesium-requiring reaction,
pyrophosphate is liberated.
Pyrophosphatase quickly converts the
pyrophosphate to inorganic phosphate.
(DNA)n + dNTP  > (DNA)n+1 +PPi

Since addition is at the free 3’-hydroxyl terminus,

elongation of the new DNA chain is in the
5’  3’ direction. DNA polymerase III prefers
double-stranded DNA templates and is
present as a multisubunit complex known
as DNA polymerase III holoenzyme.
Puromycin is a structural analogue of the aminoacyl
end of the tRNA. It irreversibly reacts with the
peptidyl-tRNA, thereby terminating protein
synthesis.

Streptomycin, like tetracycline and

chloramphenicol, inhibits ribosomal activity.

Mitomycin covalently cross-links DNA, preventing

cell replication.

Rifampicin is an inhibitor of DNA-dependent RNA

polymerase.
Like bacterial DNA, eukaryotic DNA is replicated in a
semiconservative manner. However, in contrast to most
bacterial DNA, which is circular in structure, nuclear
chromosomal DNA is a single, uninterrupted
molecule that is linear and unbranched. A
eukaryotic chromosome contains a strand of DNA
at least 100 times as large as the DNA molecules
found in prokaryotic. Eukaryotic, but not
Xeroderma pigmentosum appears to be due to the
inability of an excision-repair system to remove
thymine dimers, which are formed on exposure of
DNA to ultraviolet radiation. Mutagenesis by this
mechanism is presumably the basis for the
multiple neoplasm that occur in patients who have
this disease.

The nucleolus is an organelle unique to eukaryotic

cells. It is the site where hundreds of copies of
genes repeated in tandem for the four
ribosomal RNAs are transcribed by RNA
polymerase I to give a 45S primary transcript.
Enzymatic modification and cleavage remove spacer
regions to yield 28S, 18S, and 5.8S ribosomal RNA.
The 5S subunit is synthesized by RNA polymerase III
in the nucleoplasm, rather than in the nucleolus.
Ribosomal proteins combine with the ribosomal
subunit to assemble into a 60S subunit containing the
5S, 5.8S, and 28S RNA and a 40S subunit containing
the 18S RNA. Combined, the two subunits produce
a functional eukaryotic ribosome with a
Cot ½ curves allow a comparison of the kinetics of
reassociation of thermally denatured DNA. Co is
the total concentration of DNA in the solution being
studied (moles per liter of nucleotides) and t is time
(sec).

Analysis of the kinetics of reassociation of single

fragments of thermally denatured DNA demonstrates
that the Cot ½ values of prokaryotic DNA are
substantially greater than those of eukaryotic
DNA; that is, one-half of the sheared, thermally
denatured prokaryotic DNA from an organism such as
E. coli takes longer to reassociate than similarly
treated DNA from mouse liver cells this is because,
in general, eukaryotic DNA contains repetitive
sequences, of DNA, while prokaryotic DNA does
not.

The rate of reassociation of single strands of DNA

into double strands is dependent upon the
concentration of complimentary fragments in the
solution under study. Centromeres, genes for
The signal hypothesis attempts to explain the
biosynthesis of secretory and membrane
proteins. Since all eukaryotic ribosomes are
intrinsically the same, whether or not particular
ribosome attaches to endoplasmic reticulum
depends on the synthesis of a single peptide
from 15 to 30 amino acid residues near the
amino end of a nascent polypeptide chain. Most
secretory and membrane proteins are
synthesized continuously with such a signal
peptide that binds the ribosome to a receptor
site on the ER. The protein passes through the
membrane into the lumen during its synthesis, and
the signal peptide is excised by a specific
peptidase on the luminal surface of the ER.

The attachment of ribosomes to mRNA, either

singly or in multiples on long messengers
(polysomes), and the release of proteins from
Bromouracil is incorporated into DNA in place of thymidine, to
yield a denser DNA. The newly synthesized DNA fragments can
then be quantitated by centrifugation through density
gradients of cesium chloride. 5-Bromouracil is neither more
reactive nor more sensitive to cleavage than thymidine. It
does not cause frameshift mutations, as do the acridine dyes.

The introduction of recombinant DNA into the host bacteria is

usually accomplished by packaging it into infectious λ
bacteriophages. Another method is simply to allow that bacteria
bacteri
cells to take up naked recombinant DNA molecules (e.g.,
plasmids) from growth medium. The latter process occurs with
a frequency of about one of every 106 DNA molecules.
Conjugation is not used to introduce artificially produced
recombinant DNA. Although a variety of methods exist,
recombinant DNA may be formed by splitting plasmid or
bacteriophage vehicle DNA as well as donor (e.g. Eukaryotic
DNA) with specific restriction endonucleases. The foreign donor
and vehicle DNA is then annealed together with DNA ligase and
introduced into the bacterial host cell.

Marker genes, such as those for antibiotic resistance, may be include

in the vehicle DNA so that the host cells containing the introduced
recombinant DNA may be specifically selected and cloned.
Acridines induce frameshift mutations, in which a
nucleotide is either added to or removed from
the DNA, and the mRNA is translated as triplets
of nucleotides without punctuation, the
sequence of amino acids laid down after the
point of the frameshift will bear no
resemblance to the unmutated sequence. The
mechanism of this frameshift appears to be
intercalation of the dye between adjacent base
pairs in the DNA double the helix. If the dye
remains intercalated, reading is advanced; but if the
dye and adjacent base are deleted, reading is
retarded.

Infection of an E. coli cell by T4 bacteriophage

very rapidly leads to loss of host DNA and other
macromolecular synthesis. Viral genome-
specified deoxyribonucleases are formed
immediately on infection, and these destroy host
DNA. The bacteriophage DNA is not destroyed
because it contains hydroxymethylcytosine in
The lac operon is shut off by a repressor protein
that is inactivated by an inducer- i.e, negative
control of induction. In the absence of an
inducer, transcription of the galactosidase
structural gene into mRNA cannot proceed. The
combination of repressor protein and inducer
constitutes a repressor-induced complex.

A palindromic sequence in base pairs of double-

stranded DNA demonstrates twofold rotational
symmetry; that is, the strand reads the same
form 3’ to 5’ as its complement reads from 5’ to
3’. Many restriction enzymes recognizes
specified palindromic sequences of from four to
six base pairs and hydrolyze the phosphodiester
bonds central to the symmetrical axis in each
strand. The cleavage site of the given palindrome is
shown:
G-T-C-A-T-G-A-C
X
In the positive-control-of induction
model, it is postulated that the
regulation gene specifies an inducer
protein that requires activation by an
inducer molecule before it will
interact with the operon to switch it
on. An example of this type of control is
found in the arabinose ara operon of E.
coli.

The repressor protein binds to a specific

operator on the operon. This prevents
the RNA polymerase from
transcribing the subsequent
structural genes into mRNA. Repressor
protein does not act distally in this
Transduction involves the use of bacterial
viruses to transfer small regions of a
bacterial chromosome from a donor to a
recipient. Although of little use for
detecting linkage between genes that
are not close together, transduction can
be a valuable method for the analysis of
genetic fine structure.

During purine ring biosynthesis, the amino

acid glycine is completely incorporated
to provide C-4, C-5, and N-7. Glutamine
contributes N-3 and N-9, asparate
provides N-1, and derivatives of
tetrahydrofolate furnish C-2 and C-8.
Point mutations that are frameshift
mutations put the normal reading-
frame out of register by one base
pair. The insertion of an extra base
pair or the deletion of one or more
base pairs falls into this category.

Transitions and transversions are not

frameshift mutations; they are
substitutions of one base pair for
another. Substitutions are the most
common type of mutation. In transitions,
a purine is replaced by a purine or a
pyrimidine or vice versa. It has been
suggested that transitions occur
spontaneously owing the tautomeric
changes in base-hydrogen-bond locations.
Transversions can be caused by defective
The genetic deficiency that results in the absence of
hypoxanthine phosphoribosyltransferase (HGPRT)
is the Lesch-Nyhan syndrome, a disease producing
symptoms of self-destructive behavior, mental
retardation, and spasticity. In normal individuals, the
enzyme catalyzes the salvage synthesis of inosine
5—monophophate (IMP) or guanosine
5’-monophosphate (GMP) from the reaction of
hypoxanthine or guanine with
phosphoribosylpyrophosphate (PRPP).
Although gout can be caused by a variety of less
devastating biochemical lesions that lead to the
precipitation of sodium urate crystals from elevated
serum levels of urate, in the Lesch-Nyhan syndrome
gout is but one of the many symptoms caused by
elevated levels of urate and PRPP. In some forms of
gout, as well as in the Lesch-Nyhan syndrome,
allopurinol treatment inhibits xanthine oxidase
and thereby prevents urate overproduction from excess
hypoxanthine. However, the drug has no effect on
Bacteria acquire drug resistance by spontaneous
mutation, recombination, or transduction.
Resistance to penicillin in gram-positive
staphylococci is plasmid determined. However,
conjugal transfer does not occur in gram-
positive, and the plasmids are transferred by
transducing bacteriophages.

Plasmids are one class of mobile genetic elements

called transposons that are found in bacteria.
They are small, circular accessory chromosomes
that can replicate autonomously of the host
chromosome or insert into the host
chromosome. In contrast to general genetic
recombination, where homologous sequences
(alleles) are exchanged, plasmids containing
insertion elements can join unrelated genes
and this effect recombination of non-
homologous DNA.
Plasmids may also carry genes that confer maleness
and allow conjugation (F factor) carry drug-
resistance genes (R factor), or carry genes for
toxin production (colicinogenic factors).
Experimental insertion of foreign DNA into plasmids
allows cloning of recombinant DNA.

Bacitracin is a bacteriolytic cyclic peptide that

blocks cell-wall synthesis by inhibiting
phosphorylation of undecaprenol-PP formed
during the transfer of subunits from carrier to
cell wall.

Penicillin blocks cross-linking of the peptidoglycan

(transpeptidation reaction).

Chloramphenicol inhibits protein synthesis.

Sulfonamides are competitive analogues of p-

Bacteria contain extrachromosomal genetic
elements known as plasmids that can
mediate chromosome transfer via conjugation.
The most widely studied plasmid is F, the
sex factor of E. coli K12. Bacteria containing
the sex factor may act as females or males
and are able conjugate with males or
females.

Prokaryotic ribosomes have a sedimentation

coefficient of 70S, being composed of 50S
and 30S subunits. Ribosomes in
chloroplasts and mitochondria of
eukaryotic cells are more similar to
prokaryotic ribosomes than to eukaryotic
cytosolic ribosomes. Like bacteria ribosomes,
chloroplast and mitochondrial ribosomes use a
formylated tRNA. In addition they are
The operon, as proposed by Jacob and Monod, can be
described as a group of functionally related
structural genes that map close to each other in
the chromosome and can turned on or off
together through the same regulatory loci. The
DNA elements of the model are the regulator, the
promoter, the operator, and the structural genes. The
regulator codes for the synthesis of a repressor
protein that binds the operator, preventing
attachment of RNA polymerase to the promoter
and initiation of synthesis. Inducers, such as
isopropylthiogalactoside in the case of the lac
operon, bind the repressor, thereby inactivating
it and allowing synthesis to proceed.

Chloramphenicol, gentamycin, streptomycin, and

tetracycline block peptide synthesis by binding to
ribosomes. The modes of action vary in detail.
Tetracycline, for example, inhibits binding of aminoacyl-
tRNA to mRNA by interference at the level of the 30S
subunit of the ribosomes, decreasing the rate of protein
synthesis and also interfering with the reading of mRNA
.
The biosynthesis of
deoxyribonucleotides occurs by
reduction of the 2’-hydroxyl group
of the pentose moiety in all four
nucleoside diphosphates. The
immediate hydrogen donor is reduced
thioredoxin, which is regenerated by
thioredoxin reductase and the reduced
form of nicotinamide adenine
dinucleotide phosphate (NADPH)-
coupled flavoprotein. Thus NADPH is
the ultimate source of reducing
Feedback inhibition of purine nucleotide
biosynthesis occurs at several sites in the
pathway.

AMP, GMP, and IMP inhibit both the

formation of PRPP from ribose 5-
phosphate and the conversion of PRPP to
phosphoribosylamine.

AMP inhibits the conversion of IMP to

adenylosuccinate (AMP precursor), and
GMP inhibits the conversion of IMP to
xanthylate (GMP precursor).

Uric acid is not a feedback inhibitor of purine

Xanthine oxidase catalyzed the conversion of
hypoxanthine or xanthine to uric acid.
Purine nucleotides, such as AMP, GMP and
IMP, are catabolized to uric acid via
hypoxanthine and xanthine.

Since there are four different bases that can

specify amino acids as triplet codons,
sixty-four codons are possible. Thus, most
amino acids are specified by more than
one codon, making the code degenerate.
Only UAG, UAA, and UGA do not specify amino
acids, they are termination signals or stop
codons. In the example used- UCCUCUU- the
degeneracy of the code is seen, since UCC and
UCU both specify Ser.
DNA ligase catalyzes the formation of a
phosphodiester bond between two DNA chains
that must be part of a double-stranded DNA
molecule; the enzyme cannot link single-stranded
DNA molecules. The energy source for the bond
formation in E. coli is NAD+; in some animal cells
and bacteriophages, ATP serves this purpose.
DNA ligase functions in DNA synthesis and repair
and in recombination.

The primary function of DNA polymerase I from E. coli is

to catalyze the step-by-step addition of a
deoxyribonucleotide from a deoxyribonucleoside
triphosphate to a preexisting DNA chain, for
which a free 3’- OH in the DNA template is
required. DNA polymerase I also can hydrolyze DNA
progressively from the 3’- OH terminus, but the
nucleotide removed must not be part of a double
helix. The activity probably has an editing function
during replication. The enzyme also has 5’ - 3’
nuclease activity. The cleaved bond must be in a
The word “kilobase” refers to a unit of length
equivalent to either 1000 bases of a single-
stranded DNA molecule or 1000 base pairs of
double-stranded molecule. A kilobase (kb) of
double-stranded DNA corresponds to a mass
of approximately 660 kilodaltons and a
contour length of 0.34 um. Single-stranded
DNA has about the same length and about
half the weight. Viral DNA ranges from 5 to
200 kb, prokaryotic DNA from 760 for
mycoplasma to 4,000 for E. coli, and
eukaryotic DNA from 13,000 for yeast to
about 3,000,000 for man.

Operator genes are the loci for binding of

repressor or inducer molecules.
According to whether or not the operator
gene locus is occupied, the structural
The structural hallmark of the DNA helix is the
hydrogen bond. When hydrogen bonds between
paired bases of the double-stranded helix are
disrupted, the strands come apart or melt.

Acids, alkalis, heating or specific enzymes melt

DNA. When a solution of DNA is heated, the melting
temperature is that temperature at which half of the
helical structure is lost. The melting temperature
can be precisely measured by following the increase
in absorbance at 260 nm that occurs as double
stranded DNA becomes single-stranded DNA. This
effect is known as hyperchromism. The melting
temperature is abrupt, demonstrating the highly
cooperative nature of the hydrogen bonds holding
together the a-helix. The base pairings of G-C are
more stable than A-T and melt later than A-T. For this
rason, DNA with a high G-C content melts at a higher
temperature than DNA with a lower G-C content.
The initiation of DNA synthesis in E. coli requires the
unwinding of parental DNA and the formation of
complementary RNA primers by DNA-directed RNA
polymerase (primase).

The actual replication of DNA is carried out by DNA

polymerase (primase).

The actual replication of DNA is carried out by DNA

polymerase III in bacteria and a DNA polymerase in
eukaryotic cells.

In both prokaryotic cells and eukaryotic cells, DNA

replication occurs at more than one site. Thus, the end
of the discontinuous pieces to create a continuous,
complete chain of DNA.

Repair of DNA is carried out by β DNA polymerase in

eukaryotic cells.
Puromycin and cycloheximide are inhibitors of
protein synthesis.

By binding to ribosomes in place of amino acids and

acting as an analogue of the aminoacyl-tRNA,
puromycin causes premature chain termination
of growing proteins. Since puromycin contains an α-
amino group, it is attached by a peptide bond to the
nascent peptide chain by peptidyl transferase. The
product prematurely released is an incomplete peptide
with puromycin residue at its carboxyl end.

Cycloheximide is an antibiotic specific for

eukaryotic cells that inhibits peptidyl transferase
activity of 60S ribosomal subunits. Its counterpart,
chloramphenicol, has the same effect on
prokaryotic 50S ribosomal subunits. Another
antibiotic that inhibits protein synthesis in
prokaryotes by binding to the 50S subunits is
erythromycin. However, erythromycin exerts its
effect by preveting translocation. Some antibiotics
are useful as clinical and research drugs because of
Both rifamycin and actinomycin are
inhibitors of transcription and are
obtained from Streptomyces.

Rifamycin prevents initiation of RNA

synthesis by blocking formation of the
first phosphodiester bond. Thus, only new
RNA chain initiation is affected.

In contrast, actinomycin does not allow DNA

to serve as a template for RNA
synthesis. At low enough-concentration,
actinomycin can inhibit RNA synthesis
without greatly affecting DNA or protein
synthesis, making it a powerful tool for
mechanistic studies in both eukaryotic
and prokaryotic cells.
Fertilization of eggs by sperm and bacterial
conjugation are both instances of cells fusion
that allow transfer of chromosomal material
between cells.

In transduction, a small portion of the

chromosome of the donor bacterium becomes
incorporated into the DNA of a virus. The
bacteriophage carries the donor DNA to the
host bacterium it infects.

When a bacterium growing in medium picks up

soluble DNA in the medium and incorporates it
into its genome, this is known as transfomation.
Transformation occurs only in certain bacterial,
such as E. coli, Pneumococcus, Neisseria,
Haemophilus, and Bacillus.