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Chemical Constituents

of Organisms: Part IV

DNA
Nucleic acids store and transmit hereditary information

The amino acid sequence of a polypeptide is


programmed by a unit of inheritance called a gene
Genes are made of DNA, a nucleic acid
DNA polymers are very long molecules
– DNA is supercoiled and bundled into 23 chromosomes
for packaging in the cell nucleus
The sequence of heterocyclic amine bases in DNA
encodes the genetic information required to
synthesize proteins
– Only four different bases are used for the code in DNA
– A section of DNA that encodes for a specific protein is
called a gene
– The set of all genetic information coded by the DNA in an
organism is its genome
– The set of all proteins encoded in the genome of an
organism and expressed at any given time is its
proteome
The sequence of the human genome is providing
valuable information related to human health
The Roles of Nucleic Acids
There are two types of nucleic acids:
– Deoxyribonucleic acid (DNA)
– Ribonucleic acid (RNA)
DNA provides directions for its own
replication
DNA directs synthesis of messenger RNA
(mRNA) and, through mRNA, controls
protein synthesis
Protein synthesis occurs in ribosomes

Copyright © 2008 Pearson Education, Inc., publishing as Pearson Benjamin Cummings


The Structure of Nucleic Acids
Nucleic acids are polymers called
polynucleotides
Each polynucleotide is made of monomers
called nucleotides
Each nucleotide consists of a nitrogenous
base, a pentose sugar, and a phosphate
group
The portion of a nucleotide without the
phosphate group is called a nucleoside

Copyright © 2008 Pearson Education, Inc., publishing as Pearson Benjamin Cummings


5 end
Nitrogenous bases
Pyrimidines
5 C

3 C

Nucleoside

Nitrogenous
base Cytosine (C) Thymine (T, in DNA) Uracil (U, in RNA)

Purines

Phosphate
group Sugar
5 C (pentose)
Adenine (A) Guanine (G)
3 C (b) Nucleotide

Sugars
3 end
(a) Polynucleotide, or nucleic acid

Deoxyribose (in DNA) Ribose (in RNA)

(c) Nucleoside components: sugars


5' end

5'C

3'C

Nucleoside

Nitrogenous
base

5'C

Phosphate 3'C
group Sugar
5'C (pentose)

3'C (b) Nucleotide

3' end
(a) Polynucleotide, or nucleic acid
Nitrogenous bases
Pyrimidines

Cytosine (C) Thymine (T, in DNA) Uracil (U, in RNA)

Purines

Adenine (A) Guanine (G)

(c) Nucleoside components: nitrogenous bases


Sugars

Deoxyribose (in DNA) Ribose (in RNA)

(c) Nucleoside components: sugars


Nucleotide Monomers
Nucleoside = nitrogenous base + sugar
There are two families of nitrogenous bases:
– Pyrimidines (cytosine, thymine, and uracil)
have a single six-membered ring
– Purines (adenine and guanine) have a six-
membered ring fused to a five-membered ring
• In DNA, the sugar is deoxyribose; in RNA,
the sugar is ribose
• Nucleotide = nucleoside + phosphate group

Copyright © 2008 Pearson Education, Inc., publishing as Pearson Benjamin Cummings


A nucleoside of RNA contain the sugar D-
ribose and one of the four bases adenine,
guanine, cytosine or uracil
Nucleosides that can be obtained from DNA
Nucleosides that can be obtained from RNA
Nucleotides can be named in several ways
– Adenylic acid is usually called AMP (adenosine
monophosphate)
– It can also be called adenosine 5’-monophosphate or 5’-
adenylic acid
Adenosine triphosphate (ATP) is an important energy
storage molecule
The molecule 3’,5’-cyclic adenylic acid (cyclic AMP)
is an important regulator of hormone activity
– This molecule is biosynthesized from ATP by the enzyme
adenylate cyclase
Laboratory Synthesis of Nucleosides and Nucleotides
Silyl-Hilbert-Johnson Nucleosidation
An N-benzoyl protected base reacts with a benzoyl
protected sugar in the presence of tin chloride and BSA (a
trimethylsilylating agent)
The trimethylsilyl protecting groups are removed with
aqueous acid in the 2nd step
The benzoyl groups can be removed with base
Unnatural nucleotide derivatives can be
synthesized from nucleosides bearing a
substitutable group on the heterocyclic ring
Dibenzyl phosphochloridate is a
phosphorylating agent for converting
nucleosides to nucleotides

The 5’-OH is phosphorylated selectively if


the 2’- and 3’-OH groups are protected
Nucleotide Polymers
Nucleotide polymers are linked together to
build a polynucleotide
Adjacent nucleotides are joined by covalent
bonds that form between the –OH group on
the 3′ carbon of one nucleotide and the
phosphate on the 5′ carbon on the next
These links create a backbone of sugar-
phosphate units with nitrogenous bases as
appendages
The sequence of bases along a DNA or
mRNA polymer is unique for each gene
Copyright © 2008 Pearson Education, Inc., publishing as Pearson Benjamin Cummings
The DNA Double Helix
A DNA molecule has two polynucleotides
spiraling around an imaginary axis, forming a
double helix
In the DNA double helix, the two backbones
run in opposite 5′ 3′ directions from each
other, an arrangement referred to as
antiparallel
One DNA molecule includes many genes
The nitrogenous bases in DNA pair up and
form hydrogen bonds: adenine (A) always with
thymine (T), and guanine (G) always with
cytosine (C)
Copyright © 2008 Pearson Education, Inc., publishing as Pearson Benjamin Cummings
Base Pairings
5'end 3'end

Sugar-phosphate
backbones

Base pair (joined by


hydrogen bonding)

Old strands

Nucleotide
about to be
added to a
new strand
3'end

5'end

New
strands

3'end 5'end

5'end 3'end
!
"#
Secondary Structure
The secondary structure of DNA was proposed by
Watson and Crick in 1953
E. Chargaff noted that in DNA the percentage of
pyrimidine bases was approximately equal to the
percentage of purine bases
Also the mole percentage of adenine Is nearly
equal to that of thymine
The mole percentage of guanine is nearly equal to
cytosine
Chargaff also noted that the ratio of A and T versus G
and C varies by species but the ratio is the same for
different tissues in the same organism
X-ray crystallographic data showed the bond lengths
and angles of purine and pyrimidine bases
X-ray data also showed DNA had a long repeat distance (34
Å)
Based on this data, Watson and Crick proposed the
double helix model of DNA (next slide)
Two nucleic acid chains are held together by hydrogen
bonding between the bases on opposite strands
The double chain is wound into a helix
Each turn in the helix is 34Å long and involves 10 successive
nucleotide pairs
Each base pair must involve a purine and a pyrimidine to
achieve the proper distance between the sugar-phosphate
backbones
Base pairing can occur only between thymine and adenine, or
cytosine and guanine; no other pairing has the optimum
pattern of hydrogen bonding or would allow the distance
between sugar-phosphate backbones to be regular
X-ray diffraction photograph of a DNA fibre
(Rosalind Franklin and Maurice Wilkins)

DNA has the form of a regular helix


The distance between adjacent
nucleotides is 3.4 Å
The helix makes one complete turn
every 34Å. It therefore follows that
there are 10 bp per turn
The diameter is 20Å
The density of DNA suggests the
helix must contain two polynucleotide
chains
Specific pairing of bases means the two chains of
DNA are complementary
–Knowing the sequence of one chain allows
one to also know the sequence of the other
Replication of DNA
The DNA strand begins to unwind just prior to
cell division
Complementary strands are formed along
each chain (each chain acts as a template for
a new chain)
Two new DNA molecules result; one strand
goes to each daughter cell
Absorption of UV light by nucleic acids and hyperchromicity

Nucleic acid bases are capable of absorbing UV light - the


sugar-phosphate backbone does not contribute greatly to absorption

Maximum absorption of light ( max) by DNA and RNA is 260nm, however,


the extinction coefficient depends on the environment of the bases. Therefore,
the absorbance at 260nm (A260) for nucleic acids is:

Isolated nucleotides > single stranded DNA or RNA > double stranded DNA

When DNA denatures, the A260 increases by ~40% - Hyperchromic effect


ELECTRON MICROGRAPHS OF CIRCULAR DUPLEX DNA
EXHIBITING VARIOUD DEGREES OF SUPERCOILING

Increasing supercoiling
Flow of Genetic Information

Transcription Translation
RNA and Protein Synthesis
“The central dogma of molecular genetics”

A gene is the portion of a DNA molecule which codes for


one protein
– Proteins have many critical functions, e.g., catalysis,
structure, motion, cell signaling, the immune response,
etc.
DNA resides in the nucleus and protein synthesis occurs in
the cytoplasm
– Transcription of DNA into messenger RNA (mRNA)
occurs in the nucleus
– mRNA moves to the cytoplasm and the translation into
proteins occurs using two other forms of RNA:
ribosomal RNA (rRNA) and transfer RNA (tRNA)
DNA

1 Synthesis of
mRNA in the
nucleus mRNA

NUCLEUS
CYTOPLASM
DNA

1 Synthesis of
mRNA in the
nucleus mRNA

NUCLEUS
CYTOPLASM

mRNA
2 Movement of
mRNA into cytoplasm
via nuclear pore
DNA

1 Synthesis of
mRNA in the
nucleus mRNA

NUCLEUS
CYTOPLASM

mRNA
2 Movement of
mRNA into cytoplasm Ribosome
via nuclear pore

3 Synthesis
of protein

Amino
Polypeptide acids
Transcription: Synthesis of Messenger RNA
(mRNA)
In the nucleus a DNA molecule partially unwinds
to expose a portion corresponding to at least one
gene
Ribonucleotides with complementary bases
assemble along the DNA strand
Base-pairing is the same in RNA, except that
in RNA uracil replaces thymine
Ribonucleotides are joined into a chain of mRNA
by the enzyme RNA polymerase
An intron (intervening sequence) is a segment of DNA
which is transcribed into mRNA but not actually used
when a protein is expressed
An exon (expressed sequence) in the part of the DNA
gene which is expressed
Each gene usually contains a number of introns and
exons
Introns are excised from mRNA after transcription
Ribosomes - rRNA
Protein synthesis is catalyzed in the cytoplasm by
ribosomes
–A ribosome consists of approximately two thirds
RNA and one third protein
–A ribosome is a ribozyme ( an reaction catalyst
made of ribonucleic acid)
A ribosome has 2 large subunits
–The 30S subunit binds the mRNA that codes for
the protein to be translated
–The 50S subunit catalyzes formation of the amide
bond in protein synthesis
Transfer of an amino acid to the growing peptide
chain is aided by acid-base catalysis involving an
adenine in the 50S subunits
Transfer RNA (tRNA)
Transfer RNAs (tRNAs), specific to each amino acid,
transport amino acids to complimentary binding sites on
the mRNA bound to the ribosome
–More than one tRNA codes for each amino acid
tRNA is comprised of a relatively small number of
nucleotides whose chain is folded into a structure with
several loops
–One arm of the tRNA always terminates in the
sequence cytosine-cytosine-adenine, and it is here the
amino acid is attached
–On another arm is a sequence of three bases called
the anticodon, which binds with the complementary
codon on mRNA
The mRNA genetic code is shown on the next slide
The Genetic Code
The genetic code is based on three-base sequences in
mRNA
Each three-base sequence corresponds to a particular
amino acid
–The fact that three bases are used to code for each
amino acid provides redundancy in the overall code
and in the start and stop signals
–N-formyl methionine (fMet) is the first amino acid
incorporated into bacterial protein and appears to be
the start signal
–fMet is removed from the protein chain before its
synthesis is complete
Translation
Translation is peptide synthesis by a ribosome
using the code from an mRNA
The polypeptide begins to acquire its secondary
and tertiary structure as it is being synthesized
Several ribosomes can be translating the same
mRNA molecule simultaneously
Protein molecules are synthesized only when they
are needed
–Regulator molecules determine when and if a
particular protein will be expressed i.e.
synthesized
Amplifying DNA in Vitro: The
Polymerase Chain Reaction (PCR)
The polymerase chain reaction, PCR, can
produce many copies of a specific target
segment of DNA
A three-step cycle—heating, cooling, and
replication—brings about a chain reaction
that produces an exponentially growing
population of identical DNA molecules
$ % &'() 5′′ 3′′
TECHNIQUE
Target
sequence

Genomic DNA 3′′ 5′′

1 Denaturation 5′′ 3′′

3′′ 5′′

2 Annealing

Cycle 1
yields Primers
2
molecules

3 Extension

New
nucleo-
tides

Cycle 2
yields
4
molecules

Cycle 3
yields 8
molecules;
2 molecules
(in white
boxes)
match target
sequence
$ % &'()

5′′ 3′′
TECHNIQUE
Target
sequence

Genomic DNA 3′′ 5′′


$ % &'()

1 Denaturation 5′′ 3′′

3′′ 5′′
2 Annealing

Cycle 1
yields Primers
2
molecules

3 Extension

New
nucleo-
tides
$ % &'()

Cycle 2
yields
4
molecules
$ % &'()

Cycle 3
yields 8
molecules;
2 molecules
(in white
boxes)
match target
sequence
Gel Electrophoresis
One indirect method of rapidly analyzing and
comparing genomes is gel electrophoresis
This technique uses a gel as a molecular
sieve to separate nucleic acids or proteins by
size
A current is applied that causes charged
molecules to move through the gel
Molecules are sorted into “bands” by their size
$ % &'(*
TECHNIQUE

Mixture of Power
DNA mol- source
ecules of – Cathode Anode +
different
sizes

Gel
1

Power
source
– +
Longer
molecules

2 Shorter
molecules
$ % &'(*

RESULTS
DNA and Proteins as Tape
Measures of Evolution
The linear sequences of nucleotides in DNA
molecules are passed from parents to
offspring
Two closely related species are more similar
in DNA than are more distantly related
species
Molecular biology can be used to assess
evolutionary kinship

Copyright © 2008 Pearson Education, Inc., publishing as Pearson Benjamin Cummings


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