DNA, RNA, GEN DAN

KROMOSOM

Dr. dr. Mgs. Irsan Saleh, M.Biomed

Bagian Farmakologi Fakultas Kedokteran
Universitas Sriwijaya
Palembang
 Module
description
Analysis of each cellular
component in the human body to
the molecular level in order
study pathogenesis of disease,
diagnosis diseases, develop
treatment strategies including
develop new modalities of
treatment

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 Nucleoside
Nucleotide
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Pentose
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 Pentose
The chemical structure of pentose which
contains five carbon atoms, labeled as
C1' to C5'.
The pentose is called RIBOSE in RNA and
DEOXYRIBOSE in DNA, because the DNA's
pentose lacks an oxygen atom at C2'.
Recalling that RNA stands for
"ribonucleic acid", and DNA for
"deoxyribonucleic acid".

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 Nucleotide
Composed of three parts: PENTOSE,
BASE, and PHOSPHATE GROUP.
In DNA or RNA, a pentose is
associated with only one phosphate
group, but a cellular free nucleotide
(such as ATP) may contain more than
one phosphate group.
If all phosphate groups are removed,
a nucleotide becomes a
NUCLEOSIDE. 8
 Bases of Nucleic
acids
DNA RNA
Adenine Adenine
Guanine Guanine
Cytosine Cytosine
Thymine Uracil

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Deamination may change the cytosine to uracil, or the methylated cytosine to
thymine.

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 Why has DNA Thymine and
RNA Uracil ?
Cytosine is one of four bases in DNA molecules.
Cytosine may be mutated to uracil by
deamination.
Since uracil is not part of DNA, this mutation
can easily be detected and repaired by base
excision.
Suppose DNA, like RNA, were made up of
uracil, then the cytosine to uracil mutation
could be corrected only by mismatch repair
which is very inefficient.
This may explain why DNA chooses thymine,
instead of uracil, even though the chemical
structure of uracil is simpler than thymine.

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 Pairing of DNA
strands
A DNA molecule has two strands, held
together by the hydrogen bonding between
their bases.
Adenine can form two hydrogen bonds with
thymine; cytosine can form three hydrogen
bonds with guanine.
Although other base pairs [e.g., (G:T) and (C:T)
] may also form hydrogen bonds, their
strengths are not as strong as (C:G) and (A:T)
found in natural DNA molecules.
Due to the specific base pairing, DNA's two
strands are complementary to each other the
nucleotide sequence of one strand determines
the sequence of another strand.
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B form, the helix
makes a turn
every 3.4 nm, and
the distance
between two
neighboring base
pairs is 0.34 nm.
Hence, there are
about 10 pairs per
turn. The
intertwined
strands make two
grooves of
different widths,
referred to as the
MAJOR GROOVE
and the MINOR
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GROOVE, which
 Z form, because its bases
The normal right-
seem to zigzag. Z DNA is
handed "double helix"
left-handed. One turn spans
structure of DNA, also
4.6 nm, comprising 12 base
known as the B form.
pairs. The DNA molecule
with alternating G-C
sequences in alcohol or high
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salt solution tends to have
Most cellular RNA molecules are single
stranded. They may form secondary structures
such as stem-loop and hairpin.
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Classes of RNA
The major role of RNA is to
participate in protein
synthesis, which requires
three classes of RNA:
messenger RNA (mRNA)
transfer RNA (tRNA)
ribosomal RNA (rRNA)
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The secondary structure of
tRNA.
Blue color indicates
modified nucleotides, with
"m" representing
"methylated".
Anticodon is the
trinucleotides
complementary to a codon
on mRNA.

tRNA
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 tRNA
The major role of tRNA is to translate mRNA
sequence into amino acid sequence.
A tRNA molecule consists of 70-80 nucleotides.
Some nucleotides in tRNA have been modified,
such as dihydrouridine (D),pseudouridine (),
and inosine (I).
In dihydrouridine, a hydrogen atom is added to
each C5 and C6 of uracil.
In pseudouridine, the ribose is attached to C5,
instead of the normal N1.
Inosine plays an important role in codon
recognition. In addition to these modifications,
a few nucleosides are methylated.

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 Translation
Translation is carried out by tRNA through the
relationship between its anticodon and the
associated amino acid.
When a tRNA is brought to the ribosome by the
pairing between its anticodon and the mRNA's
codon, the amino acid attached at its 3' end will be
added to the growing peptide.
In bacteria, there are 30-40 tRNAs with different
anticodons.
In animal and plant cells, about 50 different tRNAs
are found.
However, there are 61 codons coded for amino
acids. Suppose each codon can pair with only a
unique anticodon, then 61 tRNAs would be needed.

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Wobble pairing
The reason why less than 61 tRNAs are
required is because of the "wobble
pairing" between anticodon and codon.
Base pairing does not obey the
standard Watson-Crick pairing at the
wobble position.
One base can pair with several other
bases. For example, guanine (G) can
pair with both cytosine (C) and uracil
(U) ; inosine (I) can pair with cytosine,
adenine and uracil.
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The non-
standard
base pairing
at the
wobble
position.

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 Aminoacyl-tRNA
The tRNA together with the amino acid
attached to its 3' end is called an
aminoacyl-tRNA.
The attached amino acid is encoded by
the codon which matches the tRNA's
anticodon.
The process is catalyzed by a class of
enzymes called aminoacyl-tRNA
synthetases, which recognize the
anticodon and its compatible amino acid.
A cell has 20 different aminoacyl-tRNA
synthetases, each can add only one of 20
amino acids to a compatible tRNA.
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 Notation of aminoacyl-
tRNA
"Arg-tRNA" denotes the tRNA
with arginine attached while
"tRNAArg" specifies the tRNA
which contains the anticodon
for the codon of arginine.
"Arg-tRNAArg" represents the
arginine-specific tRNA with
arginine attached.
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 Ribosomal RNA (rRNA)
(1)
In prokaryotes, the ribosomal RNA (rRNA) has three
types: 23S, 5S, and 16S.
In mammals, four types of rRNA have been found :
28S, 5.8S, 5S and 18S.
The unit "S" stands for Svedberg, which is a measure
of the sedimentation rate.
After rRNA molecules are produced in the nucleus,
they are transported to the cytoplasm, where they
combine with tens of specific proteins to form a
ribosome.
In prokaryotes, the size of a ribosome is 70S,
consisting of two subunits: 50S and 30S.
The size of a mammalian ribosome is 80S,
comprising a 60S and a 40S subunit.
Proteins in the larger subunit are designated as L1,
L2, L3, etc. (L = large). In the smaller subunit,
proteins are denoted by S1, S2, S3, etc. 29
 Ribosomal RNA (rRNA)
(2)
During protein synthesis, the ribosome binds to
mRNA and tRNA as shown in the following figure.
Only the tRNA containing the anticodon which
matches mRNA's codon may join the complex.

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 Other classes of
RNA
Ribozymes:
The RNA molecules with
catalytic activity.
They were discovered in early
1980s by Thomas Cech and
Sidney Altman who shared the
1989 Nobel Prize in Chemistry.
Small RNA molecules:
RNA interference and other
functions.
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 Major types of small RNA
molecules
Small nuclear RNA (snRNA) - involved in
mRNA splicing.
Small nucleolar RNA (snoRNA) - directs
the modification of ribosomal RNAs.
Micro-RNA (miRNA) - regulates gene
expression (only 20-25 nucleotides long):
It may be divided into two subtypes: small
interfering RNA (siRNA) and small temporally
regulated RNA (stRNA).
siRNA mediates RNA interference (RNAi)
which has been widely used in functional
genomics and also has therapeutic potential.

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 Chromatin
The substance which becomes visible
chromosomes during cell division.
Its basic unit is NUCLEOSOME, composed of 146
bp DNA and eight HISTONE proteins.
The structure of chromatin is dynamically
changing, at least in part, depending on the
need of transcription
In the metaphase of cell division, the chromatin
is condensed into the visible chromosome.
At other times, the chromatin is less condensed,
with some regions in a "BEADS-ON-A-STRING"
CONFORMATION.
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 Structure of chromatin
fibers
The structure of chromatin fibers is dynamically
changing.
In the condensed state, each nucleosome is
associated with an H1 (or H5) to form a
SOLENOID structure. H1 and H5 are called
LINKER HISTONES.
They are essential in stabilizing the solenoid
conformation. This can be demonstrated by
extracting the condensed chromatin at a low
salt concentration, which will release H1 or H5.
Then, the "beads-on-a-string" conformation
may be observed by the atomic force
microscopy
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Each
nucleosome
consists of
146 bp DNA
and 8
histones: two
copies for
each of H2A,
H2B, H3 and
H4.
The DNA is
wrapped
around the
histone core,
making nearly
two turns per
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nucleosome.
 Structure of
chromatin
a. The 30 nm chromatin fiber is associated with
scaffold proteins (notably topoisomerase II)
to form loops. Each loop contains about 75
kb DNA. Scaffold proteins are attached to
DNA at specific regions called scaffold
attachment regions (SARs), which are rich in
adenine and thymine.
b. The chromatin fiber and associated scaffold
proteins coil into a helical structure which
may be observed as a chromosome. G bands
are rich in A-T nucleotide pairs while R bands
are rich in G-C nucleotide pairs.

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 Histone Acetylation
(1)
Acetylation of the lysine residues at the
N terminus of histone proteins removes
positive charges, thereby reducing the
affinity between histones and DNA.
This makes RNA polymerase and
transcription factors easier to access the
promoter region.
Therefore, in most cases, HISTONE
ACETYLATION ENHANCES TRANSCRIPTION
while HISTONE DEACETYLATION
REPRESSES TRANSCRIPTION.
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 Histone Acetylation
(2)
Histone acetylation is catalyzed by
histone acetyltransferases (HATs) and
histone deacetylation is catalyzed by
histone deacetylases (denoted by HDs
or HDACs).
Several different forms of HATs and HDs
have been identified.
Among them, CBP/p300 is probably the
most important, since it can interact
with numerous transcription regulators.
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Acetylation and
deacetylation of
the lysine residue.

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 The standard genetic code
(1)
Genetic code was deciphered by Marshall
Nirenberg and his colleagues in early 1960s
Synthesis of a peptide always starts from
methionine (Met), coded by AUG.
The stop codon (UAA, UAG or UGA) signals
the end of a peptide.
In a DNA molecule, the sequence from an
initiating codon (ATG) to a stop codon (TAA,
TAG or TGA) is called an OPEN READING
FRAME (ORF), which is likely (but not
always) to encode a protein or polypeptide.

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 The standard genetic code
(2)
Amino acids with close physical properties have
similar codons. For example, both Asp and Glu
are negatively charged. Their codons all contain
"GA" in the first two positions. The physical
properties of Ser and Thr are also very close.
They are coded by UCN and ACN (N = any),
respectively.
One advantage for this assignment is to reduce
the damage caused by replication errors or other
mutations. For instance, if the third nucleotide of
Ser codon is mutated, the produced amino acid
remains Ser. If the first nucleotide of Ser codon is
mutated to A, it will produce Thr, which is similar
to Ser.
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 The standard genetic code
(3)
Such mutation will not have significant
effect on the protein's structure and
function.
Let us consider a hypothetical case that
codons are randomly assigned. Suppose
Ser were coded by GAG, GUA, GCU and
UGG; Thr were coded by AAA, AGU,
UUU and CCC. Then, any mutation
would produce an amino acid different
from Ser and Thr.
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 Cracking the genetic code
(1)
Approach used by Marshall Nirenberg and his
colleagues to crack the genetic code:
1. Synthesize a trinucleotide (e.g. UUU) which
mimics a codon in mRNA.
2. Prepare various types of aminoacyl-tRNA,
e.g., Thr-tRNA, Phe-tRNA, Lys-tRNA, etc.
3. Radioactively label an aminoacyl-tRNA (e.g.
Phe-tRNA) which might contain the
anticodon for the synthesized trinucleotide.
4. Place the trinucleotide, aminoacyl-tRNA
and ribosome on a nitrocellulose filter.

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 Cracking the genetic code
(2)
5. Individual trinucleotide and aminoacyl-tRNA can
pass through the filter, but the ribosome is too
big to pass through.
6. Therefore, if the labeled aminoacyl-tRNA contains
the anticodon for the trinucleotide, it will bind to
the trinucleotide and ribosome on the filter.
7. The radioactivity can be detected on the filter and
the amino acid in the labeled aminoacyl-tRNA is
likely to be encoded by the trinucleotide.
8. If no radioactivity was detected, the trinucleotide
is unlikely to be the codon of the amino acid.
Most of the 64 possible codons can be determined
by repeating this procedure for different
trinucleotides and labellings.

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The standard genetic code applies to most, but
not all, cases. Exceptions have been found in the
mitochondrial DNA of many organisms and in the
nuclear DNA of a few lower organisms.
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 Repetitive DNA
sequences
A stretch of DNA sequence often repeats
several times in the total DNA of a cell.
DNA reassociation kinetics is used to classify
the repetitive DNA sequences. The total DNA
is first randomly cleaved into fragments with
an average size of about 1000 bp. Then, they
are heated to separate the complementary
strands of each fragment. Subsequently,
temperature is reduced to allow strand
reassociation. If a fragment contains a
sequence which is repeated many times in the
total DNA, it will have greater chance to find
a complementary strand and reassociate
more quickly than other fragments with less
repetitive sequences.
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 A stretch of DNA sequence often
repeats several times in the total
DNA of a cell.
An entire telomere of each
human chromosome of about 15
kb, is constituted by thousands
of the repeated sequence
"GGGTTA".

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 Classes of repetitive DNA
sequences
Based on the reassociation rate, DNA sequences
are divided into three classes:
HIGHLY REPETITIVE: About 10-15% of
mammalian DNA fragments reassociate very
rapidly. This class includes tandem repeats.
MODERATELY REPETITIVE: Roughly 25-40% of
mammalian DNA fragments reassociate at an
intermediate rate. This class includes
interspersed repeats (also known as mobile
elements or transposable elements).
SINGLE COPY (OR VERY LOW COPY NUMBER):
This class accounts for 50-60% of mammalian
DNA.
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 Interspersed
repeats
Repeated DNA sequences located at dispersed
regions in a genome.
They are also known as MOBILE ELEMENTS or
TRANSPOSABLE ELEMENTS.
A stretch of DNA sequence may be copied to a
different location through DNA RECOMBINATION.
After many generations, such sequence (the
repeat unit) could spread over various regions.
Mobile elements were first discovered by Barbara
McClintock in 1940s from the studies of corn.
Subsequently, they were found in all kinds of
organisms. In mammals, the most common
mobile elements are LINEs (Long Interspersed
Nuclear Elements) and SINEs (Short Interspersed
Nuclear Elements).

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Basic organization of LINEs:
ORF1 and ORF2 are open reading
frames.
The protein product of ORF1 is called
p40, with unknown function.
ORF2 encodes a reverse transcriptase
which is necessary for copying the
element to other locations.
The red color regions on both ends are
direct repeats.
All mobile elements contain direct
repeats 56
Comparison between direct repeats and
inverted repeats:
Direct repeats have the same sequence
along a given direction.
Inverted repeats have complementary
sequences along opposite directions.
The most common LINEs in humans is
the L1 family.
A human genome contains about
60,000 to 100,000 L1 elements. 57
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