Download 3.1.1 Pentose sugars

Principles of Genetic Engineering
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3. Sructure and synthesis of DNA and RNA
3.1 Nucleic acids structure
When a German chemist, Friedrich Miescher, isolated ‟nuclein” from
human cells and discovered DNA in 1869( only four years after Mendel’s
work) , he did not realise the full importance of this component of cells.
Since this component was found to be acidic, it was renamed as nucleic
acid.
Biochemists like Levene then analysed the constitution of the nucleic
acids. It was found that the nucleic acids had three major constituents,
namely pentose sugar, nitrogenous bases and phosphate moiety.
3.1.1 Pentose sugars
There are two types of pentose sugars found in the nucleic acids, the
ribose sugar and the deoxyribose sugar (Fig 3.1).
Figure 3.1 Nucleic acids pentose sugars
Based on this observation, the nucleic acids have been called as ribose
nucleic acid (RNA) and deoxyribose nucleic acid (DNA) respectively.
These sugars actually form the back bone of the nucleic acids.
3.1.2 Nitrogenous bases
There are two groups of nitrogenous bases present in the nucleic acids.
Pyrimidines are six member carbon and nitrogen ring structures, while
purines are nine member carbon and nitrogen ring structures (Fig 3.2c).
Figure 3.2 Nucleic acids nitrogenous bases
Three species of pyrimidines generally found in nucleic acids, they are
(1) Uracil (2) Cytosine and (3) Thymine. Two species of purines
generally found in the nucleic acids, they are (1) Guanine and (2)
Adenine. One nitrogenous base is linked to first carbon of one pentose
sugar. This is then called as the nucleoside. When a phosphate group is
attached to carbon 5 of the nucleoside, then it is called as nucleotide
(figure 3.2a).
3.1.3 The fundamental building block of DNA
The nucleotide consists of a phosphate joined to a sugar, known as
2′-deoxyribose, to which a base is attached (figure 3.3).The sugar is
called 2′-deoxyribose because there is no hydroxyl at position 2′ (just two
hydrogens).
The base is joined to the 2′-deoxyribose by removal of a molecule of
water between the hydroxyl on the 1′ carbon of the sugar and the base to
form a glycosidic bond (figure 3.3).
Figure 3.3 Formation of Nucleotide by Removal of Water. The numbers
of the carbon atoms in 2’ deoxyribose are labeled in red.
Thus, by making a glycosidic bond between the base and the sugar,
and by making a phosphoester bond between the sugar and the
phosphoric acid, we have created a nucleotide. Phosphodiester linkage
create the repeating, sugar-phosphate backbone of the polynucleotide
chain, which is a regular feature of DNA.
Thus, a nucleotide containing ribose sugar, adenine base and
triphosphate is called as ribose adenosine triphosphate, (ATP) or
adenylate (figure 3.4), and the one containing deoxyribose sugar, adenine
base and triphosphate is called as deoxyribose adenosine triphosphate
(dATP) or deoxyadenylate. Depending upon the number of phosphates
attached to carbon 5 of the pentose sugar, these may be monophosphates,
diphosphates or triphosphates.
The nucleotides have many functions in the cell. They act as chemical
energy storage devices. They may also act as co-enzymes. When
polymerised, they produce nucleic acids.
Figure 3.4 Adenosine triphosphate (ATP) simply structure
3.2 Polynucleotide
When a series of nucleotides are linked to produce an un-branched
chain of nucleotides, it is called a polynucleotide. Two successive
nucleotides are linked through a phosphodiester bond formed between the
3′ OH group of one nucleotide and the 5′ phosphate group of the second
nucleotide (Fig 3.5). The two successive nucleotides are not in the same
plane and are positioned at an angle of twist with respect to each other.
This results in a helical structure of the polynucleotide chain. This angle
of twist may vary resulting into different conformations of the DNA.
Figure 3.5 Nucleic acids Polynucleotide
3.4 DNA double helix
Although single stranded DNA as genetic material is also known,
bulk of the DNA in a living cell is double stranded. Thus there are two
anti-parallel strands held together with the help of hydrogen bonds
between pairs of nitrogenous bases. The A=T base pair forms two
hydrogen bonds while the G≡C base pair forms three hydrogen bonds
(Fig 3.5). The double helical DNA structure described by Watson and
Crick. Since A pairs with T and G pairs with C, the two strands become
complementary to each other. Since the two strands are held together with
weak hydrogen bonds, they can easily separate out and become single
stranded. If a solution of double stranded DNA is gradually heated, there
is a thermal disruption of hydrogen bonds, converting the double stranded
structure into two single stranded entities. This is referred to as melting or
denaturation. Similarly, alkaline pH also disrupts hydrogen bonds.
Naturally, depending upon the number of GC base pairs and AT base
pairs present, different double stranded DNAs would have different
melting temperatures. Higher the ratio of GC base pairs, larger is the
number of hydrogen bonds and hence higher would be the melting
temperature. This property is utilized in characterizing DNAs of different
organisms. Higher GC percentage tends to better radio-protection. On the
other hand AT rich regions in DNA are often involved in initiation of
denaturation when the DNA needs to be opened up, as in case of DNA
replication.
The bio-physical parameters defined for Watson and Crick model are not
exclusive. The DNA is a dynamic molecule that keeps on acquiring
different conformations or three dimensional structures. The Watson and
Crick structure pertains to the B conformation (Fig 3.6a).
Depending on the angle of twist and its direction (negative or positive),
the DNA may become left handed helix or right handed helix. Bulk of the
DNA is right handed. However, at certain GC rich or AT rich regions, the
DNA may acquire left handed conformation (Fig 3.6c).
While DNA is more versatile and can acquire several conformations,
RNA is restricted by the presence of –OH group at second carbon of
ribose sugar. Hence it is not able to acquire B conformation. Therefore,
when DNA and RNA have to interact, it is the DNA that acquires A
conformation. Otherwise, it generally stays in B conformation, which
seems to be energetically most favorable conformation of DNA.
Relative humidity and ionic compositions significantly affect the
conformations and become the determining factors for the conformation
acquired by the DNA. The living system is able to manipulate the
conformation acquired by the DNA according to its needs. It seems the
acquisition of Z conformation could help DNA unwind during various
processes.
Figure 3.6 DNA three dimensional structures
DNA may be linear or circular. Circular double stranded DNA is
found in lower life forms such as bacteria and in cell organelles. Higher
life forms tend to have linear nuclear DNA. Circularisation of DNA
makes it less prone to damage, but has its own problems at the time of
separation of the two strands during replication or transcription.
In higher life forms the DNA tends to get stabilized with the help of
associated proteins. It is for this reason that the chromosomes of higher
life forms have a higher order of organization.
3.5 RNA structure
Like DNA, RNA also is a polynucleotide . However, it is comprised
of only ribose containing nucleotides. Most of the RNA is single
stranded. However, small interfering RNAs (siRNA) are double stranded.
The RNA is right handed coiled and predominantly stays in A
conformation. RNA mostly contains uracil in place of thymine.
3.5.1 RNA types and functions
DNA is involved in two major functions, namely conservation and
transmission of genetic information. On the other hand, the RNA has a
number of varied functions in the living system. To name a few:
(1) RNA brings genetic information from the DNA for protein synthesis.
(2) It helps in the process of protein synthesis by helping in association of
ribosomes with the messenger molecule.
(3) It functions as carrier of amino-acids.
(4) It plays a significant role in the preparation of messenger molecule.
(5) It is involved in post-transcriptional editing of genetic message.
(6) It is involved in the process of translation.
RNA is classified according to the function in which it is involved. For
example, the RNA involved in the first function above is aptly called as
messenger RNA (mRNA). The one involved in second function is called
as ribosomal RNA (rRNA). The RNA involved in carriage of amino acids
is called as transfer RNA (tRNA). Small nuclear RNA (snRNA) are
involved in preparation of messenger RNA, while guide RNA (gRNA) is
involved in post-transcriptional alteration in the genetic message
(RNA editing). The RNA species involved in a given function tends to
have its own structural peculiarities.
3.5.1.1 mRNA
Messenger RNA is one of the most unstable molecules of life.
At the same time it is one of the most significant molecules. The genetic
message stored in the DNA is passed on to the site of protein synthesis in
the form of messenger RNA. Therefore, at any given time, there are
hundreds of different messenger RNA molecules present in the cell, one
for each genetic message. The half life of different mRNAs varies from
minutes to days. In prokaryotes, often intact mRNA may not be present at
all. While on one end it may be under synthesis, its degradation at the
other end may start. This is possible because the site of transcription and
translation are not separated by any membrane. In eukaryotes, the site of
transcription is within nucleus, while protein synthesis occurs outside the
nucleus. Thus the half life of the eukaryotic messenger RNA is
prolonged. This is achieved by several structural modifications that take
place after RNA transcription .
Structurally, the prokaryotic mRNA is simplest. It is single stranded
linear molecule. Its size varies from gene to gene. It tends to be a perfect
complement of the DNA template used for its synthesis. On the other
hand, the eukaryotic mRNA is rarely a perfect complement of the DNA
template. It has a specialized structure at the 5′ end, called as the cap. It
has a long tail of A residues at the 3′ end, called as the polyA tail
(Fig. 3.7). These structural features are involved in transport of the
mRNA across the nuclear membrane, recognition of mRNA by the
protein synthesizing machinery and increased half life of the mRNA.
However, like prokaryotic mRNA, even eukaryotic mRNA is a single
stranded linear molecule.
Figure 3.7 mRNA structure
3.5.1.2 rRNA
As the name suggests, the rRNA is the RNA found as integral part of
the ribosome. There are three types of ribosomal RNA. One of these is
found in the small sub-unit of the ribosome. In prokaryotes it is about
1500 bases long and tends to sediment at 16S (S is called as Svedberg's
coefficient after the inventor of ultracentrifuge and represents size; larger
the value of S, larger is the size of sedimenting entity during
centrifugation). Therefore, it is also called as the 16S rRNA. In
eukaryotes, it tends to sediment at 18S and hence called as 18S rRNA.
The small sub-unit rRNA undergoes secondary folding due to internal
hydrogen bond formation (Fig 3.8).
Figure 3.8 Prokaryotic and Eukaryotic rRNA structures
This folding leads to formation of several functional domains. For
example the 3′ domain is involved in recognition of mRNA for formation
of correct small sub-unit ribosome-mRNA complex during initiation of
protein synthesis in prokaryotes. Like small sub-unit rRNA, the larger
sub-unit of ribosome has two to three rRNA species associated with it.
One of these is the 23S rRNA in prokaryotes and 28S rRNA in
eukaryotes. These also undergo folding in the same manner as the
16S/18S rRNA. They also add to the function of the larger subunit of
ribosome. For example, this rRNA species is involved in relative
movement of the mRNA and ribosome during protein synthesis. The
smaller rRNAs sediment at about 5S/5.8S. They are associated with the
larger sub-unit of the ribosome.
3.5.1.3 tRNA
There are about 22 different transfer RNA molecules ranging between
70-80 bases in length and sedimenting at about 4S. Each transfer RNA
specializes in carrying a specific amino acid to the site of protein
synthesis. However, except for minor differences, all tRNAs have a
generalized three dimensional structure. The three dimensional structure
is generated by two orders of internal hydrogen bonding. The first order
generates a transient secondary structure that might resemble a clover leaf
(Fig 3.9). In a typical clover leaf secondary form, the tRNA has four
specific arms. The first one is the acceptor arm having a CCA tail at the 3′
end. This domain of the tRNA that forms covalent linkage with the
amino-acid for carrying it to the site of protein synthesis. The second arm
is called the D-arm, which has a D-loop and a D-stem. This arm is
involved in the second order of hydrogen bond formation with some
bases in the third arm, the T arm. The fourth arm is called as anticodon
arm because it has the three bases making up the anticodon that pairs with
the codons on the mRNA at the time of protein synthesis. It is for this
reason that tRNA is also called as translational adaptor (Fig 3.9).
Figure 3.9 tRNA structure