Nucleic acid structure and function

1. NUCLEIC ACID
STRUCTURE AND
FUNCTION
Dr. Roshan Kumar Mahat
(PhD Medical Biochemistry)
Assistant Professor
Department of Biochemistry

2. DeoxyribonucleicAcid(DNA)

3. Structure of DNA
 DNA is a polymer of deoxyribonucleoside
monophosphates covalently linked by 3'→5'–
phosphodiester bonds.
 With the exception of a few viruses that contain
single-stranded (ss) DNA, DNA exists as a double
stranded (ds) molecule, in which the two strands
wind around each other, forming a double helix.

4.  In eukaryotic cells, DNA is found associated
with various types of proteins (known
collectively as nucleoprotein) present in the
nucleus, whereas in prokaryotes, the protein–
DNA complex is present in a non-membrane
bound region known as the nucleoid.

5. Purine and pyrimidine bases in DNA

6. Figure: Deoxyribose and ribose, the sugars of DNA and
RNA. The carbon atoms are numbered from 1 to 5.
When the sugar is attached to a base, the carbon atoms
are numbered from 1 to 5 to distinguish it from the
base. In deoxyribose, the X = H; in ribose, the X =OH.

7. 3'→5'-Phosphodiester bonds:
 Phosphodiester bonds join the 3'-hydroxyl group
of the deoxy pentose of one nucleotide to the 5'-
hydroxyl group of the deoxy pentose of an
adjacent nucleotide through a phosphate group.
 The resulting long, unbranched chain has polarity,
with both a 5'-end and a 3'-end that are not
attached to other nucleotides.

9.  Phosphodiester linkages between nucleotides
(in DNA or RNA) can be cleaved hydrolytically
by chemicals, or hydrolyzed enzymatically by a
family of nucleases: deoxyribonucleases for
DNA and ribonucleases for RNA. [Note: Only
RNA is cleaved by alkali.]

10. Concept of base paring:
Figure: Base pairs of DNA

11.  As a consequence of base pairing, the two
strands of DNA are complementary; that is,
adenine on one strand corresponds to
thymine on the other strand, and guanine
corresponds to cytosine.
Figure: Two complementary
DNA sequences.
 Hydrogen bonds, plus the
hydrophobic interactions
between the stacked bases,
stabilize the structure of
the double helix.

12.  Note: The specific base pairing in DNA leads to
the Chargaff Rule: In any sample of dsDNA,
the amount of adenine equals the amount of
thymine, the amount of guanine equals the
amount of cytosine, and the total amount of
purines equals the total amount of
pyrimidines.

13.  The concept of base pairing proved to be
essential for determining the mechanism of DNA
replication and the mechanisms of transcription
and translation.
 Base pairing allows one strand of DNA to serve as
a template for the synthesis of the other strand.
 Base pairing also allows a strand of DNA to serve
as a template for the synthesis of a
complementary strand of RNA.

14. FIG: DNA strands serve as templates.
During replication, the strands of the helix
separate in a localized region. Each
parental strand serves as a template for
the synthesis of a new DNA strand.

15. DNA Strands Are Antiparallel:
Fig: Antiparallel strands of DNA.
 This concept of directionality
of nucleic acid strands is
essential for understanding the
mechanisms of replication and
transcription.

16. The Double Helix:
 Because each base pair contains a purine
bonded to a pyrimidine, the strands are
equidistant from each other throughout.
 If two strands that are equidistant from each
other are twisted at the top and the bottom,
they form a double helix.
 In the double helix of DNA, the base pairs that
join the two strands are stacked like a spiral
staircase along the central axis of the
molecule.

17.  The spatial relationship between the two strands
in the helix creates a major (wide) groove and a
minor (narrow) groove.
 These grooves provide access for the binding of
regulatory proteins to their specific recognition
sequences along the DNA chain.
Note: Certain anticancer drugs, such as dactinomycin (actinomycin D),
exert their cytotoxic effect by intercalating into the narrow groove
of the DNA double helix, thus interfering with DNA and RNA
synthesis.

18. Fig: Two DNA strands twist to form a
double helix.

19. Structural forms of double helix
 The DNA molecule exist in various forms
depending on the base composition and
physical conditions:
1. The A form
2. the B form, described by Watson and Crick
3. The C form
4. The D form
5. the E form, and
6. the Z form.

20. Property A-DNA B-DNA Z-DNA
Type of helix Right handed Right handed Left handed
Base pairs/turn 11 10 12
Distance between
adjacent bases
2.3 A0 3.4 A0 3.8 A0
Helix pitch 28.6 A0 34 A0 44.4 A0
Helical diameter 23 A0 20 A0 18 A0
Major groove Narrow and very
deep
Wide and deep Wide and flat
Minor groove Wide and shallow Narrow and deep Very narrow
Comparison of A-DNA, B-DNA and Z-DNA

21. Figure: Z, B, and A forms of DNA. The solid red lines connect one phosphate
group to the next.

22. Denaturation of DNA
 The term denaturation refers to disruption of
native conformation of a biomolecule, so that
it does not retain its higher order structure.
 In case of DNA, denaturation refers to
separation of the double strands of DNA into
two complementary strands.
 The double-stranded structure of DNA can be
separated into two component strands in
solution by increasing the temperature or
decreasing the salt concentration.

23. Melting temperature:
• The strands of a given molecule of DNA
separate over a temperature range.
• The midpoint is called the melting
temperature, or Tm.
• When DNA is heated, the temperature at
which one half of the helical structure is
lost is defined as the melting temperature
(Tm).

24.  The Tm is influenced by the base composition
of the DNA and by the salt concentration of
the solution.
 At the physiological pH and ionic strength, the
melting temperature of DNA is between 850C
to 950C.
 DNA rich in G–C pairs, which have three
hydrogen bonds, melts at a higher
temperature than that rich in A–T pairs, which
have two hydrogen bonds.
 For every 10% increase in GC content, the
melting temperature increases by 50C.

25. Monitoring of strand separation:
 Denaturation affects properties of DNA and
these can be used to monitor strand
separation.
1. Hyperchromic effect: the heterocyclic DNA
bases absorb UV light of wavelength 260nm.
Absorbance of this light increases by 40 %
upon denaturation. This is called
hyperchromic effect.
2. Viscosity: viscosity of DNA solution decreases
on melting because the single strands are far
more flexible than the stiff, resilient double
helix.

26. Figure: Melting temperatures (Tm) of DNA molecules with different
nucleotide compositions. (At a wavelength of 260 nm, single-
stranded DNA has a higher relative absorbance than does double-
stranded DNA.)

27. Denatured DNA can undergo renaturation:
 Under appropriate conditions, complementary
DNA strands can reform the double helix by
the process called renaturation (or
reannealing).
 It is a rapid process, even the large DNA
molecules make take few seconds to minutes
for renaturation.

28. Figure: Reversible denaturation and annealing (renaturation) of DNA.

29.  The maximum buoyant density of DNA is
1.70±0.01.
 The DNA molecule having higher GC
content have more compact structure
and hence greater buoyant density.
 For every 10% increase in the GC
content, the density rises by 0.12 units.
Buoyant density:

30. RibonucleicAcid(RNA)

31. General features of RNA
 RNA is a polymer of purine and pyrimidine
ribonucleotides linked together by 3′,5′-
phosphodiester bonds analogous to those in
DNA.

32.  Although sharing many features with DNA,
RNA possesses several specific differences:
1. In RNA, the sugar moiety to which the
phosphates and purine and pyrimidine bases are
attached is ribose rather than the 2′-deoxyribose
of DNA.
2. Although RNA contains the ribonucleotides of
adenine, guanine, and cytosine, it does not
possess thymine except in the rare case. Instead
of thymine, RNA contains the ribonucleotide of
uracil.

33. 3. RNA typically exists as a single strand. However,
given the proper complementary base sequence
with opposite polarity, the single strand of RNA
is capable of folding back on itself like a hairpin
and thus acquiring double-stranded
characteristics: G pairing with C, and A pairing
with U.

34. Figure: Diagrammatic
representation of the
secondary structure of a
single-stranded RNA
molecule in which a stem
loop, or “hairpin,” has been
formed.

35. 4. Since the RNA molecule is a single strand
complementary to only one of the two strands
of a gene, its guanine content does not
necessarily equal its cytosine content, nor does
its adenine content necessarily equal its uracil
content.
5. RNA can be hydrolyzed by alkali to 2′, 3′ cyclic
diesters of the mononucleotides, compounds
that cannot be formed from alkali-treated DNA
because of the absence of a 2′-hydroxyl group.
The alkali lability of RNA is useful both
diagnostically and analytically.

36. Types of RNA
 In all prokaryotic and eukaryotic organisms,
three main classes of RNA molecules exist:
1. Messenger RNA(m RNA)
2. Transfer RNA (t RNA)
3. Ribosomal RNA (r RNA)
 The other are:
1. small nuclear RNA (SnRNA),
2. micro RNA(mi RNA) and
3. small interfering RNA(Si RNA) and
4. heterogeneous nuclear RNA (hnRNA).

37. Ribosomal RNA
 rRNAs are found in association with several
proteins as components of the ribosomes—
the complex structures that serve as the sites
for protein synthesis.
 There are three distinct size species of rRNA
(23S, 16S, and 5S) in prokaryotic cells.
 In the eukaryotic cytosol, there are four rRNA
species (28S, 18S, 5.8S, and 5S).
 Together, rRNAs make up about 80% of the
total RNA in the cell.

38. Figure: Comparison of prokaryotic and eukaryotic
ribosomes. The cytoplasmic ribosomes of eukaryotes are
shown. Mitochondrial ribosomes are similar to prokaryotic
ribosomes, but they are smaller (55S rather than 70S).

39. Transfer RNA
 tRNAs are the smallest (4S) of the three major
types of RNA molecules.
 tRNAs serve as adapters for the translation of
the information in the sequence of
nucleotides of the mRNA into specific amino
acids.
 There are at least 20 species of tRNA
molecules in every cell, at least one (and often
several) corresponding to each of the 20
amino acids required for protein synthesis.

40.  The primary structure—that is, the nucleotide
sequence—of all tRNA molecules allows
extensive folding and intrastrand
complementarity to generate a secondary
structure that appears in two dimensions like
a cloverleaf.
 tRNAs make up about 15% of the total RNA in
the cell.
 The tRNA molecules contain a high percentage
of unusual bases

41.  tRNA has 5 arms:
1. Acceptor arm- for the attachment of amino acid to form
aminoacyl tRNA.
2. D arm
3. TΨC arm
4. Variable arm
5. Anticodon arm: contain sequence of 3 bases that
are complementary to codon mRNA.
Help define a specific tRNA

42. Figure: The tRNA cloverleaf

43. Figure: The three-dimensional folding of tRNA.

44. Messenger RNA
 This class is the most heterogeneous in
abundance, size and stability.
 mRNA comprises only about 5% of the RNA in
the cell.
 All members of this RNA class function as
messengers conveying the information in a
gene to the protein-synthesizing machinery,
where each mRNA serves as a template on
which a specific sequence of amino acids is
polymerized to form a specific protein
molecule, the ultimate gene product.

45. Figure: The expression of genetic information in DNA into the form of an
mRNA transcript with 5’ to 3’ polarity shown.

46.  Eukaryotic mRNA consists of a leader sequence at
the 5-end, a coding region, and a trailer sequence
at the 3-end.
• The leader sequence begins with a guanosine cap
structure at its 5-end.
• The coding region begins with a trinucleotide
start codon that signals the beginning of
translation, followed by the trinucleotide codons
for amino acids, and ends at a termination signal.

47.  The trailer sequence terminates at its 3-end with a
poly(A) tail that may be up to 200 nucleotides long.
 Most of the leader sequence, all of the coding region,
and most of the trailer are formed by transcription of
the complementary nucleotide sequence in DNA.
 However, the terminal guanosine in the cap structure
and the poly(A) tail do not have complementary
sequences; they are added after transcription has
been completed (posttranscriptionally).

48. Figure: The regions of eukaryotic mRNA. The wavy line
indicates the polynucleotide chain of the mRNA and the A’s
constituting the poly(A) tail. The 5-cap consists of a
guanosine residue linked at its 5-hydroxyl group to three
phosphates, which are linked to the 5-hydroxyl group of the
next nucleotide in the RNA chain (a 5–5 triphosphate
linkage). The start and stop codons represent where protein
synthesis is initiated and terminated from this mRNA.

49. Figure: The cap structure attached to the
5’ terminal of most eukaryotic messenger
RNA molecules.

50.  The cap is involved in the recognition of mRNA by
the translation machinery, and also helps stabilize
the mRNA by preventing the nucleolytic attack by
5′-exonucleases.
 The poly(A) “tail” at the 3′-hydroxyl terminal of
mRNAs maintains the intracellular stability of the
specific mRNA by preventing the attack of 3′-
exonucleases and also facilitate translation.

51. Small RNA
 A large number of discrete, highly conserved,
and small RNA species are found in eukaryotic
cells; some are quite stable.
 Most of these molecules are complexed with
proteins to form ribonucleoproteins and are
distributed in the nucleus, the cytoplasm, or
both.
 They range in size from 20 to 1000 nucleotides
and are present in 100,000 to 1,000,000
copies per cell, collectively representing ≤5%
of cellular RNA.

52. Table: Some of the Species of Small-Stable RNAs Found in Mammalian Cells

53. Small Nuclear RNAs (snRNAs)
 snRNAs, a subset of the small RNAs, are
significantly involved in mRNA processing
(splicing) and gene regulation.
 Of the several snRNAs, U1, U2, U4, U5, and U6
are involved in intron removal and the
processing of hnRNA into mRNA.
 The U7 snRNA is involved in production of the
correct 3' ends of histone mRNA—which lacks
a poly(A) tail.

54. Micro RNAs (miRNAs) and
Small Interfering RNAs (siRNAs)
 These two classes of RNAs represent a subset
of small RNAs; both play important roles in
gene regulation.
 miRNAs and siRNAs cause inhibition of gene
expression by decreasing specific protein
production by targeting mRNAs through one
of several distinct mechanisms

55. Micro RNAs (miRNAs)
 miRNAs are typically 21–25 nucleotides in
length and are generated by nucleolytic
processing of the products of distinct
genes/transcription units.
 The small processed mature miRNAs typically
hybridize, via the formation of imperfect RNA-
RNA duplexes within the 3'- untranslated
regions of specific target mRNAs, leading to
translation arrest.

56. microRNAs, short non-coding RNAs present in all living
organisms, have been shown to regulate the expression of
at least half of all human genes. These single-stranded RNAs
exert their regulatory action by binding messenger RNAs
and preventing their translation into proteins.

57. Small Interfering RNAs (siRNAs)
 siRNAs are generated by the specific
nucleolytic processing of large dsRNAs that
are either produced from other endogenous
RNAs, or dsRNAs introduced into the cell, by
for example, RNA viruses.
 These short siRNAs usually form perfect RNA-
RNA hybrids with their distinct targets
potentially anywhere within the length of the
mRNA where the complementary sequence
exists.

58.  Formation of such RNA-RNA duplexes
between siRNA and mRNA results in
reduced specific protein production
because the siRNA-mRNA complexes are
degraded by dedicated nucleolytic
machinery.
 some or all of this mRNA degradation
occurs in specific organelles termed P
bodies.

59. Small interfering RNA (siRNA) are 20-25 nucleotide-long double-
stranded RNA molecules that have a variety of roles in the cell.
siRNA starts off as a double strand RNA, is processed by Dicer and
the RISC and then binds to its target mRNA leading to
degradation and 'knockdown' of gene expression.

60. Significance of miRNAs and siRNAs
 Both miRNAs and siRNAs represent exciting
new potential targets for therapeutic drug
development in humans.
 In addition, siRNAs are frequently used to
decrease or "knock-down" specific protein
levels in experimental procedures in the
laboratory, an extremely useful and
powerful alternative to gene-knockout
technology.

61. Sr. No. RNA DNA
1. Single stranded mainly
except when self
complementary sequences
are there it forms a double
stranded structure (Hair pin
structure)
Double stranded (Except for
certain viral DNAs which are
single stranded)
2. Ribose is the main sugar The sugar moiety is deoxy
ribose
3. Pyrimidine components
differ. Thymine is never
found(Except tRNA)
Thymine is always there but
uracil is never found
4. Being single stranded
structureIt does not follow
Chargaff’s rule
It does follow Chargaff's rule.
Difference between RNA and DNA

62. Sr. No. RNA DNA
5. RNA can be easily destroyed
by alkalies to cyclic diesters
of mono nucleotides.
DNA resists alkali action due
to the absence of OH group at
2’ position
6. RNA is a relatively a labile
molecule, undergoes easy
and spontaneous
degradation
DNA is a stable molecule. The
spontaneous degradation is
very 2 slow. The genetic
information can be stored for
years together without any
change.
7. Mainly cytoplasmic, but also
present in nucleus (primary
transcript and small nuclear
RNA)
Mainly found in nucleus, extra
nuclear DNA is found in
mitochondria, and plasmids
etc
8. The base content varies
from 100- 5000. The size is
variable.
Millions of base pairs are
there depending upon the
organism

63. Sr. No. RNA DNA
9. There are various types of RNA –
mRNA, r RNA, t RNA, Sn RNA, Si
RNA, mi RNA and hn RNA. These
RNAs perform different and
specific functions.
DNA is always of one type and
performs the function of
storage and transfer of genetic
information.
10. No variable physiological forms of
RNA are found. The different
types of RNA do not change their
forms
There are variable forms of
DNA (A to E and Z)
11. RNA is synthesized from DNA, it
can not form DNA(except by the
action of reverse transcriptase). It
can not duplicate (except in
certain viruses where it is a
genomic material )
DNA can form DNA by
replication, it can also form
RNA by transcription.
12. Many copies of RNA are present
per cell
Single copy of DNA is present
per cell.