Download DNA and RNA

DNA and RNA
Dr. Sugandhika Suresh
Department of Biochemistry
Features of the DNA double helix
1. 2 DNA strands per molecule
2. Right-handed helix
3. 2 chains are antiparallel
4. Sugars and phosphates –outside
5. Bases inside -- “stacked like pennies”
6. Bases are bonded together by H-bonds
7. Specific base pairings are observed - complementary
8. A pairs with T
9. C pairs with G
10. 10 base pairs per turn
11. Spacing causes a major and a minor groove
Two strands are twisted together around
a common axis
 Right handed

Right-Handed vs.
Left-Handed Helices

The helix is right-handed

As it spirals away from you, the helix turns in a clockwise
direction
Right- and Left-handed DNA

The two strands are antiparallel

One runs in the 5’ to 3’ direction and the other 3’ to
5’
• Deoxyribose phosphate backbone -hydrophilic
Antiparallel
DNA Can Form Alternative Types of
Double Helices

The DNA double helix can form different types of
secondary structure

The predominant form found in living cells is B-DNA

However, under certain in vitro conditions, A-DNA and
Z-DNA double helices can form
FORMS OF DNA
DNA forms
B-form
A-form
Z-form

A-DNA





Right-handed helix
11 bp per turn
Occurs under conditions of low humidity
Little evidence to suggest that it is biologically important
Z-DNA



Left-handed helix
12 bp per turn
Its formation is favored by



GG-rich sequences, at high salt concentrations
Cytosine methylation, at low salt concentrations
Evidence from yeast suggests that it may play a role in
transcription and recombination
Denaturation of DNA
Denaturation by
heating.
How is it observed?
The T at which ½ the DNA
sample is denatured is
called the melting
temperature (Tm)
A260
For dsDNA,
A260=1.0 for 50 µg/ml
For ssDNA and RNA
A260=1.0 for 38 µg/ml
For ss oligonuleotides
A260=1.0 for 33 µg/ml
Hyperchromic shift
If the temperature is
lowered, the strands
recombine.
The rate of reassociation is
inversely proportional to the
complexity of the DNA.
% Denatured
The two strands of the double helix separate
reversibly at high temperatures
The temperature at which
this “denaturation” or
100
“melting” occurs depends on
the pH and salt
80
concentration, and increases
with the GC content of the
60
DNA. (The curves drawn
here are schematic.)
40
40 50 60 70% GC
20
0
70
80
90
100
o
Temperature / C
110
Double-stranded and single-stranded DNA differ in their
optical absorption at 260 nm
dA
dG
dU
dC
The conjugated p-electron systems of
the purine & pyrimidine bases absorb
strongly in the UV.
(That’s why UV light is mutagenic and
carcinogenic.)
nucleotides
ssDNA
dsDNA
The absorbance of double-stranded
DNA (dsDNA) at 260 nm is less than
that of either single-stranded DNA
(ssDNA) or the free bases. This is called
“hypochromism.”
Importance of Tm
Critical importance in any technique that
relies on complementary base pairing
Designing PCR primers
Southern blots
Northern blots
Colony hybridization
Factors Affecting Tm
G-C content of sample
Presence of intercalating agents (anything that
disrupts H-bonds or base stacking)
Salt concentration
pH
Length
Renaturation
Strands can be induced to renature (anneal)
under proper conditions. Factors to consider:
Temperature
Salt concentration
DNA concentration
Time
DNA packaging in chromosomes
DNA wound around
histone proteins
Packaging DNA
Histone
octamer
Histone proteins
B DNA Helix
2 nm
Packaging DNA
Histone
octamer
Histone proteins
B DNA Helix
2 nm
Packaging DNA
11 nm
Histone
octamer
Histone proteins
Nucleosome
B DNA Helix
2 nm
DNA-histone octamer
H1 Links Nucleosomes together
Nucleosomes:
+H1
-H1
Packaging DNA
Packaging DNA
Packaging DNA
“Beads on
a string”
11 nm
30 nm
Tight helical
fibre
Looped
200 nm Domains
Protein scaffold
Packaging DNA
Nucleosomes
11 nm
30 nm
Tight helical fibre
Metaphase
Chromosome
700 nm
200 nm Looped Domains
2 nm
B DNA Helix
Protein scaffold
Chromosomes, Chromatids and
Centromeres
A packaged
chromosome
Chromatid
Identical
chromatid
Chromosome
arm
Centromere
Chromosome
arm
Two identical
chromosomes
Replication
Anaphase
RNA structure and function
Objectives
The differences between DNA and RNA
The structure and function of RNAs
RNA & DNA: Similarities
Both RNA & DNA:
Unbranched polymers
Polynucleotides
Contain phosphodiester bonds
RNA & DNA: Differences
RNA
DNA
•Single-Strand (mostly)
•Cytoplasm (mainly)
•AGCU
•Modified bases
•Ribose
•Protein Biosynthesis
•Post-transcriptional events
•Double
•Nucleus
•d AGCT
•Deoxyribose
•Storage &transfer
•DNA Repair
Biological roles of RNA
1. RNA is the genetic material of some viruses
2. RNA functions as the intermediate (mRNA)
between the gene and the proteinsynthesizing machinery.
3. RNA functions as an adaptor (tRNA)
between the codons in the mRNA and amino
acids.
4.Through sequence complementarity, RNA
serves as a regulatory molecule to bind to and
interfere with the translation of certain mRNAs; or as
a recognition molecule to guide many posttranscriptional processing steps.
5.Through the tertiary structures, some RNAs function as
enzymes to catalyze essential reactions in the cell
(RNase P ribozyme, large rRNA in ribosomes, selfsplicing introns, etc).
RNA Structure

The primary structure of an RNA strand is much
like that of a DNA strand

RNA strands are typically several hundred to
several thousand nucleotides in length

In RNA synthesis, only one of the two strands of
DNA is used as a template
Components unique to RNA
Replaces
Deoxyribose
Replaces
Thymine
40
RNA Primary Structure
(-e)
5'
Structure of
RNA backbone
(-e)
(-e)
(-e)
3'
• RNA chain directionality: 5'3'
• Backbone carries charge (-e) on each nucleotide
• Formation of an RNA structure requires cations

Although usually single-stranded, RNA molecules can
form short double-stranded regions

This secondary structure is due to complementary basepairing



This allows short regions to form a double helix
RNA double helices typically



A to U and C to G
Are right-handed
Have the A form with 11 to 12 base pairs per turn
Different types of RNA secondary structures are
possible
Structures of RNA
1. Primary structure
2.Sequence complementarity: base pairing as
DNA
3.Secondary structure
4. Tertiary structure
1. Primary structure
RNA
RNA contains ribose and uracil and is
usually single-stranded
STRUCTURE
RNA
2.Sequence complementarity: inter- and
intra-molecular base pairing
STRUCTURE (1)
Watson-Crick base pairing
G-C
U
A-U
3.Secondary structures and
interactions
RNA
RNA chains fold back on themselves to
form local regions of double helix
similar to A-form DNA 2 structure elements
nd
STRUCTURE (2)
RNA helix are the basepaired segments between
short stretches of
complementary sequences,
which adopt one of the
various stem-loop
structures
hairpin
bulge
loop
Complementary regions
Held together by
hydrogen bonds
Noncomplementary regions
Have bases projecting away from
double stranded regions
Also called
hair-pin
The double helical structure of RNA resembles
the A-form structure of DNA.
•
The minor groove is wide and shallow,
but offers little sequence-specific
information.
•
The major groove is so narrow and
deep that it is not very accessible to
amino acid side chains from interacting
proteins.
4. RNA can fold up into complex
tertiary structures
Why?
RNA has enormous rotational freedom
in the backbone of its non-base-paired
regions.
Some RNAs with tertiary structures can
catalyze
• Ribozymes are RNA molecules that adopt
complex tertiary structure and serve as
biological catalysts.
• RNase P and self-splicing introns are
ribozymes
The Central Dogma
transcription
splicing
mRNA
tRNA
translation
ribosome
DNA
pre mRNA
mRNA
protein
•
•
•
•
RNA Molecules
mRNA -messenger
tRNA - transfer
rRNA - ribosomal
Other types of RNA
-RNaseP –trimming 5’ end of pre tRNA
-telomerase RNA- maintaining the chromosome ends
-Xist RNA- inactivation of the extra copy of the x
chromosome
- hn RNA- hetero nuclear
- sn RNA – small nuclear
• Messenger RNA (mRNA)
– codes for protein
• Small nuclear RNAs (snRNA)
– splice mRNA in nucleus
• Transfer RNA (tRNA)
– carries amino acid to ribosome
• Ribosomal RNA (rRNA)
– is the integral part of the ribosome
• Small interfering RNA (siRNA)
– mRNA turn-over, defense mechanism
• Micro RNA (miRNA)
– Gene expression regulation
RNA: Types
Major types:
Ribosomal RNA (rRNA)
80%
Transfer RNA (tRNA)
15%
Messenger RNA (mRNA)
5%
The rRNA
Nucleoprotein
complexes
of ribosomes
Svedberg Unit:
Related to
Molecular weight
& Shape
The tRNA
Smallest RNA
4S (74 – 95)
At least 20 species
Unusual bases
Secondary structure
Intra-chain base pairing
Adaptor molecule
Carries its sp. a.a. to site
of protein biosynthesis
tRNA
The mRNA
Size:Heterogeneous
(500 – 6000)
Primary (precursor):
hnRNA
Post-transcriptional
Processing of
Euokaryotic mRNA
Carries genetic information
from nucleus to cytoplasm
(Template of protein synthesis)




Reads mRNA
Carries the correct
amino acid
Essential in
translation
Has ‘dual specificity’
since it can read the
mRNA and bring the
correct AA as well
Molecule contains singleand double-stranded
regions
These spontaneously
interact to produce this
3-D structure
tertiary structure of tRNAphe

The transfer RNA that
carries phenylalanine