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DNA and RNA. Dr. Sugandhika Suresh Department of Biochemistry. Features of the DNA double helix. 2 DNA strands per molecule Right-handed helix 2 chains are antiparallel Sugars and phosphates –outside Bases inside -- “stacked like pennies” Bases are bonded together by H-bonds - PowerPoint PPT Presentation
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DNA and RNA
Dr. Sugandhika SureshDepartment of Biochemistry
Features of the DNA double helix1. 2 DNA strands per molecule2. Right-handed helix3. 2 chains are antiparallel4. Sugars and phosphates –outside5. Bases inside -- “stacked like pennies”6. Bases are bonded together by H-bonds7. Specific base pairings are observed - complementary8. A pairs with T9. C pairs with G10. 10 base pairs per turn11. 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
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
DNA Can Form Alternative Types of Double Helices
FORMS OF DNA
B-form
A-form
Z-form
DNA forms
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 DNADenaturation by
heating.How is it observed?
A260 For dsDNA, A260=1.0 for 50 µg/mlFor ssDNA and RNA
A260=1.0 for 38 µg/mlFor ss oligonuleotides A260=1.0 for 33 µg/mlHyperchromic shift
The T at which ½ the DNA sample is denatured is called the melting temperature (Tm)
The two strands of the double helix separate reversibly at high temperatures
The temperature at which this “denaturation” or “melting” occurs depends on the pH and salt concentration, and increases with the GC content of the DNA. (The curves drawn here are schematic.)
100
80
60
40
20
0
% D
enat
ured
110100908070Temperature / o
C
40 50 70% GC60If the temperature is lowered, the strands recombine.
The rate of reassociation is inversely proportional to the complexity of the DNA.
dsDNAssDNAnucleotides
dA
dC
dGdU
The conjugated p-electron systems of the purine & pyrimidine bases absorb strongly in the UV.
(That’s why UV light is mutagenic and carcinogenic.)
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.”
Double-stranded and single-stranded DNA differ in their optical absorption at 260 nm
Importance of Tm
Critical importance in any technique that relies on complementary base pairing Designing PCR primersSouthern blotsNorthern blotsColony hybridization
Factors Affecting Tm
G-C content of samplePresence of intercalating agents (anything that
disrupts H-bonds or base stacking)Salt concentrationpH Length
Renaturation
Strands can be induced to renature (anneal) under proper conditions. Factors to consider:TemperatureSalt concentrationDNA concentrationTime
DNA packaging in chromosomes
DNA wound around histone proteins
A TT AG CC G
G C
TA
T
AG
C
C G
G C
T A
A T
Packaging DNA
Histone proteins
Histoneoctamer
B DNA Helix 2 nm
A TT AG CC G
G C
TA
T
AG
C
C G
G C
T A
A T
Packaging DNA
Histone proteins
B DNA Helix
Histoneoctamer
2 nm
A TT AG CC G
G C
TA
T
AG
C
C G
G C
T A
A T
Packaging DNA
Histone proteins
Histoneoctamer
Nucleosome
11 nm
B DNA Helix 2 nm
DNA-histone octamerH1 Links Nucleosomes together
Nucleosomes:
+H1
-H1
Packaging DNA
A TT AG CC G
C G
G C
T A
A T
Packaging DNA
A TT AG CC G
C G
G C
T A
A T
Packaging DNA
A TT AC G
C G
G C
T A
A T
Protein scaffold
11 nm“Beads on a string”
30 nm
Tight helical fibre
Looped Domains200 nm
Packaging DNA
G
C
A
T
Protein scaffold
Metaphase Chromosome
700 nm
11 nm
30 nm200 nm
2 nm
Looped Domains
Nucleosomes
B DNA Helix
Tight helical fibre
Replication
Chromosomes, Chromatids and Centromeres
Centromere
Chromosome arm
Chromosome arm
Identicalchromatid
Chromatid
Anaphase
A packaged chromosome
Two identical chromosomes
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•Single-Strand (mostly)•Cytoplasm (mainly)•AGCU •Modified bases•Ribose•Protein Biosynthesis•Post-transcriptional events
DNA•Double•Nucleus•d AGCT
•Deoxyribose•Storage &transfer
•DNA Repair
Biological roles of RNA1. RNA is the genetic material of some viruses
2. RNA functions as the intermediate (mRNA) between the gene and the protein-synthesizing machinery.
3. RNA functions as an adaptor (tRNA) between the codons in the mRNA and amino acids.
4.Through sequence complementarity, RNAserves as a regulatory molecule to bind to and interfere with the translation of certain mRNAs; or as a recognition molecule to guide many post-transcriptional 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, self-splicing introns, etc).
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
RNA Structure
40
ReplacesDeoxyribose
ReplacesThymine
Components unique to RNA
RNA Primary Structure
• RNA chain directionality: 5'3'• Backbone carries charge (-e) on each nucleotide• Formation of an RNA structure requires cations
(-e)
(-e)
(-e)
(-e)5'
3'
Structure of RNA backbone
Although usually single-stranded, RNA molecules can form short double-stranded regions This secondary structure is due to complementary base-
pairing A to U and C to G
This allows short regions to form a double helix
RNA double helices typically 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
RN
A STR
UCTU
RE
RNA contains ribose and uracil and is usually single-stranded
1. Primary structure
RN
A STR
UCTU
RE (1)
Watson-Crick base pairing
U A-U
G-C
2.Sequence complementarity: inter- and intra-molecular base pairing
3.Secondary structures and interactions
RNA chains fold back on themselves to form local regions of double helix similar to A-form DNA
RN
A STR
UCTU
RE (2)
hairpin
bulge
loop
RNA helix are the base-paired segments between short stretches of complementary sequences, which adopt one of the various stem-loop structures
2nd structure elements
Also called hair-pin
Complementary regions
Noncomplementary regions
Held together by hydrogen bonds
Have bases projecting away from double stranded regions
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.
RNA has enormous rotational freedom in the backbone of its non-base-paired regions.
Why?
4. RNA can fold up into complex tertiary structures
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
DNA pre mRNA mRNA protein
transcription
splicing
translation
mRNA
ribosome
tRNA
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: TypesMajor types:
Ribosomal RNA (rRNA) 80%
Transfer RNA (tRNA) 15%
Messenger RNA (mRNA) 5%
Nucleoprotein complexes
of ribosomes
Svedberg Unit:
The rRNA
Related toMolecular weight
& Shape
The tRNASmallest RNA 4S (74 – 95)
At least 20 species
Unusual basesSecondary structure Intra-chain base pairingAdaptor molecule Carries its sp. a.a. to site of protein biosynthesis
tRNA
The mRNASize:Heterogeneous (500 – 6000)
Primary (precursor): hnRNA
Post-transcriptionalProcessing of
Euokaryotic mRNACarries 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 tertiary structure of tRNAphe
The transfer RNA that carries phenylalanine
Molecule contains single- and double-stranded
regions
These spontaneously interact to produce this
3-D structure