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