PTT 207 Biomolecular and Genetic Engineering Semester 1 2012/2013 - - PowerPoint PPT Presentation

PTT 207 Biomolecular and Genetic Engineering

Semester 1 2012/2013

BY: PUAN NURUL AIN HARMIZA ABDULLAH

Chapter 2

The Structure of DNA

“Imagine yourself inside one

• f those cooling DNA

solutions, observing the rebirth of beautifully undulating, semirigid, double-helical threads from the jumble of billions of intertwisted single strands. It is a mind reeling spectacle”

Christian de Duve, A Guided Tour

• f the Living Cell (1984), p. 292

2.2 Primary structure:

the components of nucleic acids

Components of nucleotides

• Five-carbon sugars
• Nitrogenous bases
• The phosphate

functional group

Edwin Chargaff’s “rules”

• [A] = [T]
• [G] = [C]
• [A] + [G] = [T] + [C]

Nucleosides and nucleotides

DNA and RNA chains are formed through a series

• f three steps:

1.A base attached to a sugar is a nucleoside. 2.A nucleoside with one or more phosphates attached is a nucleotide. 3.Nucleotides are linked by 5′ to 3′ phosphodiester bonds between adjacent nucleotides to form a DNA or RNA chain.

• The components of a DNA or RNA chain

are joined by covalent bonds.

• A covalent bond is a strong chemical bond

formed when electrons are shared between two atoms.

• These bonds are very stable and do not

break spontaneously within cells.

Nomenclature of nucleotides

Example: the base cytosine (C) DNA deoxycytidine 5′-triphosphate (dCTP) RNA cytidine 5′-triphosphate (CTP) Generic deoxynucleoside 5′-triphosphate (dNTP) nucleoside 5′-triphosphate (NTP)

The length of RNA and DNA

• RNA

The number of nucleotides (nt) or bases is used as a measure of length.

• Double-stranded DNA

The number of base pairs (bp) is used as a measure of length.

1000 bp = 1 kilobase pair (kb or kbp) 1,000,000 = 1 megabase pair (Mb or Mbp)

• Natural RNAs come in sizes ranging from

less than one hundred to many thousands of nucleotides

• DNA can be as long as several kb to

thousands of Mb

• Oligonucleotides are short chains of

single-stranded DNA (< 50 bases)

Significance of 5′ and 3′

• The 5′-PO4 and 3′-OH ends of a DNA or RNA

chain are distinct and have different properties

• By convention, a DNA sequence is written with

the 5′ end to the left and the 3′ end to the right

• The two ends are designated by the symbols 5’

and 3’:

• 5’  carbon to a PO4 is attached.
• 3’  carbon to a OH is attached.

Why it is important to understand this 5’3’ polarity?

2.3 Secondary structure of DNA

Hydrogen bonds form between the bases

• Two common “Watson-Crick” or

“complementary” base pairs:

• 1. Adenine (A) is joined to thymine (T) by two

hydrogen bonds.

• 2. Guanine (G) is joined to cytosine (C) by three

hydrogen bonds.

The two common Watson-Crick base pairs of DNA.

1.08nm Why aren’t G with A or C with T? Find out…

Base stacking provides chemical stability to the DNA double helix

• The nitrogenous bases are nonpolar and thus

“hydrophobic”.

• The hydrophobic nitrogenous bases stack onto each
• ther without a gap by means of a helical twist.
• A double-stranded DNA molecule has a hydrophobic core

composed of stacked bases.

• Because of sugars and phosphates are soluble in water,

they orient toward the outside of the helix, where the polar phosphate groups can interact with the polar environment.

• Hydrophobic bonding is an example of weak van

der Waals interactions.

• A large number of weak van der Waals

interactions can significantly increase the stability of a structure, such as the DNA double helix.

Base stacking.

Structure of the Watson-Crick DNA double helix

• Polarity in each strand: 5′ 3′
• 5’P  3’OH
• Two strands are antiparallel
• 5’P  3’OH
• 3’OH  5’P
• Major and minor grooves
• The sugar-phosphate backbone is not equally spaced

Key features of the DNA double helix. The major groove occurs where the backbones are far apart, the minor groove occurs where they are close together.

Major and minor grooves

• The major groove carries a “message” that

can be read by DNA binding proteins..

• In the major groove, the pattern of

hydrogen-bonding groups is different for AT, TA, GC, and CG base pairs.

• In the minor groove, there is only one

difference in the pattern between AT and GC base pairs.

(why at major grooves and not at minor grooves?)

Distinguishing between features of alternative double-helical structures

• B-DNA (Watson-Crick DNA)
• A-DNA
• Z-DNA

• The predominant form of DNA in vivo is B-DNA.
• But, there is evidence for a role of Z-DNA in vivo:
• Z-DNA binding proteins.
• Short sections of Z-DNA within a cell are

energetically favorable and stable.

• Role in regulating gene expression?

• A region of Z-DNA is connected to B-DNA

through a junction in which one base pair is flipped out, or extruded, from the DNA helix.

• This process is called base flipping.

DNA can undergo reversible strand separation

Significance of complementary base pairing:

• Fidelity of DNA replication, transcription, and

translation.

• Ability to manipulate in the lab by denaturation,

renaturation, and hybridization.

Denaturation, renaturation, and hybridization of dsDNA.

Denaturation or “melting”

• f DNA
• Denaturation/melting refers to the unwinding/separation of

DNA strands (by heat or salt).

• Base stacking in duplex DNA quenches the capacity of bases to

absorb UV light.

• Hyperchromicity: As DNA “melts” its absorption of UV light

increases.

• Tm (melting temperature): The temperature at which half of the

bases in a dsDNA sample have denatured.

• The G+C content has significant effect on Tm

Find out HOW? Find out WHY?

DNA Denaturation Curve

Renaturation or “reannealing”

• f DNA
• The capacity to renature denatured DNA permits

hybridization.

• Hybridization is the complementary base pairing
• f strands from two different sources.
• The rate at which DNA reanneals is a function of

the length of the DNA and the initial concentration in the sample.

DNA Renaturation Curve

A DNA renaturation Cot curve

• C/C0 = 1/[1 + KC0t]
• The expression C0t is called “Cot.”
• Cot ½ is when renaturation is half completed.
• A plot of C/C0 versus C0t is called a Cot curve.

Comparison of Cot curves for E. coli and calf thymus DNA

• The Cot ½ of calf thymus DNA is greater than the Cot ½ of E. coli

DNA.

• Explanation: The larger the genome size, the longer it will take for

any one sequence to encounter its complementary sequence.

2.4 Unusual DNA Secondary Structure

2.5 Tertiary structure of DNA

• Supercoils form a twisted, 3-D structure which is

more favorable energetically.

• Less stable than relaxed DNA.

Negative (left-handed) supercoil: underwound Positive (right-handed) supercoil: overwound

Supercoiling of DNA

Consider a short linear dsDNA molecule of 10 complete turns/twists, T=10) with 10.5 bp/turn. If the ends of the DNA molecule are sealed together, the result is an energetically relaxed circle that lies flat. Since each chain is seen to cross the other 10 times, this relaxed circle has a linking number (L) of 10. But if the double helix is underwound by one full turn to the left and then the ends are sealed together, the result is a strained circle with 11.67 bp/turn, where L=9 and T=9.

• The strain present within supercoiled

DNA sometimes leads to localized denaturation.

• B-DNA→ Z-DNA transitions may be

triggered by negative supercoiling.

• Topoisomerases are enzymes that

introduce transient breaks in DNA strands and release the strain of supercoiling.

DNA supercoiling plays an important role in many processes, such as replication, transcription, and recombination

• Genome of some viruses: small circle

Relaxed circle: reduced activity Negatively supercoiled circle: increased activity

• Bacterial genome: very large circle

Form independent DNA loop domains

• Eukaryotic genomes: linear

Form independent DNA loop domains

• Negative supercoiling makes it easier to separate

the DNA strands during replication and transcription.

• The DNA of thermophilic Archaea exists in a

positive supercoiled state that protects the DNA from denaturation at high temperatures.

THANK YOU