The biochemistry of nucleic acids

Tommer Ravid

Room 1-523Tel: 658 4349e-mail: travid@cc.huji.ac.il

Metabolism of nucleic acids (DNA and RNA)

1. In many cases requires proofreading.

2. In many cases requires a plan (template) in addition to substrates.

3. Unusual definition of substrate.

4. Different substrates at different times, at different cells.

5. Unusual structural organization. Unusual problems for enzymes and regulators to act on the nucleic acid.

6. Utilizing nucleotides as building blocks.

A generic scheme of a nucleotide.Three chemical components connectedwith covalent bonds:1. A nitrogenous base.2. A pentose mostly a ribose in its ring form.3. A phosphate (one or more).

Nucleoside: a pentose and a base with no phosphate group

Deoxyribonucleotides

Building Block for RNA and DNA

Not a building block for DNA

The name of the nucleotide is determined by the base(simply because it is the variable component in the molecule)

Numbers of carbon atoms on the ribose are marked with ‘ to distinguish from the atom numbers of the nitrogenous base.

Bases come from two families, purines and pyrimidines.

How many purines and how many pyrimidines do we know?

Specific to mammals andhigher plants.

Specific for bacteria(involved in repair)

Found in bacteria infectedby phages.

Some less known (but not rare at all) nitrogenous bases

Some more of the less known bases (found in tRNAs)

I: Inosine: PseudouridineT: RibothimidineD: 5,6-dihydrouridineM1l: 1-methylinosineM1G: 1-methylguanosineM2G: dimethylguanosine

Nucleotide sequence of yeast tRNAAla

General secondary structure of tRNAs

To remember by heart:

Formulas of:

ATP, GTP, CTP, UTP

dATP,dGTP,dCTP,dTTP

Some cellular functions of nucleotides1. Building blocks of nucleic acids.

2. Energy carrier (ATP, GTP).

3. Building parts of enzymes co-factors (e.g., NAD, FAD, CoenzymeA, S-adenosylmethionine).

4. Regulators in signal transduction processes.

5. Second messengers in signal transduction (cAMP, cGMP).

6. Phosphate donors in phosphorylation reactions.

7. Serve as structural molecules (rRNA).

8. Activators of carbohydrates for synthesis (glycogen for example).

Adenosine 5’-triphosphate (ATP)High concentrations in cells - a problem of balance in nucleotidesconcentration.

ThioesterModified ADPHigh free energy of hydrolysisHigh acyl group transfer potential (to many acceptor molecules)Vitamin required (pantothenate)

Coenzyme A

C

CCH

C

C

HC

NC

CN

NC

NHC

H3C

H3C

O

O

CH2

HC

HC

HC

H2C

OH

O P O P O

O

O-

O

O-

Ribose

OH

OH

Adenine

C

CCH

C

C

HC

NC

C

HN

NH

C

NHC

H3C

H3C

O

O

CH2

HC

HC

HC

H2C

OH

O P O P O

O

O-

O

O-

Ribose

OH

OH

AdenineFAD FADH2

2 e + 2 H+

dimethylisoalloxazine

Some cellular functions of nucleotides1. Building blocks of nucleic acids.

2. Energy carrier (ATP, GTP).

3. Building parts of enzymes co-factors (e.g., NAD, FAD, CoenzymeA, S-adenosylmethionine).

4. Regulators in signal transduction processes.

5. Second messengers in signal transduction (cAMP, cGMP).

6. Phosphate donors in phosphorylation reactions. Involved in many more pottranslational modifications.

7. Serve as structural molecules (rRNA).

8. Activators of carbohydrates for synthesis (glycogen for example).

Some cellular functions of deoxynucleotides1. Building blocks of nucleic acids (DNA).

2. Energy carrier (ATP, GTP).

3. Building parts of enzymes co-factors (e.g., NAD, FAD, CoenzymeA, S-adenosylmethionine).

4. Regulators in signal transduction processes (GTP).

5. Second messengers in signal transduction (cAMP, cGMP).

6. Phosphate donors in phosphorylation reactions.

7. Serve as structural molecules (rRNA).

8. Activators of carbohydrates for synthesis (glycogen for example).

Chemical properties of nucleotides1. Components of nucleotides are independently reacting with other molecules. Hydroxyls are nucleophils. Phosphate nuclei are electrophiles. The components of a given nucleotide rarely react with each other.

2. All components are covalently linked - the bonds are saturated and can rotate freely. The ribose can also acquire several conformations.

3. Nitrogen bases are planar (allowing close contacts that eliminate water molecules for example).

4. Ribose and phosphate groups are hydrophylic while bases are hydrophobic.

5. Electronegative atoms in the bases rings. These atoms are capable of forming hydrogen bonds.

6. The bases are ‘conjugated molecules’ in which the atoms are closed and the orbitals are delocalized over the entire ring. As a result the bases are planar. They also absorb UV light (pick at about 260nm).

7. The glycosidic bond is always in a configuration.

Conformations of ribose

Where the cells obtain nucleotides from ?

De novo synthesis provides nucleotidesin the mono-phosphate form.

Specific kinases provide nucleotidesdi-phosphate and tri-phosphate.

Deoxyribonucleotides

Reduction of ribonucleotides to deoxyribonucleotides by ribonucleotide reductase

Ribonucleotide reductase

The basic reaction in nucleotide polymerization.

Catalyzed by all known polymerases.

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The driving force towardssynthesis is the breakdown ofPyrophosphate (PPi).

Phosphodiester bond

PPi+H2O 2Pi Pyrophosphatase

Hydrolysis of RNA under alkaline conditions

DNA/RNA structure

Some biologically important hydrogen bonds

Secondary structure of RNAs

-Planar bases (resonance)-Hydrophobic/hydrophilic elements -Van der vals forces + dipole moment

Stacking forces imposing a right helix

The strength of the stacking forces isdetermined by the particular sequence.Purines have stronger stacking forces than pyrimidines.

Base stacking

Structural principles of the double helixes of nucleic acidsThe two strand are anti-parallel.

Base-pairing by hydrogen bonds (A-T(U), C-G).

The two strands circles around a common pivot.

Diameter of the double helix is essentially constant.

Formation of the double helix is reflected in a hypochromic effect.

“We wish to suggest a structure for the salt of deoxyribose nucleic acid (DNA). ThisStructure has novel features which are of considerable biological interest”.

As a result of the double helical nature of

DNA, the molecule has two asymmetric

grooves. One groove is smaller than the other.

This asymmetry is a result of the geometrical

configuration of the bonds between the

phosphate, sugar, and base groups that forces

the base groups to attach at 120 degree angles

instead of 180 degree. The larger groove is

called the major groove, occurs when the

backbones are far apart; while the smaller one

is called the minor groove, occurs when they

are close together.

The homeodomain of the Engrailed protein binds to a particular site in the DNA. Helix 3 contacts the base pairs in the major groove, while the amino-terminal portion of the homeodomain enters the minor groove.

Structural parameters of B-DNA (Watson & Crick) optimal/average model1. Right handed helix.

2. A-T and C-G pairing (only)3. 10 bases per one complete round.

4. 36o angle between one pair to the neighbor pair.5. Creating a major and minor grooves.

DimensionsDiameter: 2.0 nm.

Space between pairs: 0.34 nm.Length of one circle (10bp): 3.6 nm.

Deviations from the optimal B-DNA structure1. Angle between one pair to the neighbor pair could be 28o-42o.2. Bases could be propelled (not fully planar).3. Left-handed helix??

A-DNA B-DNA Z-DNA

Helical sense right handed right handed left handed

Diameter ~26 Å ~20 Å ~18 Å

Base pairs/turn 11 10.5 12

Helix rise/base pair 2.6Å 3.4Å 3.7Å Base tilt normal to helix axis 20o 6o 7o

• Most DNA in most organisms is double-stranded and most doubled-stranded DNA is in the B-form.

• Molecular structure suggests that B-form is found naturally because it, but not A-form, can accommodate a spine of water molecules (green) lying in minor groove.

• The hydrogen bonds contributed by the water give added stability to the B form.

•B-DNA structure (B_DNA) • "Watson and Crick" double helix, the form favored for random-sequence DNA under physiological conditions • right-handed • Strands are complementary in nucleotide sequence: where one strand has a T, it's base-paired (by hydrogen bonds) to an A on the other strand; if first strand has a G, it's base-paired to a C on the other strand. • Strands are antiparallel: if 1 strand runs 5'-->3' left to right, its partner runs 3'-5' left to right. • Major groove is wide and deep; minor groove is narrow and deep. • Tightly packed atoms in core (along helix axis), no hole

•A-DNA structure (A_DNA) • Favored for DNA-DNA duplex under dehydrating conditions • Right-handed, double-stranded (complementary strands) • favored under physiological conditions for RNA-RNA ( (A_RNA) or RNA-DNA duplexes, because the 2¢ hydroxyl of ribose sterically inhibits formation of the B conformation • Strands antiparallel and complementary in sequence • Major groove narrow and deep; very little minor groove (not much of a "groove" -- wide and shallow) • Wider diameter than B-DNA, with hole down the helix axis

•Z-DNA structure (Z_DNA) • Left-handed form of double-stranded DNA (complementary strands) • backbone phosphates "zig-zag" • Favored by alternating purine-pyrimidine sequences, and high salt concentrations (which minimize the electrostatic repulsion between backbone phosphates) • Strands antiparallel and complementary in sequence • almost no major groove (flat); minor groove narrow and deep • atoms very tightly packed • physiological role uncertain -- does occur in short tracts in vivo in both prokaryotes & eukaryotes, and may have something to do with regulation of expression of some genes, or in genetic recombination

A non enzymatic transformation

Tautomeric forms of uracil

Free pyrimidine and purine bases may exist in two or more tautomeric forms depending on the pH. Uracil, for example, occurs in lactam, lactim, and double lactim forms.

As a result of resonance, all nucleotide bases absorb UV light, and nucleic acids are characterized by a strong absorption at wavelengths near 260 nm.

Absorption spectra of the common nucleotides

Denaturation of DNA results in an increase in optical density

Heat denaturation of DNA

Relationship between tm and the G+C content of a DNA.