Nucleotides and Nucleic AcidsJM

Nucleic Acids Objectives

Nucleotide Base Sugar (Base + Sugar = Nucleoside) Phosphate (Nucleoside + phosphate = Nucleotide)

Nucleic Acids contain all the CHNOPS elements except sulfur (S). 6 C Carbon 12.0107 7 N Nitrogen 14.0067 8 O Oxygen 15.9994 1 H Hydrogen 1.00794 15 P Phosphorus 30.97376

Nucleotide Structure - 1 Sugars O HOCH2 OH Generic Ribose Structure Ribose O HOCH2 5’ 1’ 4’ 3’ 2’ O HOCH2 OH H N.B. Carbons are given numberings as a prime Deoxyribose

Important Pyrimidines Pyrimidines that occur in DNA are cytosine and thymine. Cytosine and uracil are the pyrimidines in RNA. HN N H O HN N H O CH3 NH2 HN O N H Uracil Thymine Cytosine

Pyrimidines NH N O T H H3C Thymine N 5 6 1 2 3 4 C N NH2 O H Cytosine

Pyrimidines U N Uracil Thymine is found ONLY in DNA. In RNA, thymine is replaced by uracil Uracil and Thymine are structurally similar Uracil NH N O U H N 5 6 1 2 3 4

Important Purines Adenine and guanine are the principal purines of both DNA and RNA. N NH2 N H O HN N H N H2N Purine = adenine and guanine Pure silver Pure = AG Adenine Guanine

Purines N H NH2 Adenine A N 1 2 3 4 5 6 7 8 9 N H O NH2 G Guanine

Chargaff's Rules 1950's: Erwin Chargaff studies heterocyclic base ratios in DNA from various organisms Species G A C T (G+A)/(C+T) A/T G/C S. aureus 21.0 30.8 19.0 29.2 1.11 1.05 E. coli 24.9 26.0 25.2 23.9 1.08 1.09 0.99 Wheat germ 22.7 27.3 22.8 27.1 1.00 1.01 Bovine thymus 21.5 28.2 22.5 27.8 0.96 Human thymus 19.9 30.9 19.8 29.4 Human liver 19.5 30.3 0.98 Chargaff's Rules: In DNA of all organisms... (G+A)/(C+T) = purines/pyrimidines ratio ~1:1 A/T ratio ~1:1 G/C ratio ~1:1 A+G=T+C. A/T and G/C ratios random in RNA

} } The Problem Solved James Watson and Francis Crick combine... 1953: Rosalind Franklin: x-ray studies of DNA show helical structure } Not compatible with single helix Diameter = 20 Å Length = 34 Å per 360o turn Calculated density James Watson and Francis Crick combine... Franklin's x-ray data Chargaff's rules Examination of molecular models } DNA is a base-paired double helix Franklin's Photo 51. The X pattern is characteristic of a helical structure Watson and Crick made extensive use of models to study molecular structure. Follow their example!

DNA replication “It has not escaped our notice that the specific pairing we have postulated immediately suggests a possible copying mechanism for the genetic material.” James Watson Francis Crick 1953 The greatest understatement in biology!

Base Pairing Watson and Crick proposed that A and T were equal because of complementary hydrogen bonding. 2-deoxyribose 2-deoxyribose A T

Base Pairing Likewise, the amounts of G and C were equal because of complementary hydrogen bonding. 2-deoxyribose 2-deoxyribose G C

The DNA Duplex Watson and Crick proposed a double-stranded structure for DNA in which a purine or pyrimidine base in one chain is hydrogen bonded to its complement in the other.

Watson-Crick Base Pairs Adenine-Thymine Guanine-Cytosine Heterocyclic bases associate via two or three hydrogen bonds Base pairs similar size and shape  efficient packing into double helix

Two antiparallel strands of DNA are paired by hydrogen bonds between purine and pyrimidine bases.

Based-Paired Double Helix 5' end 3' end Space-filling model: atoms represented at their van der Waals radii (electron cloud volumes) Aromatic stacking Hydrogen bonds easily disassembled Strong 3' end 5' end DNA strands are antiparallel

Helical structure of DNA Helical structure of DNA. The purine and pyrimidine bases are on the inside, sugars and phosphates on the outside.

The DNA Space Problem Human genome = 3 x 109 base pairs (bp) (3 x 109 bp) x (34 Å per 10 bp) x (10-10 m per Å) = ~1 meter in length Solution: DNA tertiary structure = supercoiling

Nucleosides The classical structural definition is that a nucleoside is a pyrimidine or purine N-glycoside of D-ribofuranose or 2-deoxy-D-ribofuranose. Informal use has extended this definition to apply to purine or pyrimidine N-glycosides of almost any carbohydrate. The purine or pyrimidine part of a nucleoside is referred to as a purine or pyrimidine base.

(a pyrimidine nucleoside) Uridine and Adenosine Uridine and adenosine are pyrimidine and purine nucleosides respectively of D-ribofuranose. O N HOCH2 HN OH HO N HOCH2 O OH HO NH2 Uridine (a pyrimidine nucleoside) Adenosine (a purine nucleoside)

Nucleotides Nucleotides are phosphoric acid esters of nucleosides.

Phosphate Groups Phosphate groups are what makes a nucleoside a nucleotide Phosphate groups are essential for nucleotide polymerization Basic structure: P O X

Naming Conventions Nucleosides: Nucleotides: Purine nucleosides end in “-sine” Adenosine, Guanosine Pyrimidine nucleosides end in “-dine” Thymidine, Cytidine, Uridine Nucleotides: Start with the nucleoside name from above and add “mono-”, “di-”, or “triphosphate” Adenosine Monophosphate, Cytidine Triphosphate, Deoxythymidine Diphosphate

Adenosine 5'-Monophosphate (AMP) Adenosine 5'-monophosphate (AMP) is also called 5'-adenylic acid. N O OH HO NH2 OCH2 P

Adenosine 5'-Monophosphate (AMP) Adenosine 5'-monophosphate (AMP) is also called 5'-adenylic acid. N O OH HO NH2 OCH2 P 5' 1' 4' 3' 2'

Adenosine Diphosphate (ADP) OH HO NH2 OCH2 P

Adenosine Triphosphate (ATP) OH HO NH2 OCH2 P

Major Compounds of Life Macromolecule Simple Molecule Structure 4. Nucleic Acids nucleotides (DNA + RNA) (sugar + phosphate + N-base) NH2 N N N adenine (N-base) ribose (sugar) N O O HO - P - O O phosphoric acid OH OH

Nucleotide nomenclature

Nucleotide Base Sugar (Base + Sugar = Nucleoside) Phosphate (Nucleoside + phosphate = Nucleotide)

Nucleotides in nucleic acids Bases attach to the C-1' of ribose or deoxyribose The pyrimidines attach to the pentose via the N-1 position of the pyrimidine ring The purines attach through the N-9 position Some minor bases may have different attachments.

Roles of nucleotides Building blocks of nucleic acids (RNA, DNA) Analogous to amino acid role in proteins Energy currency in cellular metabolism (ATP: adenosine triphosphate) Allosteric effectors Structural components of many enzyme cofactors (NAD: nicotinamide adenine dinucleotide)

Roles of nucleic acids DNA contains genes, the information needed to synthesize functional proteins and RNAs DNA contains segments that play a role in regulation of gene expression (promoters) Ribosomal RNAs (rRNAs) are components of ribosomes, playing a role in protein synthesis Messenger RNAs (mRNAs) carry genetic information from a gene to the ribosome Transfer RNAs (tRNAs) translate information in mRNA into an amino acid sequence RNAs have other functions, and can in some cases perform catalysis

ATP Perhaps the best known nucleotide is adenosine triphosphate (ATP), a nucleotide containing adenine, ribose, and a triphosphate group. ATP is often mistakenly referred to as an energy-storage molecule, but it is more accurately termed an energy carrier or energy transfer agent.

ATP is a nucleotide - energy currency Base (adenine) triphosphate Ribose sugar DG = -50 kJ/mol

ATP diffuses throughout the cell to provide energy for other cellular work, such as biosynthetic reactions, ion transport, and cell movement. The chemical potential energy of ATP is made available when it transfers one (or two) of its phosphate groups to another molecule. This process can be represented by the reverse of the preceding reaction, namely, the hydrolysis of ATP to ADP.

NAD is an important enzyme cofactor nicotinamide NADH is a hydride transfer agent, or a reducing agent. Derived from Niacin

Nucleotides play roles in regulation

Nucleic Acids Nucleic acids are polymeric nucleotides (polynucleotides). 5' Oxygen of one nucleotide is linked to the 3' oxygen of another.

A section of a polynucleotide chain.

Nucleic Acid Structure Polymerization Nucleotide Sugar Phosphate “backbone”

Essential for replicating DNA and transcribing RNA Two separate strands Antiparellel (5’3’ direction) Complementary (sequence) Base pairing: hydrogen bonding that holds two strands together 3’ 5’ Sugar-phosphate backbones (negatively charged): outside Planner bases (stack one above the other): inside 3’ 5’

Nucleic acids Nucleotide monomers can be linked together via a phosphodiester linkage formed between the 3' -OH of a nucleotide and the phosphate of the next nucleotide. Two ends of the resulting poly- or oligonucleotide are defined: The 5' end lacks a nucleotide at the 5' position, and the 3' end lacks a nucleotide at the 3' end position.

Helical turn: 10 base pairs/turn 34 Ao/turn

C1 Nucleic Acid Structure-6 A, B and Z helices A-form B-form Z-form

Nucleic acids B form - The most common conformation for DNA. A form - common for RNA because of different sugar pucker. Deeper minor groove, shallow major groove. A form is favored in conditions of low water. Z form - narrow, deep minor groove. Major groove hardly existent. Can form for some DNA sequences; requires alternating syn and anti base configurations. 36 base pairs Backbone - blue; Bases- gray

B-DNA

A, B and Z DNA A form – favored by RNA B form – Standard DNA double helix under physiological conditions Z form – laboratory anomaly, Left Handed Requires Alt. GC High Salt/ Charge neutralization A, B & Z DNA Kinemages

DNA vs. RNA 4 bases: guanine (G), cytosine (C), adenine (A), thymine (T) G-C A-T deoxyribose sugar double stranded nucleus 4 bases: guanine (G), cytosine (C), adenine (A), uracil (U) G-C A-U ribose sugar single stranded rRNA, mRNA, tRNA synthesized in nucleus (nucleoi for rRNA); function in cytosol 5

RNA rRNA mRNA tRNA RNA + protein form ribosomes carries DNA code to cytosol for protein synthesis on the ribosomes read in triplets called codons (5’ to 3’) tRNA carries amino acids to mRNA and matches up by base-pairing of triplet anti-codon with mRNA codon triplet 8

Stability of Nucleic Acids Hydrogen bonding Does not normally contribute the stability of nucleic acids Contributes to DNA double helix, RNA secondary structure 2. Stacking interaction/hydrophobic interaction between aromatic base pairs/bases contribute to the stability of nucleic acids. It is energetically favorable for the hydrophobic bases to exclude waters and stack on top of each other This stacking is maximized in double-stranded DNA

Effect of Acid Strong acid + high temperature: completely hydrolyzed to bases, riboses/deoxyrobose, and phosphate pH 3-4 : apurinic nucleic acids [glycosylic bonds attaching purine (A and G) bases to the ribose ring are broken ], can be generated by formic acid

Effect of Alkali & Application High pH (> 7-8) has subtle (small) effects on DNA structure High pH changes the tautomeric state of the bases enolate form enolate form keto form keto form Base pairing is not stable anymore because of the change of tautomeric states of the bases, resulting in DNA denaturation

RNA hydrolyzes at higher pH because of 2’-OH groups in RNA 2’, 3’-cyclic phosphodiester alkali OH free 5’-OH RNA is unstable at higher pH

Disrupting the hydrogen bonding of the bulk water solution Chemical Denaturation Urea (H2NCONH2) : denaturing PAGE Formamide (HCONH2) : Northern blot Disrupting the hydrogen bonding of the bulk water solution Hydrophobic effect (aromatic bases) is reduced Denaturation of strands in double helical structure

Buoyant density (DNA) 1.7 g cm-3, a similar density to 8M CsCl Purifications of DNA: equilibrium density gradient centrifugation Protein floats RNA pellets at the bottom

C3 Spectroscopic and Thermal Properties of Nucleic Acids UV absorption: nucleic acids absorb UV light due to the aromatic bases The wavelength of maximum absorption by both DNA and RNA is 260 nm (lmax = 260 nm) Applications: detection, quantitation, assessment of purity (A260/A280) 2. Hypochromicity: caused by the fixing of the bases in a hydrophobic environment by stacking, which makes these bases less accessible to UV absorption. dsDNA, ssDNA/RNA, nucleotide

3. Quantitation of nucleic acids Extinction coefficients: 1 mg/mL dsDNA has an A260 of 20 ssDNA and RNA, 25 The values for ssDNA and RNA are approximate The values are the sum of absorbance contributed by the different bases (e : purines > pyrimidines) The absorbance values also depend on the amount of secondary structures due to hypochromicity. Purity of DNA A260/A280: dsDNA--1.8 pure RNA--2.0 protein--0.5

5. Thermal denaturation/melting: heating leads to the destruction of double-stranded hydrogen-bonded regions of DNA and RNA. RNA: the absorbance increases gradually and irregularly DNA: the absorbance increases cooperatively. melting temperature (Tm): the temperature at which 40% increase in absorbance is achieved.

6. Renaturation: Rapid cooling: only allow the formation of local base paring. Absorbance is slightly decreased Slow cooling: whole complementation of dsDNA. Absorbance decreases greatly and cooperatively. Annealing: base paring of short regions of complementarity within or between DNA strands. (example: annealing step in PCR reaction) Hybridization: renaturation of complementary sequences between different nucleic acid molecules. (examples: Northern or Southern hybridization)

DNA Supercoiling Closed circular molecule Supercoiling & energy Topoisomer & topoisomerase

Almost all DNA molecules in cells can be considered as circular, and are on average negatively supercoiled. Counter helical turn

2. Negative supercoiled DNA has a higher torsional energy than relaxed DNA, which facilitates the unwinding of the helix, such as during transcription initiation or replication Topoisomer: A circular dsDNA molecule with a specific linking number which may not be changed without first breaking one or both strands.

Topoisomerases exist in cell to regulate the level of supercoiling of DNA molecules. Type I topoisomerase: breaks one strand and change the linking number in steps of ±1. TypeII topoisomerase: breaks both strands and change the linking number in steps of ±2. Gyrase: introduce the negative supercoiling (resolving the positive one and using the energy from ATP hydrolysis.

Ethidium bromide (intercalator): locally unwinding of bound DNA, resulting in a reduction in twist and increase in writhe. Topoisomerases Type I: break one strand of the DNA , and change the linking number in steps of ±1. Type II: break both strands of the DNA , and change the linking number in steps of ±2.