Nucleotides and Nucleic Acids Lehninger Chapter8 8.1 Basics 8.2 Structure 8.3 Chemistry 8.

4 Nucleotide Function

8.1 Basics Building Blocks Canonical and Minor Bases Phosphodiester bonds Naming and Drawing Base Stacking and Pairing

Building Blocks
Nucleotides = Base + Sugar + Phosphate Nucleosides = Base + Sugar Nitrogen Bases
Purines (5 + 6 membered rings) numbering
Adenine Guanine

Pyrimidines (6 membered ring) numbering

Thymine Cytosine Uracil

Pentose Sugars (numbering)

Ribose Deoxy Ribose

Ribose

Sugar Pucker

Canonical and Minor Bases

DNA A, G, C, T RNA A, G, C, U Modified bases Methylation in DNA Lots of Mods in RNA

Purines

Pyrimidines

Phosphodiester bonds
Formed by Polymerase and Ligase activities C-5' OH carries the phosphate in nucleotides C5' - O - P - O - C3' Phosphate pKa ~ 0 Natural Oligonucleotides have 5' P and 3' 0H Base hydrolysis due to ionizaiton of 2' OH in RNA

Oligonucleotide naming / drawing conventions

5 - Left to Right - 3 pACGTOH ACGT

Base Stacking and Base Pairing

Bases are very nearly planar Aromaticity => large absorbance at 260nm Epsilon 260 10,000 (M-1 cm-1 ) The A260 50 g /ml for DS DNA The A260 40 g /ml for SS DNA or RNA Flat surfaces are hydrophobic Dipole-Dipole and Van Der Waals interactions also stabilize stacked structures Bases have hydrogen bond donors and acceptors H-bonding potential satisfied in paired structures

8.2 Structure
DNA contains genetic Information Distinctive base composition foretells base pairing patterns Double helical structures Local structures mRNAs - little structure Stable RNAs - complex structures

DNA contains genetic Information

Purified DNA can "transform" Bacteria
Avery, MacLeod & McCarty transferred the virulence trait to pneumococci

The genetic material contains 32P (DNA) and not 35S (protein C, M)
Hershey and Chase grew bacteriophage on either 32P or 35S Bacteriophage infection resulted in transfer of 32 P and not 35S

Distinctive Base composition foretell base pairing patterns

Hydrolysis of DNA and analysis of base composition
Same for different individuals of a given species Same over time Same in different tissues %A = %T and %G = %C (Chargaff's Rules)

Amino acid compositions vary under all three conditions No quantitative relationships in AA composition

Structural Basis of Chargaffs Rules

Two Strands have complementary sequences 2 logical operations to obtain complementary strand 5' to 3'
1. Reverse: Rewrite the sequence, back to front 2. Complement: Swap A with T, C with G

Double helical structures

Potentially Right or Left Handed
Actually Mostly Right Handed

Potentially Parallel or Anti-parallel

Actually anti-parallel

Sugar Pucker + 6 rotatable bonds gives 3 families A, B, Z structures

http://www.olemiss.edu/depts/chemistry/courses/chem47 1_10/ABZ_DNA.kin

KING 3D display software:

http://kinemage.biochem.duke.edu/software/king.php

B-DNA

Semiconservative Replication

DNA Backbone Flexibility

Multiple Degrees of Rotational Freedom

Glycosidic Bond Configurations

3 Canonical Helical DNA Structures

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

Local structures
Palindromes Inverted repeats
Not quite the same as (Madam Im Adam) Symmetrical Sequence Elements Match Symmetry of Protein Homo-Oligomers Symmetry often incomplete/imperfect

Direct Repeats Hairpin and Cruciform Structures

Hoogstein Pairing in Base Triples

Messenger RNAs
Contain protein coding information
ATG start codon to UAA, UAG, UGA Stop Codon A cistron is the unit of RNA that encodes one polypeptide chain Prokaryotic mRNAs are poly-cistronic Eukaryotic mRNAs are mono-cistronic

Base pairing/3D structure is the exception

Can be used to regulate RNA stability termination, RNA editng, RNA splicing

The Genetic Code

G
G A C U G glu ala val A C U
met

A
arg asp lys thr ile A C U G G ser asn gln

C
arg his pro leu A C U leu G A trp

U
cys tyr ser phe C U

gly

GG[GACU] code for Glycine UGG codes for Tryptophan UGA, UAG, UAA are stop codons AG[CU] and UC[GACU] code for Serine

mRNA coding patterns

Stable RNAs with complex structures

RNA Helices are short, bulges, loops

RNA Secondary Structure Maps

Calculated from helix thermodynamic parameters Loop entropy considerations

tRNA-Phe 2r Structure

tRNA - the prototype structure

tRNA Phe Kinemage

http://www.olemi ss.edu/depts/chem istry/courses/che m471/6tna.kin

8.3 Chemistry
Denaturation and reannealing Hybridization Spontaneous Chemical Reactions Methylation Sequencing Chemical Synthesis

Denaturation and reannealing

Tm (transition midpoint) as a function of base composition

Salt dependence is more dramatic

Hybridization
DNA sequences can spontaneously reanneal and form helices Basis for many of molecular biology techniques.
PCR, DNA sequencing

Spontaneous Chemical Reactions

Pyrimidine Dimers

Keto Enol Tautomerization

Methylation Reactions

DNA Polymerase Mechanism

Di-deoxy NTPs Chain terminators

Sanger Sequencing

Dye-Terminator (one-tube rxns)

Next-generation DNA sequencing

Millions of DNA fragments isolated, amplified by PCR and monitored in parallel
Jay Shendure & Hanlee Ji Nature Biotechnology 26, 1135 1145 (2008) Published online: 9 October 2008 doi:10.1038/nbt1486

DNA Solid-Phase Synthesis

8.4 Nucleotide Function

Energetic Intermediates Adenine Enzyme Cofactors Regulatory Molecules

Nucleotides

Phospho-Anhydrides and Phosphate Esters High Energy Bonds

Co-Enzyme A Carrier for Acetyl units in intermediary metabolism, fatty acid synthesis and oxidation

+/NADH, NAD

+/NADPH NADP

and FAD/FADH2
Redox Cofactors

Regulatory/Signalling Molecules