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

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 μg /ml for DS DNA The A260 ≈ 40 μg /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 Chargaff’s 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/chem471_10/ABZ_DNA.kin KING 3D display software: http://kinemage.biochem.duke.edu/software/king.php

B-DNA

Semi-conservative Replication

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 Direct Repeats Not quite the same as (Madam I’m Adam) Symmetrical Sequence Elements Match Symmetry of Protein Homo-Oligomers Symmetry often incomplete/imperfect Direct Repeats Hairpin and Cruciform Structures

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 A C U GG[GACU] code for Glycine arg ser trp   cys glu asp lys asn gln his tyr ala thr pro val met ile leu phe 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

tRNA-Phe 2° Structure

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 re-anneal and form helices Basis for many of molecular biology techniques. PCR, DNA sequencing