ATGCU H H A - pKa=3.5 C- pKa= 4.2 T- pKa= 9 G- pKa= 9.2 U- pKa=9.2 Protonated Form (red) dominates below pKa At pH 7, Some bases (U, T, G) will be protonated and others (A, C) will be deprotonated H H
Nucleotides come from aminoacids Glycine CO2 C N Aspartic acid N C Formate C C C Formate N N Glutamine Glutamine
Nomenclature nucleoside = sugar +base nucleotide = sugar + base + phosphate
Linear to ring In solution, the straight-chain (aldehyde) and ring (b-furanose) forms of free ribose are in equilibrium. RNA contains only the ring form, b-D-ribofuranose. Deoxyribose undergoes a similar interconversion in solution, but in DNA exists solely as β-2′-deoxy-D-ribofuranose.
Methyl nucleotides Adenine or cytosine methylation is part of the restriction modification system in bacteria, in which DNA is methylated. Foreign DNAs which are not methylated are degraded by sequence-specific restriction enzymes. Neurospora crassa has a well characterized methylation system. Genome has very little repeated DNA, methylation occurs in repeated DNA –transposon 60% and 90% of all CpGs are methylated in mammals. Unmethylated CpGs are grouped in clusters called CpG islands that are present in the promoters of genes.
Nucleotide functions Energy for metabolism in cells ATP Cofactors for enzymes NAD Signal transduction cAMP
Cyclic nucleotides Cyclic adenosine monophosphate (cAMP, cyclic AMP or 3'-5'-cyclic adenosine monophosphate) is a second messenger important in many biological processes. cAMP is derived from adenosine triphosphate (ATP) cAMP is a second messenger, used for intracellular signal transduction, such as transferring the effects of hormones like glucagon which cannot pass through the cell membrane. It is involved in the activation of protein kinases
Asthma and bronchodilation The b-adrinergic receptors are the targets for treatment of asthma. They are located in many organs of the body, but the ones that are pertinent to asthma are the b-receptors located in the bronchial smooth muscle and arterioles of the lungs which are especially important in the body’s airflow to and from the lungs. When these receptors are stimulated they cause smooth muscle relaxation resulting in bronchial dilation and vasodilation. Beta2 receptors are serpentine receptors, meaning the protein crosses the cellular membrane seven times. They are activated primarily by epinephrine. The carboxy-terminal end is on the intracellular side and the amino-terminal end is on the extracellular side. These are coupled to G proteins which have three subunits a,b, g. The alpha subunit of the G protein is activated by GTP, and the GTP activated a-subunit activates adenylate cyclase. Adenylate cyclase converts ATP to cAMP which serves as a second messenger leading to physiologic effects. Beta-2 adrenergic receptor agonists These drugs work to dilate the bronchial airways during an acute asthma attack. The b2-adrenergic receptor agonists work by binding to the receptor and activating adenylyl cyclase. Adenylyl cyclase, in turn, increases the production of cyclic adenosine monophosphate (cAMP). Bronchodilation is supported by this increase in cAMP.
RNA hydrolysis (alkaline) Hydrolysis of RNA under alkaline conditions. The 2’ hydroxyl acts as a nucleophile in an intramolecular displacement. The 2’,3’-cyclic monophosphate derivative is further hydrolyzed to a mixture of 2’- and 3’-monophosphates. DNA, which lacks 2’ hydroxyl, is stable under similar conditions.
DNA Base pairing Anti-parallel strands Major groove is 22A wide. Minor groove is 13A wide
Rotation b a g d e c z
Endo and Exo four of the five atoms are in a single plane. The fifth atom (C-2′ or C-3′) is on either the same (endo) or the opposite (exo) side of the plane relative to the C-5′ atom.
Stacked Twist Adjacent Bases Slide Roll
Replication The magic of anti-parallel strands- Perfect duplication of DNA Synthesis of DNA chain ONLY occurs in 5’ to 3’ direction
5’ to 3’ 3’ 5’ 3’ 5’ 5’ 3’ 3’ 5’ 3’ 5’ 5’ Direction of replication
Leading and lagging strands 3’ 5’ Lagging strand Leading strand Okazaki fragments 5’ 3’
Semi conservative Dispersive Semi conservative Conservative
Semi conservative Semi-conservative Conservative Dispersed
T antigen double hexamer Binds to origin Unwinding origin T ag recruit RP-A T ag and RP-A recruit polPrimase Replication initiates RNA synthesis followed by DNA synthesis Steps in SV40 replication RF-C, PCNA, polrecruited polto polswitch Replication elongation
The single chromosome of the prokaryote Escherichia coli is about 1.3 mm of DNA. A human cell contains about 2 m of DNA (1 m per chromosome set) The human body consists of approximately 1013 cells and therefore contains a total of about 2 × 1013 m of DNA. Distance from the earth to the sun is 1.5 × 1011 m The DNA in your body could stretch to the sun and back about 50 times. The diameter of the nucleus is 5x10-6 meters How is the DNA packaged? Chromatin= DNA +histones +non-histones 1g +1g +1g Chromatin
Four histone proteins H2A H2B H3 H4 Very highly conserved DNA is wrapped around the outside of the histone octamer 166 bp of DNA wraps around the histones Linker DNA connects nucleosomes 7 fold compaction Histone H1 Nucleosome- Histones
Nucleosomes 2 mol H2A 2 mol H2B 2 mol H3 2 mol H4 1 mol H1 ~200 bp DNA Ionic interactions between basic positively charged histones and negatively charged phosphates in DNA
Sequence recognition Each base pair can be identified by characteristic chemical groups that lie along the edge of the base pair exposed in the major or minor groove
Lambda repressor The lambda repressor is a dimer also called cI protein. It binds DNA via helix-turn-helix motif. Regulates transcription of cI and Cro protein. Absence of cI protein, cro gene may be transcribed. In the presence of cI, only cI gene may be transcribed.
Lambda repressor Sequence recognition Diameter of major groove=22A Diameter of minor grooe =13A Diameter of alpha helix= 12A
Why a dimer? Co-operativity! A single operator binds one dimer Non-cooperative would be hyperbolic curve Cooperative would be sigmoid curve