nucleotides and nucleic acidsjm
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Nucleic Acids ObjectivesTRANSCRIPT
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 7 N Nitrogen 8 O Oxygen 1 H Hydrogen 15 P Phosphorus
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 guaninePure
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 Length =
34 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 ' end Space-filling model: atoms represented at their van
der Waals radii (electron cloud volumes) Aromatic stacking Hydrogen
bonds easily disassembled Strong 3' end ' 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 per 10 bp) x (10-10 m per ) = ~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
MacromoleculeSimple Molecule Structure 4. Nucleic Acidsnucleotides
(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 (53 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-CA-T
deoxyribose sugar double stranded nucleus 4 bases: guanine (G),
cytosine (C), adenine (A), uracil (U) G-CA-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 forthe 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) hassubtle (small) effectson 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 anA260 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 torsionalenergy 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 strandsand
change the linking number in steps of 2. Gyrase:introduce the
negative supercoiling (resolving the positive one and using the
energy fromATP 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.