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Ch 8: Nucleotides and Nucleic Acids, 8.1 Some Basic Definitions and…
Ch 8: Nucleotides and Nucleic Acids
Nucleic acids are both repositories and functional expressions of biological information.
Nucleoside triphosphates occupy a central role in cellular metabolism, serving as an energy currency and as important regulatory signals.
8.1 Some Basic Definitions and Conventions
The functions of DNA
storage of biological information
transmission of that information to the next generation
gene: a segment of a DNA molecule that contains the information required for the synthesis of a functional biological product, whether protein or RNA
Several Classes of RNA:
Ribosomal RNAs (rRNAs): components of ribosomes
messenger RNAs (mRNAs) intermediates in protein synthesis
transfer RNAs (tRNAs): adapter molecules that translate the information in mRNA into a specific amino acid sequence
noncoding RNAs (ncRNAs): wide variety of functions
nucleotides and nucleic acids have characteristic bases and pentoses
nucleotides have three components:
a nitrogenous base (purine or pyrimidine)
types:
major purine bases:
guanine (G)-- found in DNA and RNA
adenine (A)--found in DNA and RNA
major pyrimidine bases:
cytosine (C)--found in DNA and RNA
thymine (T)--only found in DNA
uracil (U)--only found in RNA
the properties of nucleotide bases affect the 3D structure of nucleic acids
weakly basic compounds
aromatic molecules
because most bonds in the ring have partial double-bond character:
pyrimidines are planar
purines have a slight pucker
they absorb light
strong absorption near 260 nm
may exist in readily interconverted forms called tautomers (lactan, lactim, and double lactim) that depend on pH
a pentose
1+ phosphates
nucleoside: the nucleotide molecule without a phosphate group
nucleotide pentoses
two kinds:
2'-deoxy-d-ribose found in DNA
D-ribose, found in RNA
in beta-furanose form
structure and names of the four major deoxyribonucleotides
deoxyribonucleotides: structural units of DNA; also called deoxyribonucleoside 5'-monophosphates, deoxynucleotides, and deoxynucleoside triphosphates
lowercase d indicates deoxy-version
structure and names of the four major ribonucleotides
ribonucleotides: structural units of RNA; also called ribonucleoside 5'-monophosphates
nucleotides with phosphate groups in different positions:
adenosine 3',5'-cyclic monophosphate (cAMP) is sourced from ATP
guanosine 3',5'-cyclic monophosphate is sourced from cGMP
phosphodiester linkage: covalent bond that joins successive nucleotides of both RNA and DNA
occur between 5'-phosphate group of one nucleotide unit and the 3'-hydroxyl group of the next nucleotide
hydrolysis of DNA and RNA
under alkaline conditions:
RNA is rapidly hydrolyzed due to the presence of 2'-hydroxyl groups
DNA is not rapidly hydrolyzed
solubility of nucleotides
hydrophobic and relatively insoluble in pH 7.0 water
leads to stacking interactions via van der Waals and dipole-dipole
charged and more soluble at acidic or alkaline pH values
Base pairing permits the duplication of genetic information
base pairs: hydrogen-bonding patterns between complementary strands of nucleic acids
A bonds specifically to T and U
G bonds specifically to C
8.3 Nucleic Acid Chemistry
The role of DNA as a repository of genetic information
depends in part on its inherent stabilty
chemical transformations are generally very slow in the absence of enzyme catalysts
carcinogenesis and aging may be linked to slowly accumulating, irreversible DNA alterations
Double-helical DNA and RNA can be denatured
due to pH extremes or high temperatures
denaturation temperature, tm: temperature at which 1/2 of DNA is present as separated single strands
Increases with content of G-=C base pairs
disrupts hydrogen bonds and base-stacking interactions
in partially denatured DNA....
denatured regions form bubbles
often rich in A=T base pairs
denaturation of double-stranded RNA and RNA-DNA hybrids:
RNA duplexes are more stable to heat denaturation than DNA duplexes
RNA-DNA hybrid stability is generally intermediate
Double-helical DNA and RNA can anneal
anneal: two step process by which two strands spontaneously rewind when temperature or pH is returned to its normal range
hypochromic and hyperchromic effects:
hypochromic effect: the observed decrease in the absorption of UV light when complementary strands are paired
hyperchromic effect: the observed increase in the absorption of UV light when a double-stranded nucleic acid is denatured
monitoring UV absorption at 260 nm can detect the transition from double-stranded to single-stranded DNA
nucleotides and nucleic acids undergo nonenzymatic transformations
mutations: alterations in DNA structure that produce permament changes in the genetic information encoded
linked to aging and carcinogenesis
deamination reactions
deamination: spontaneous loss of exocyclic amino groups
deamination of cytosine to uracil occurs about 100 times/day
recognized as foreign in DNA and removedQ
almost certainly why DNA contains thymine rather than uracil
depurination reactions
depurination: hydrolysis of the N-Beta-glycosyl bond between the base and the pentose
creates an AP (apurinic, apryimidinic) site or abasic site
most common among purines
UV radiation can result in a 6-4 photoproduct, a type of pyrimidine dimer
Ionizing radiation can cause ring opening and fragmentation of bases and breaks in the covalent backbone of nucleic acids
DNA Damage by reactive chemicals
nitrous acid precursors: deaminating agents
alkylating agents: generate modified nucleotides nonenzymatically
can methylate guanine to O6-methyl-guanine, which cannot base-pair with cytosine
DNA damage by oxidative damage
reactive oxygen species (hydrogen peroxide, hydroxyl radicals, superoxide radical) damage DNA
hydroxyl radicals are responsible for most oxidative DNA damage
cells have an elaborate defense system to destroy reactive oxygen species
Some bases of DNA are methylated
A and C are methylated more frequently than G and T
all known DNA methylases use S-adenosylmethionine as a methyl group donor
in eukaryotes, 5% of cytidine residues are methylated
affects DNA metabolism and gene expression
The chemical synthesis of DNA has been automated
synthesis of DNA by phosphoramidite method is highly efficient
gene sequences can be amplified with the polymerase chain reaction
polymerase chain reaction (PCR): method of amplifying DNA segments of interest
relies on DNA polymerases (enzymes that synthesize DNA form deoxyribonucleotides (dNTPs) using a DNA template)
DNA polymerases add nucleotides to teh 3' ends of preexisting strands called primers
Process:
Heat to separate strands.
Add synthetic DNA oligo-nucleotide primers and cool.
Add thermostable Taq DNA polymerase to catalyze 5'-3' DNA synthesis.
Repeat steps 1-3. After 20 cycles, the target sequences has been amplified about 10^6 fold.
PCR tech is highly sensitive
can detect and amplify just one DNA molecule in almost any sample type
uses:
cloning of rare, undegraded DNA segemtns from 40,000+ years ago
tracing evolution
potent tool in forensic medicine
detecting viral infections and cancers before they cause symptoms
prenatal diagnosis of genetic diseases
the sequences of long DNA strands can be determined
sanger sequencing: dideoxy chain-termination sequencing
nucleotide analogs called dideoxynucleoside triphosphates (ddNTPs) interrupt synthesis
each of the four is labeled with a different-colored fluorescent tag
8.2 Nucleic Acid Structure
hierarchical levels of nucleic acid structure
primary structure: covalent structure and nucleotide sequence
secondary structure: regular, stable structure taken up by some or all the nucleotides
Tertiary structure: complex folding of large chromosomes or the elaborate folding of tRNA or rRNA structures
DNA is usually a double helix that stores genetic information
x-ray diffraction pattern revealed that DNA molecules are helical
DNA can occur in different 3D forms
The Three forms of DNA:
B-Form DNA
Watson-Crick Model: offset pairing of the two DNA strands creates a major groove and a minor groove
most stable for a random-sequence DNA molecule under physiological conditions
A-Form DNA
right handed double helix with a wider helix, 11bp/turn, and a tilted plane
favored in solutions devoid of water
Z-form DNA
left-handed helix with 12 bp/turn and a backbone with a zig-zag appearance
appears more slender and elongated
found in streches
structural variation in DNA reflects:
different possible conformations of the deoxyribose
rotation about the contiguous bonds making up the phosphodeoxyribose backbone
free rotation about the C-1'-N-glycosyl bond
DNA strands are antiparallel, with 3',5'-phosphodiester bonds running in opposite directions
double-helical DNA strands are complementary:
when A occurs in one chain, T is found in the other
when G occurs in one chain, C is found in the other
the double helix is stabilized by:
NOT hydrogen bonding
metal cations that shield the negative charges of backbone phosphates
base stacking interactions between successive base pairs
successive G-C or C-G are stronger than successive A-T or T-A
duplexes with higher G-C context are more stable
replication process:
Preexisting or "parent" strands become separated
Each "parent" strand serves as a template for the biosynthesis of a complementary "daughter strand"
Certain DNA sequences adopt unusual structures
palindrome: region of DNA that is identical when read either forward or backward
applied to regions of DNA with inverted repeats
can form hairpin and cruciform structures from the self-complementarity within each strand
Mirror repeat: sequence when the inverted repeat occurs wihtin each individual strand
does not form hairpins or cruciform structures
DNA Structures containing three DNA strands
Hoogsteen pairing: non-Watson-Crick pairing that results in triplex DNAs
Hoogsteen Positions: N-7, )6, and N6 of purines; participate in the hydrogen bonding with a third DNA strand
Plays functional roles such as telomere maintenance
DNA structures containing four DNA strands
tetraplex DNAs: occur when four DNA strands pair
occur readily only for DNA sequences with a very high proportion of G residues
G tetraplex: very stable form
Structure of RNAs
Messenger RNAs code for polypeptide chains
mRNA can be monocistronic or polycistronic
monocistronic: codes for only one polypeptide; true of most mRNAs in eukaryotes
polycistronic: codes for 2+ different polypeptides; occurs in bacteria and archaea
3D structure:
right handed helical conformation
always single-stranded
dominated by base-stacking interactions, strongest between two purines
can base pair with complementary regions of DNA or RNA in antiparallel form
transcription: process by which mRNAs are formed on a DNA template
secondary structure of RNAs
structure of complementary RNA strands is an A-form right-handed double helix
breaks caused by mismatched or unmatched bases result in bulges or internal loops
internal loops form between palindromic sequences
base-paired helical structures in RNA result in extensive helical segments, with hairpins being the most common type of secondary structure
8.4 Other Functions of Nucleotides
nucleotides carry chemical energy in cells
hydrolysis of nucleoside triphosphates provides chemical energy
ATP is the most widely used
when coupled to a reaction with a positive free-energy change, ATP hydrolysis shifts the equilibrium to favor product formation
hydrolysis of the ester linkage yields about 14 kJ/mol under standard conditions
hydrolysis of each anhydride bond yield about 30 kJ/mol
Adenine nucleotides are components of many enzyme cofactors
adenosine does not participate directly in the primary function, but its removal reduces cofactor activities
nucleotide-binding fold: single protein domain that binds adenosine
Some nucleotides are regulatory molecules
second messengers: compounds produced inside the cell following the interaction of extracellular chemical signals with receptors
often a nucleotide, like cAMP
ppGpp: produced in bacteria during amino acid starvation to inhibit the synthesis of the rRNA and tRNA molecules
ATP and ADP serve as:
neurotransmitters in a variety of signaling pathways
signals for receptors that mediate pain sensation
blood clotting signals
