chapter 8 Flashcards
Functions of Nucleotides and Nucleic Acids
Nucleotide Functions:
Energy for metabolism (ATP)
Enzyme cofactors (NAD+)
Signal transduction (cAMP)
Nucleic Acid Functions:
Storage of genetic info (DNA)
Transmission of genetic info (mRNA)
Processing of genetic information (ribozymes)
Protein synthesis (tRNA and rRNA)
Nucleotides and Nucleosides
Nucleotide =
Nitrogeneous base
Pentose
Phosphate
Nucleoside =
Nitrogeneous base
Pentose
Nucleobase =
Nitrogeneous base
Phosphate Group
-Negatively charged at neutral pH
-Typically attached to 5’ position
–Nucleic acids are built using 5’-triphosphates
—ATP, GTP, TTP, CTP
-Nucleic acids contain one phosphate moiety per nucleotide
-May be attached to other positions
Pentose in Nucleotides
β-D-ribofuranose in RNA
β-2’-deoxy-D-ribofuranose in DNA
Different puckered conformations of the sugar ring are possible
___puckered conformations for
4 puckered conformations for
ribofuranose rings in nucleotides
Nucleobases
-Derivatives of pyrimidine or purine
-Nitrogen-containing heteroaromatic molecules
-Planar or almost planar structures
-Absorb UV light around 250–270 nm
Pyrimidine Bases
-Cytosine is found in both DNA and RNA
-Thymine is found only in DNA
-Uracil is found only in RNA
All are good H-bond donors and acceptors
-Cytosine pKa at N3 is 4.5
-Thymine pKa at N3 is 9.5
-Neutral molecules at pH 7
Purine Bases
-Adenine and guanine are found in both RNA and DNA
-Also good H-bond donors and acceptors
-Adenine pKa at N1 is 3.8
-Guanine pKa at N7 is 2.4
-Neutral molecules at pH 7
β−N-Glycosidic Bond
-In nucleotides the pentose ring is attached to the nucleobase via
N-glycosidic bond
-The bond is formed to the anomeric carbon of the sugar in β configuration
-The bond is formed:
–to position N1 in pyrimidines
–to position N9 in purines
-This bond is quite stable toward hydrolysis, especially in pyrimidines
-Bond cleavage is catalyzed by acid
Conformation around N-Glycosidic Bond
-Relatively free rotation can occur around the N-glycosidic bond in free nucleotides
-The torsion angle about the N-glycosidic bond (N-C1’) is denoted by the symbol c
-The sequence of atoms chosen to define this angle is O4’-C1’-N9-C4 for purine, and O4’-C1’-N1-C2 for pyrimidine derivatives
-Angle near 0° corresponds to syn conformation
-Angle near 180° corresponds to anti conformation
-Anti conformation is found in normal B-DNA
Structural variation in DNA.
Structural variation in DNA.The conformation of a nucleotide in DNA is affected by rotation about seven different bonds. Six of the bonds rotate freely. The limited rotation about bond 4 gives rise to ring pucker. This conformation is endo or exo, depending on whether the atom is displaced to the same side of the plane as C-5′ or to the opposite side.
For purine bases in nucleotides, only
Structural variation in DNA.For purine bases in nucleotides, only two conformations with respect to the attached ribose units are sterically permitted, anti or syn. Pyrimidines occur in the anti conformation.
UV Absorption of Nucleobases
-Absorption of UV light at 250–270 nm is due to π → π* electronic transitions
-Excited states of common nucleobases decay rapidly via radiationless transitions
–Effective photoprotection of genetic material
–No fluorescence from nucleic acids
Polynucleotides
-Covalent bonds formed via phosphodiester linkages
–negatively charged backbone
-DNA backbone is fairly stable
–DNA from mammoths?
–Hydrolysis accelerated by enzymes (DNAse)
-RNA backbone is unstable
–In water, RNA lasts for a few years
–In cells, mRNA is degraded in few hours
-Linear polymers
-No branching or cross-links
-Directionality
–5’ end is different from 3’ end
–We read the sequence from 5’ to 3’
Phosphodiester linkages in the covalent backbone of DNA and RNA.
Phosphodiester linkages in the covalent backbone of DNA and RNA. The phosphodiester bonds (one of which is shaded in the DNA) link successive nucleotide units. The backbone of alternating pentose and phosphate groups in both types of nucleic acid is highly polar. The 5′ and 3′ ends of the macromolecule may be free or may have an attached phosphoryl group.
Hydrolysis of RNA
-RNA is unstable under alkaline conditions
-Hydrolysis is also catalyzed by enzymes (RNase)
-RNase enzymes are abundant around us:
–S-RNase in plants prevents inbreeding
–RNase P is a ribozyme (enzyme made of RNA) that processes tRNA -precursors
–Dicer is an enzyme that cleaves double-stranded RNA into oligonucleotides
—protection from viral genomes
—RNA interference technology
Chemical instability of RNA in alkaline pH can be used to
Chemical instability of RNA in alkaline pH can be used to purify DNA. Why and How?
Hydrogen-Bonding Interactions
-Two bases can hydrogen bond to form a base pair
-For monomers, large number of base pairs is possible
-In polynucleotide, only few possibilities exist
-Watson-Crick base pairs predominate in double-stranded DNA
-A pairs with T
-C pairs with G
-Purine pairs with pyrimidine
Discovery of DNA Structure
One of the most important discoveries in biology
Why is this important?
“This structure has novel features which are of considerable biological interest”
―Watson and Crick, Nature, 1953
Good illustration of science in action
-Missteps in the path to a discovery
-Value of knowledge
-Value of collaboration
-Cost of sharing your data too early
DNA Can Form Three Types of Helices
-Because of DNA’s flexibility it can exist in 3 forms
-The biologically active form is B form
-The A form is found when precipitating DNA
-Z form are relatively rare and may play a role in gene regulation
left handed z form, a and b right handed
Chargaff’s rules:
Chargaff’s rules:
-The base composition of DNA generally varies from one species to another.
-DNA specimens isolated from different tissues of the same species have the same base composition.
-The base composition of DNA in a given species does not change with an organism’s age,nutritional state,or changing environment.
-In all cellular DNAs,regardless of the species,the number of adenosine residues is equal to the number of thymidine residues(that is,A = T),and the number of guanosine residues is equal to the number of cytidine residues(G = C).From these relationships it follows that the sum of the purine residues equals the sum of the pyrimidine residues; that is,A + G = T + C.
Complementarity of DNA Strands
-Two chains differ in sequence
(sequence is read from 5’ to 3’)
-Two chains are complementary
-Two chains run antiparallel
Replication of Genetic Code
-Strand separation occurs first
-Each strand serves as a template
for the synthesis of a new strand
-Synthesis is catalyzed by enzymes
known as DNA polymerases
-Newly made DNA molecule has one
daughter strand and one parent strand.
“It has not escaped our notice that the specific pairing
we have postulated immediately suggests a possible
copying mechanism for the genetic material.”
―Watson and Crick, Nature, 1953
Restriction Enzyme - Action of EcoRI
used to cut out a portion of DNA, recognize sequence and cut out and add a new piece and reform bond
Palindromic sequences can form hairpins and cruciforms
Palindromes: words or phases that are the same when read backward or forward:
Saippuakuppinippukauppias
(Finnish word for “soap cup batch trader”)
Νίψον ἀνομήματα, μὴ μόναν ὄψιν
(Ancient Greek fountain text: “Wash the sin as well as the face”)
Messenger RNA: Code Carrier for the Sequence of Proteins
-Is synthesized using DNA template
-Contains ribose instead of deoxyribose
-Contains uracil instead of thymine
-One mRNA may code for more than one protein
-Together with transfer RNA (tRNA) transfers genetic information from DNA to proteins
Bacterial mRNA
Schematic diagrams show:(a)monocistronic and (b)polycistronic mRNAs of bacteria.
Bacterial mRNA
Schematic diagrams show:(a)monocistronic and (b)polycistronic mRNAs of bacteria. Red segments represent RNA coding for a gene product; gray segments represent noncoding RNA. In the polycistronic transcript, noncoding RNA separates the three genes.
RNA molecules have quite complex structures
The product of transcription of DNA is always single-stranded RNA, which tends to assume a right-handed conformation dominated by base-stacking interaction
Typical right-handed stacking pattern of single-stranded RNA (Bases in yellow, Phosphorus atoms in orange, and riboses and phosphate oxygens in green)
The paired RNA with perfectly complementary seq is often an
The paired RNA with perfectly complementary seq is often an A-form right-handed double helix. B-form has not been observed, Z-form can be made in lab with high salt or high T.
Secondary structure of RNAs.(a)Bulge, internal loop, and hairpin loop.(b)An A-form right-handed helix for a hairpin.
Even more complex structures of RNA
Base-paired helical structures in an RNA
The secondary structure of the M1 RNA component of the enzyme RNase P of E. coli, with many hairpins.
The two brackets: additional complementary sequences that may be paired in the 3D structure.
The blue dots: non-Watson-Crick G=U base pairs (boxed inset).
Note that G=U base pairs are allowed only when presynthesized strands of RNA fold up or anneal with each other. There are no RNA polymerases that insert a U opposite a template G, or vice versa, during RNA synthesis.
A t-RNA
A t-RNA
(with some unusual base-pairing pattern)
A hammerhead ribozyme
A segment of mRNA
DNA Denaturation
Covalent bonds remain intact
–Genetic code remains intact
Hydrogen bonds are broken
–Two strands separate
Base stacking is lost
–UV absorbance increases
Denaturation can be induced by high temperature, or change in pH
Denaturation may be reversible: annealing
-Native DNA at 25ºC and pH 7 is very
-When T >
-Disrupt H bonds but backbone
-Renaturation is a rapid
-IF completely denatured- 2 step process-
-annealing
-Native DNA at 25ºC and pH 7 is very viscous
-When T > 80ºC or at extreme pH, viscosity decreases sharply
-Disrupt H bonds but backbone covalent bonds remain intact
-Renaturation is a rapid one step process if a section of DNA strands is undisrupted
-IF completely denatured- 2 step process-
–Requires a section of the double helix to reform by reuniting a dozen or more residues in the same area by random collisions- slow
–Successive unpaired bases zipper to form a double helix- fast
DNA Reversible denaturation and annealing (renaturation)
Thermal DNA Denaturation (Melting)
-DNA exists as double helix at normal temperatures
-Two DNA strands dissociate at elevated temperatures
-Two strands re-anneal when temperature is lowered
-The reversible thermal denaturation and annealing form basis for the polymerase chain reaction
-DNA denaturation is commonly monitored by UV spectrophotometry at 260 nm
Factors Affecting DNA Denaturation
-The midpoint of melting (Tm) depends on base composition
–High CG increases Tm
-Tm depends on DNA length
–Longer DNA has higher Tm
–Important for short DNA
-Tm depends on pH and ionic strength
–High salt increases Tm
Two near-complementary DNA strands can hybridize
Detection of a specific DNA molecule in complex mixture
- radioactive detection
- fluorescent DNA chips
Amplification of specific DNA
- polymerase chain reaction
- site-directed mutagenesis
-Evolutionary relationships
-Antisense therapy
DNA hybridization
Two complementary strands have the ability to detect each other in solution.
The complements can come from the same source, or they can be from different sources
If they are from different sources they are hybrids
Molecular Mechanisms of Spontaneous Mutagenesis
-Mutation – alternation in DNA structure that produce permanent changes in the genetic information encoded therein
-Deamination – loss of the exocyclic amino group
-Depurination/Depyrimidination – hydrolysis of the N-glycosidic bond between the base and the pentose, to create a DNA lesion called an AP (apurinic, apyrimidinic) site or abasic site
-Cells have mechanisms to correct most of these modifications
Deamination is spontaneous- does not break the backbone
-Loss of their exocyclic amino group
-Very slow reactions
-Some can be severe such as cytosine to uracil occurring about 1 in every 107 residues every 24 hours
-Deamination of A and G: 1/100th of C to U
-This may be one reason that uracil is not used in DNA, but T
Depurination
-Process is slow and not considered physiologically significant
-Can be accelerated by dilute acid. e.g. incubation at pH3 causes selective removal of the purine bases to give the apurinic acid
-Does not break the covalent backbone but it causes a loss in information
-Occurring rate: Purines > Pyrimidines
Molecular Mechanisms of Oxidative and Chemical Mutagenesis
Oxidative damage
-Hydroxylation of guanine
-Mitochondrial DNA is most susceptible
Chemical alkylation
-Methylation of guanine
Cells have mechanisms to correct most of these modifications
Molecular Mechanisms of Radiation-Induced Mutagenesis
UV light induces dimerization of pyrimidines; this may be the main mechanism for skin cancers.
Ionizing radiation (X-rays and γ-rays) causes ring opening and strand breaking .
These are difficult to fix.
Cells can repair some of these modifications, but others cause mutations. Accumulation of mutations is linked to aging and carcinogenesis.
Other Functions of Nucleotides
Energy Source
Some Coenzymes containing adenosine
Acyl Group Transfer
Electron Transfer
Hydride Transfer
Other Functions of Nucleotides:
Regulatory Molecules
Second Messengers
Produced in bacteria in response to a slowdown in protein synthesis during AA starvation
DNA Sequenceing
Dideoxy Sequencing of DNA
DNA Sequenceing Applications & DNA Technology
Y-STR testing is useful for forensic, relationship and genealogy testing
DNA Sequenceing Applications & DNA Technology
Genomic DNA Can Be Edited Nowadays
CRISPR:
Clustered Regularly Interspaced Short Palindromic Repeats
