Nucleotides and Nucleic Acids
The average mRNA is about
1500 base pairs but varies depending on the size of the protein which it encodes and can be much larger.
Their small subunit has a
16S RNA subunit (≈1540 nucleotides) and 21 proteins.
The large subunit has a
5S RNA subunit (≈120 nucleotides), a 23S RNA subunit (≈2900 nucleotides) and 31 proteins.
• Prokaryotes have
70S ribosomes, consisting of a small (30S) and a large (50S) subunit. • Their small subunit has a 16S RNA subunit (≈1540 nucleotides) and 21 proteins. • The large subunit has a 5S RNA subunit (≈120 nucleotides), a 23S RNA subunit (≈2900 nucleotides) and 31 proteins.
• Eukaryotes have
80S ribosomes, consisting of a small (40S) and large (60S) subunit. Their 40S subunit has an 18S RNA (≈1900 nucleotides) and 33 proteins. The large subunit has 5S RNA (≈120 nucleotides), 28S RNA (≈ 4700 nucleotides), and 5.8S RNA (≈160 nucleotides) subunits and 46 proteins.
DNA-Sequencing Methodology Has Improved Over Time.
A variation of Sanger's sequencing method, in which the dideoxynucleotides used for each reaction are labeled with a differently colored fluorescent tag was used to automate large DNA-sequencing is still used for routine sequencing of genes (and still considered the "gold standard" for sequencing. However, modern sequencing now makes use of vastly more efficient methods, called next-generation or next-gen sequencing.
Nucleotides have three characteristic component:
(1) a nitrogen containing (or nitrogenous) base, (2) a ribose or deoxyribose sugar and (3) one or more phosphates.
Hydrolysis of the ester linkage yields about 14 kJ/mol under standard conditions
, whereas hydrolysis of each anhydride bond yields about 30 kJ/mol.
Nucleotides and Nucleic Acids Undergo
Nonenzymatic Transformations. Purines and pyridines as well as their nucleotides undergo spontaneous chemical changes. Although the rates are low this can be significant since can lead to permanent changes to DNA sequences called mutations.
As many as one in 105 purines per day under typical cellular conditions.
The deoxyribose remaining after depurination is readily converted from the beta-furanose to the aldehyde form, further destabilizing the DNA at this position
energy transduction
The energy of the stimulus is converted into electrical energy.
• When solutions of double-stranded DNA and RNA are subjected to extremes of pH or to temperatures above 80°C the absorbance goes up and the viscosity goes down.
This is called the hyperchromic effect and is indicative of denaturation
The nucleotide sequence of RNA molecules and the amino acid sequence of proteins is specified by
a nucleotide sequence in cell's DNA
Another type of DNA sequence is
a palindrome. A palindrome is a word, phrase, or sentence that is spelled identically read either forward or backward. The term is applied to regions of DNA with inverted repeats of base sequence. These sequences are self-complementary within each strand and can form hairpin or cruciform (cross-shaped) structures.
Transfer RNAs (tRNAs)
adapter molecules that allow translation of information from mRNA into a specific sequence of amino acids.
Often, the second messenger is
adenosine 3',5' - cyclic monophosphate (cyclic AMP, or cAMP), formed from ATP in a reaction catalyzed by adenylyl cyclase
A variety of enzyme cofactors serving a wide range of chemical functions include
adenosine as part of their structure
The significance of the central dogma is that
after the DNA model was proposed it provided a framework for carrying out further experiments to elucidate information flow from DNA to RNA to proteins.
Hoogsteen pairing
allows the formation of triplex DNAs. The triplex DNAs may contain two pyrimidine strands and one purine strand or visa versa
• Another spontaneous reactions is hydrolysis of the N--glycosyl bond between the base and the sugar, to create a DNA lesion called
an AP (apurinic, apyrimidinic) site or abasic site. This occurs at a higher rate for purines than for pyrimidines
After heating, if the temperature is then reduced to 25°C the absorbance decreases
and is related to the complementary strands rewinding or annealing.
The paired strands in RNA-RNA or RNA-DNA duplexes
are antiparallel as in DNA
Messenger RNAs (mRNAs)
are carry genetic information from the genes to a ribosome, where proteins are synthesized.
The three-dimensional structures of many RNAs, like those of proteins
are complex and unique
Ribosomal RNAs (rRNAs)
are components of ribosomes, the complexes that carry out the synthesis of proteins.
Several other classes RNAs have other functions. There
are over 20 other types of RNAs. Many associated with gene regulation.
Nucleotides are Comprised of
base, sugar, phosphate
Hydrogen bonds involving the amino and carbonyl groups between two complementary strands of nucleic acids are key to
both the structure and function of DNA and RNA.
Cells have an elaborate defense system to destroy reactive oxygen species, including
enzymes catalase and superoxide dismutase that convert reactive oxygen species to harmless products
One case is bending of the DNA helix wherever
four or more adenosine residues appear sequentially in one strand. For example, six adenosines in a row produce a bend of about 18°. The bending may be important in the binding of some proteins to DNA.
The (tm) is defined as the temperature at which
half the DNA molecules are separated into single strands.
Base pairing matches the pattern for DNA
i.e. G pairs with C and A pairs with U. This is unusual in DNA but is allowed in RNA
• Breaks in the regular A-form helix caused by mismatched bases in one or both strands are common and result
in bulges or internal loops.
DNA methylation
is a process by which methyl groups are added to the DNA molecule.
Another regulatory nucleotide, ppGpp, is
is produced in bacteria in response to a slowdown in protein synthesis during amino acid starvation
• The denaturation or melting temperature (tm)
is related to the G≡C content of the DNAmolecules.
So because DNA is comprised of 2'-deoxyribonucleotides
it is chemically more stable than RNA
Each tRNA has
its corresponding amino acid attached to its end.
The Z form (for zigzag) of DNA is
left-handed with 12 base pairs per helical turn. The DNA backbone has a zigzag appearance because the purine residues flip to the syn conformation alternating with pyrimidines in the anti. The structure appears slenderer and more elongated. The major groove is barely apparent in Z-DNA, and the minor groove is narrow and deep.
Adenine and cytosine are
methylated more often than guanine and thymine.
• Self-complementary sequences in the molecule produce
more complex structures.
RNAs have
more functions and may be divided into several classes.
Hairpin loops form between
n nearby self-complementary (palindromic) sequences
DNA and RNA Molecules Are Known as
nucleic acids
DNA and RNA molecules are comprised of chains of building blocks called
nucleotides
The covalent backbone of RNA is subject to slow, nonenzymatic, hydrolysis
of the phosphodiester bonds
The Central Dogma states that
once 'information' has passed into protein it cannot get out again. In more detail, the transfer of information from nucleic acid to nucleic acid, or from nucleic acid to protein may be possible, but transfer from protein to protein, or from protein to nucleic acid is impossible. Information means here the precise determination of sequence, either of bases in the nucleic acid or of amino acid residues in the protein
• ROS such as superoxide radical ( •O2 - ), hydrogen peroxide (H2O2 ) and hydroxyl radicals ( •OH- ), are responsible for
oxidative DNA damage
The nucleotides are named as
phosphate derivatives of the various nucleosides
The alpha,beta and beta,y linkages are .
phosphoanhydrides
Nucleotides in Nucleic Acids Are Linked by
phosphodiester bonds
Exogenous ROS can be produced from
pollutants, tobacco smoke, drugs, xenobiotics, and radiation.
Watson-Crick base pairing (i.e. A pairs with T and G pairs with C) was a key both to discovering the
structure of DNA and later to understanding its function in information storage and transfer
Avery, MacLeod and McCarty experiment (1944)
suggested that DNA and not RNA or protein was the transforming principle.
Alkylating agents
synthetic chemicals containing alkyl groups that attack DNA, causing strand breaks
Purine and pyrimidine bases can exist in
tautomeric forms (amino versus imino or lactam versus lactim).
Messenger RNA (mRNA) provides a
template for the amino acid sequence in a protein. It is single-stranded and is synthesized during transcription where a complementarity copy of a DNA (gene) sequence is formed.
Four DNA strands can also pair to form a
tetraplex, but this occurs readily only for DNA sequences with a high proportion of guanosine. The guanosine tetraplex, or G tetraplex, is stable over a wide range of conditions.
The nucleotides of both DNA and RNA are covalently linked through phosphate -group "bridges," in which
the 5' -phosphate group of one nucleotide unit is joined to the 3' -hydroxyl group of the next nucleotide, creating a phosphodiester linkage
Ribosomal ribonucleic acid (rRNA) is
the RNA component of the ribosome and is essential for protein synthesis
The chain has a free 5' -phosphate group on one end and a free 3' -hydroxyl group on the other. This provides
the bases for specifying chain directionality in DNA and RNA.
Energy
the capacity to do work
Hydrolysis of nucleoside triphosphates provides
the chemical energy to drive many cellular reactions.
Watson and Crick Model
the double-helix structure of the DNA molecule
Bioenergetics
the field of biochemistry concerned with the transformation and use of energy in living cells. , the production and utilization of ATP dominates the field.
The interaction of extracellular chemical signals such as hormones ("first messengers") with receptors on the cell surface can lead to
the formation of second messengers inside the cell
The A form is also a right-handed double helix, but
the helix is wider and the number of base pairs per helical turn is 11, rather than 10.5 as in B-DNA. • The plane of the base pairs in A-DNA is tilted about 20° relative to B-DNA.
The greater the G≡C content
the higher the melting temperature. This is due to the fact that there are three H-bonds in the G≡C base pairs and only two H-bonds in the A=T pairs.
Depurination
the loss of a purine base from a nucleotide
The storage and transmission of biological information are
the only known functions of DNA
rRNA constitutes
the predominant material within the ribosome or about 2/3 of ribosome mass
Peptidyl transferase
the primary enzymatic activity of the ribosome which catalyzes the formation of peptide bonds during the translation.
Cyclic 2',3' -monophosphate nucleotides are
the products of the action of alkali on RNA and then rapidly hydrolyzed give a mixture of 2' - and 3' nucleoside monophosphates.
Deamination
the removal of an amino group from an organism, particularly from an amino acid
, two methods for DNA sequencing, the Maxam-Gilbert method and the Sanger method, made
the sequencing of larger DNA molecules with an ease possible
• When a tRNA recognizes and binds to its corresponding codon in the ribosome
the tRNA positions the appropriate amino acid to the end of the growing amino acid chain. Then the tRNAs and ribosome continue to decode the mRNA molecule until the entire sequence is translated into a protein.
The most common hydrogen-bonding patterns are
those proposed by Watson and Crick. They suggested A bonds specifically to T (or U) and G bonds specifically to C. fundamental to both the structure and function of DNA and RNA.
The tRNA molecule has a distinctive folded structure with
three hairpin loops that form the shape of a three-leafed clover. One of these hairpin loops contains a sequence called the anticodon, which can recognize and decode an mRNA codon
Several unusual DNA structures involve
three or even four DNA strands. (ex. hoogsteen pairing)
In 1953 Watson and Crick postulated a
three-dimensional DNA structural model with some of the following characteristics.
Amino acids are specified by
three-nucleotide mRNA sequences called codons.
• DNA has 4 canonical bases as does RNA. Three of the 4 are found in both. However
thymine is unique to DNA and uracil is only in RNA (see below).
Because of steric constraints, purines in purine nucleotides are restricted to
two stable conformations called syn and anti.
Hershey and Chase experiment (1952)
using radioisotopes with bacteriophage provided additional evidence that DNA, not protein, was the genetic material
Structural variation in DNA reflects three things:
• Different possible conformations of the deoxyribose. • Rotation about the contiguous bonds that make up the phosphodeoxyribose backbone. • Free rotation about the C-1'-N-glycosyl bond.
before confirmatory data became available, Watson and Crick suggested how this structure could logically be replicated.
• First by separating the two strands. • And, second by synthesizing a complementary strand for each. • The preexisting or "parent" strands (blue) become separated, and each is the template for biosynthesis of a complementary "daughter" strand (pink)
Alkylating agents can alter certain bases of DNA
• For example, the highly reactive chemical dimethylsulfate can methylate a guanine to yield 0 6 - methylguanine, which cannot base-pair with cytosine. • Similar reactions are brought about by alkylating agents naturally present in cells, such as S-adenosyl methionine
Rosalind Franklin and Maurice Wilkins studies.
• Franklin and Wilkins carried out x-ray diffraction studies to analyze DNA fibers. They showed in the early 1950s that DNA produced a characteristic x-ray diffraction pattern. • From this analysis some of the characteristics of the DNA molecule could be deduced. • Even though Franklin and Wilkins were both at King's College in London, they found it difficult to work together. • This led to Wilkins to interact with his old friend Francis Crick at the University of Cambridge and provide valuable information to him that was critical in solving the DNA structure
The Central Dogma of Molecular Biology
• It is an explanation of the flow of genetic information in a biological system.
E. coli has two prominent methylation systems.
• One (the modification-restriction system) helps the cell to distinguish its DNA from foreign DNA by marking its own DNA with methyl groups and destroying (foreign) DNA without the methyl groups. • The other system methylates adenosine residues in a system that repairs mismatched base pairs formed during DNA replication
Why is adenosine used in these structures?
• The answer here may involve a form of evolutionary economy. • The importance of adenosine probably lies not so much in some special chemical characteristic but in the evolutionary advantage of using one compound for multiple role
Absorption Spectra of the Common Nucleotides
DNA and RNA molecules are often identified and quantified based on having an absorption maximum near 260 nm (recall proteins had an absorption maximum near 280 nm).
In the test tube, RNA is hydrolyzed rapidly under alkaline conditions, but
DNA is not because the 2'-hydroxyl groups in RNA—which are absent in DNA—are involved in the process
When located in a gene promoter
DNA methylation typically acts to repress gene transcription.
• DNA Molecules Can be Separated by
Gel electrophoresis. As with proteins, electrophoresis may be used to separate DNA molecules of differentsizes. Since DNA is a negatively charged at neutral pH it migrates toward the positive pole. Agarose gels are commonly used although smaller DNA molecules may be separated on polyacrylamide gels. Fragments of linear DNA migrate through agarose gels with a mobility that is inversely proportional to the log of their molecular weight, i.e. smaller molecule move faster and larger ones slower.
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. • And the most well known: in all cellular DNAs, regardless of the species, the number of adenosine residues is equal to the number of thymidine residues (that is, A equals T), and the number of guanosine residues is equal to the number of cytidine residues (G equals 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
model consists of (cont)
• The bases of both strands are stacked inside the double helix, are nearly perpendicular (~6o tilt) to the long axis, and also are almost planar. • The hydrophobic ring structures are very close together. • The diameter of the molecule is 20Å. • One helical turn is 34Å with 10 base pairs per turn (although subsequent measurements revealed 10.5 base pairs or 36Å per turn). • The offset pairing of the two strands creates a major groove and minor groove on the surface of the duplex.
Many RNAs Have More Complex Three-Dimensional Structures
• The product of transcription of DNA is always single-stranded RNA. • The single strand tends to assume a right-handed helical conformation dominated by base-stacking interactions, which are stronger between two purines than between a purine and pyrimidine or between two pyrimidines
Watson-Crick Model consists of
• Two helical DNA chains wound around the same axis to form a right-handed double helix. • The two chains are antiparallel (meaning they are running in opposite directions. • The hydrophilic backbones of alternating deoxyribose and phosphate groups are on the outside facing the surrounding water. • The furanose ring of each deoxyribose is in the C-2' endo conformation. • The bases are in pairs, A with T and G with C
Radiation effects on DNA
• UV light induces the condensation of two ethylene groups to form a cyclobutane ring. • In the cell, a similar reaction between adjacent pyrimidine bases in nucleic acids forms cyclobutane pyrimidine dimers (most frequently between adjacent thymidine residues). • A second type of pyrimidine dimer, called a 6-4 photoproduct, is also formed during UV irradiation. • Formation of a cyclobutane pyrimidine dimer introduces a bend or kink into the DNA • Ionizing radiation (x rays and gamma rays) can cause more extensive damage, including ring opening, fragmentation of bases and breaks in the covalent backbone of nucleic acids.
Denaturation
In proteins, a process in which a protein unravels and loses its native conformation, thereby becoming biologically inactive. In DNA, the separation of the two strands of the double helix.
nucleosides
Molecules of this type without a phosphate group
The base of a nucleotide is joined covalently (at N-1 of pyrimidines and N-9 of purines) in an
N-beta-glycosyl bond to the 1' carbon of the pentose The phosphate is esterified to the 5' carbon.
Deaminating agents
Nitrous acid (HN02 ) or compounds that can be metabolized to nitrous acid or nitrites increase deamination as described above.
DNA bases
Adenine, Thymine, Guanine, Cytosine
RNA bases
Adenine, Uracil, Cytosine, Guanine
The Watson-Crick structure is also referred to as
B-form DNA, or B-DNA. The B form is believed to be the most stable structure for most DNA sequences under physiological conditions.
DNA Can Occur in
Different Three-Dimensional Forms
• Several Types of RNAs Are Involved In
Information Transfer.
Endogenous ROS production
ROS are produced in cells through multiple mechanisms such as NADPH oxidase, the mitochondrial electron transport chain, and others.
The nucleosides and nucleotides have 2 ring different systems
The base rings numbered with nonprime numbers and the pentose ring with prime numbers
ester linkage
The bond between the ribose and the alpha phosphate
gene
The segment of a DNA molecule that contains the information required for the synthesis of a functional biological product, whether an RNA molecule or protein
• Adenosine 5' -triphosphate, ATP, is by far the most widely used for this purpose, but
UTP, GTP, and CTP are also used in some reactions
• Certain DNA Sequences Can Adopt
Unusual Three-Dimensional Structures
• ATP hydrolysis often plays an important thermodynamic role in biosynthesis
When coupled to a reaction with a positive freeenergy change, ATP hydrolysis shifts the equilibrium of the overall process to favor product formation
mirror repeat sequence
When the inverted repeat occurs within each individual strand of the DNA. But mirror repeats do not have complementary sequences within the same strand and cannot form hairpin or cruciform structures.
A-form right handed double helix
When two strands of RNA with perfectly complementary sequences are paired
Reactive oxygen species (ROS)
a group of extremely reactive peroxides and oxygen-containing radicals that may contribute to cellular damage
8-Oxoguanine is one of the most common DNA lesions resulting from reactive oxygen species and can result in
a mismatched pairing with adenine resulting in G to T and C to A substitutions in the genome
• Nucleotides Carry Chemical Energy in
cells
RNA can base-pair with
complementary regions of either RNA or DNA.
The phosphate groups, with a pKa below 1, are
completely ionized and negatively charged at pH 7.
Transfer ribonucleic acid (tRNA) function in
decoding a messenger RNA (mRNA) sequence into a protein. They are small ranging from about 76-90 base pairs.
Double-Helical DNA and RNA Can Be
denatured
The bases are derivatives of two parent compounds
pyrimidine and purine.
Certain nucleotide sequences preferentially fold into Z helices including sequences in which
pyrimidines alternate with purines, especially alternating C and G
Peptidyl transferase activity is mediated by
rRNA not ribosomal proteins
The base stacking and H-bonding in double stranded molecules is believed to
reduce UV absorption at 260 nm compared to single stranded DNA and RNA
Guanosine 3',5' -cyclic monophosphate (cGMP) also has
regulatory functions in many cells.
Griffith's transforming principle experiment (1928)
showed using Streptococcus pneumoniae which infect mice that a nonvirulent type II-R (rough) strain could be "transformed" into a virulent type III-S (smooth) when heat killed type III-S was injected into a mouse with live type II-R.
Sanger's dideoxynucleotide method proved
simpler and became the standard for DNA sequencing for many years
Weak interactions, especially base-stacking interactions, help
stabilize RNA structures, just as they do in DNA.
Pyrimidines are generally restricted to the anti conformation because of
steric interference between the sugar and the carbonyl oxygen at C-2 of the pyrimidine.
Several amino acids undergo spontaneous loss of amino groups
• Under typical cellular conditions, deamination rate of cytosine in DNA to uracil is about 1 in 107 per day. • Deamination of adenine and guanine is lower (about 1 in 109 per day). • Cytosine deamination is probably why DNA contains thymine rather than uracil. • The product of cytosine deamination (uracil) is readily recognized as foreign in DNA and is removed by a repair system. • If DNA normally contained uracil, recognition of uracils produced from cytosine deamination would be more difficult and lead to permanent sequence changes when paired with adenine. • Cytosine deamination would gradually lead to a decrease in G≡Cbase pairs and an increase in A=U base pairs in the DNA of all cells. • Establishing thymine as one of the four bases in DNA may well have been a crucial turning points in evolution and stable storage of genetic information.
