Nucleic Acids and Nucleotides

 Deoxyribonucleic acid (DNA) and ribonucleic acid (RNA), are the chemical carriers of
genetic information
 Nucleic acids are biopolymers made of nucleotides, aldopentoses linked to a purine or
pyrimidine and a phosphate

Sugars in DNA and RNA

 RNA is derived from ribose

 DNA is from 2-deoxyribose
 (the ' is used to refer to positions on the sugar portion of a nucleotide)

Heterocycles in DNA and RNA

 Adenine, guanine, cytosine and thymine are in DNA

 RNA contains uracil rather than thymine

Nucleotides

 In DNA and RNA the heterocycle is bonded to C1 of the sugar and the phosphate is
bonded to C5 (and connected to 3’ of the next unit)

The Deoxyribonucleotides

The Ribonucleotides
 Structure of Nucleic Acids
Nucleotides are join together in DNA and RNA by forming a phosphate ester bond between the
5-phosphate group on one nucleotide and the 3-hydroxyl group on the sugar of another
nucleotide. One end of the nucleic acid polymer has a free hydroxyl at C3 (the 3 end), and the
other end has a phosphate at C5 (the 5 end).

Just as the structure of a protein depends on the sequence in which individual amino acids are
connected, the structure of a nucleic acid depends on the sequence of individual nucleotides. To
carry the analogy further, just as a protein has a polyamide backbone with different side chains
attached to it, a nucleic acid has an alternating sugar-phosphate backbone with different amine
bases attached.

Differences arise from the sequence of bases on the individual nucleotides.

Describing a sequence.

 Chain is described from 5’ end, identifying the bases in order of occurrence, using the
abbreviations A for Adenosine, G for Guanosine, C for cytidine, and T for thymine (or U
for uracil in RNA)
 A typical sequence is written as TAGGCT
 Base Pairing in DNA: The Watson–Crick Model
 In 1953 Watson and Crick noted that DNA consists of two polynucleotide strands,
running in opposite directions and coiled around each other in a double helix
 Strands are held together by hydrogen bonds between specific pairs of bases
 Adenine (A) and thymine (T) form strong hydrogen bonds to each other but not to C or G
 (G) and cytosine (C) form strong hydrogen bonds to each other but not to A or T

H-Bonds in DNA

 The G-C base pair involves three H-bonds

A-T Base Pairing

 Involves two H-bonds

The differences in the strands

 The strands of DNA are complementary because of H-bonding

 Whenever a G occurs in one strand, a C occurs opposite it in the other strand
 When an A occurs in one strand, a T occurs in the other

Complementarity in base pairing in the DNA double helix. The sugar-phosphate backbone runs
along the outside of the helix, while the amine bases hydrogen bond to one another on the
inside. Both major and minor grooves are visible.
Grooves

 The strands of the DNA double helix create two continuous grooves (major and minor)
 The sugar–phosphate backbone runs along the outside of the helix, and the amine bases
hydrogen bond to one another on the inside
 The major groove is slightly deeper than the minor groove, and both are lined by potential
hydrogen bond donors and acceptors.

Nucleic Acids and Heredity

Processes in the transfer of genetic information:

 Replication: identical copies of DNA are made

 Transcription: genetic messages are read and carried out of the cell nucleus to the
ribosomes, where protein synthesis occurs.
 Translation: genetic messages are decoded to make proteins.

Replication of DNA
Replication of DNA is an enzyme-catalyzed process that begins by a partial unwinding of the
double helix. As the strands separate and bases are exposed new nucleotides line up on each
strand in a complementary manner, A to T and C to G, and two new strands begin to grow. Each
new strand is complementary to its old template strand, and two new identical DNA double
helices are produced. Since each of the new DNA molecules contains one strand of old DNA and
one strand of new DNA, the process is described as semiconservative replication. Crick
described the process best when he used the analogy of the two DNA strands fitting together
like hand in a glove. The hand and glove separate, a new hand forms inside the glove, and a new
glove is forms around the hand. Two identical copies now exist where only one existed before.

The process by which the individual nucleotides are joined to create new DNA strands involves
many steps and enzymes. Addition of new nucleotide units to the growing chains takes place in
the 5’3’ direction and is catalyzed by the enzyme DNA polymerase. The key step is the
addition of a 5’- mononucleoside triphosphate to the free 3’- hydroxyl group of the growing
chain as the 3’-hydroxyl attacks the triphosphate and expels a diphosphate leaving group.

 Begins with a partial unwinding of the double helix, exposing the recognition site on the
bases
 Activated forms of the complementary nucleotides (A with T and G with C) associate two
new strands begin to grow.

Replication of RNA Process

 Addition takes place 5’3’, catalyzed by DNA polymerase

 Each nucleotide is joined as a 5’-nucleoside triphosphate that adds a nucleotide to the
free 3’-hydroxyl group of the growing chain

Schematic representation of DNA replication. The original double-stranded DNA partially

unwinds, bases are exposed, and nucleotides line up on each strand in a complementary manner,
and two new strands begins to grow.
 Structure and Synthesis of RNA Transcription
 RNA contains ribose rather than deoxyribose and uracil rather than thymine
 There are three major kinds of RNA - each of which serves a specific function
 They are much smaller molecules than DNA and are usually single-stranded

As noted previously, RNA is structurally similar to DNA but contains ribose rather than
deoxyribose and uracil rather than thymine. There are three major kinds of RNA, each of which
serves a specific function. All three are much smaller molecules than DNA, and all remain single-
stranded rather than double-stranded.

 Messenger RNA (mRNA) carries genetic messages from DNA to ribosomes, small granular
particles in the cytoplasm of a cell where proteins synthesis takes place.
 Ribosomal RNA (rRNA) complexed with protein provides the physical makeup of the
ribosomes. Ribosomes are a complex of proteins and rRNA. The synthesis of proteins from
amino acids and ATP occurs in the ribosome. The rRNA provides both structure and
catalysis
 Transfer RNA (tRNA) transports amino acids to the ribosomes where they are joined
together to make proteins. There is a specific tRNA for each amino acid. Recognition of
the tRNA at the anti-codon communicates which amino acid is attached.

The conversion of the information in DNA into proteins begins in the nucleus of the cells with the
synthesis of mRNA by transcription of DNA. Several turns of the DNA double helix unwind,
forming a bubble and exposing the bases of the two strands. Ribonucleotides line up in the
proper order by the hydrogen bonding to their complementary bases on DNA, bond formation
occurs in the 5’3’ direction, and the growing RNA molecule unwinds from DNA.

Unlike what happens in DNA replication, where both strands are copied, only one of the two DNA
strands is transcribed into mRNA. The strand that contains the gene is called the coding strand,
or sense strand, and the strand that gets transcribed is called the template strand, or antisense
strand. Since the template strand and the coding strand are

***sa ka baghak si buduan utod ang notes***

Transcription process

 Several turns of the DNA double helix unwind, exposing the bases of the two strands
 Ribonucleotides line up in the proper order by hydrogen bonding to their complementary
bases on DNA
 Bonds form in the 5’3’ direction,
Transcription of RNA from DNA

 Only one of the two DNA strands is transcribed into mRNA

 The strand that contains the gene is the coding or sense strand
 The strand that gets transcribed is the template or antisense strand
 The RNA molecule produced during transcription is a copy of the coding strand (with U in
place of T)

Mechanism of Transcription

 DNA contains promoter sites that are 10 to 35 base pairs upstream from the beginning
of the coding region and signal the beginning of a gene
 There are other base sequences near the end of the gene that signal a stop
 Genes are not necessarily continuous, beginning gene in a section of DNA (an exon) and
then resume farther down the chain in another exon, with an intron between that is
removed from the mRNA

RNA and Protein Biosynthesis: Translation

The primary cellular function of RNA is to direct biosynthesis of the thousands of diverse
peptides and proteins required by an organism- at least 100,000 in a human. The mechanics of
protein biosynthesis appear to be catalyzed by an mRNA rather than by protein-based enzyme
and take place on ribosomes, small granular particles in the cytoplasm of a cell that consist
about 60% ribosomal RNA and 40% protein. On the ribosome, mRNA serves as template to pass
on the genetic information it has transcribed from DNA.

The specific ribonucleotide sequence in mRNA forms a message that determines the order in
which different amino acid residues are to be joined. Each “word” or codon, along the mRNA
chains consists of a sequence of three ribonucleotides that is specific for a given amino acid.
For example, the series U-U-C on mRNA is a codon directing incorporation of the amino acid
phenylalanine into the growing protein. Of the 43 = 64 possible triplets of the four bases in RNA,
61 code for specific amino acids, and 3 code for chain termination.

 RNA directs biosynthesis of peptides and proteins which is catalyzed by mRNA in

ribosomes, where mRNA acts as a template to pass on the genetic information
transcribed from DNA
 The ribonucleotide sequence in mRNA forms a message that determines the order in
which different amino acid residues are to be joined
 Codons are sequences of three ribonucleotides that specify a particular amino acid
 For example, UUC on mRNA is a codon that directs incorporation of phenylalanine into
the growing protein.
Codon Assignments of Base Triplets

The Parts of Transfer RNA

 There are 61 different tRNAs, one for each of the 61 codons that specifies an amino acid
 tRNA has 70-100 ribonucleotides and is bonded to a specific amino acid by an ester

 Each tRNA has a segment called an anticodon, a sequence of three ribonucleotides

complementary to the codon sequence

The Structure of tRNA

Processing Aminoacyl tRNA

 As each codon on mRNA is read, tRNAs bring amino acids as esters for transfer to the
growing peptide
 When synthesis of the proper protein is completed, a "stop" codon signals the end and
the protein is released from the ribosome

DNA Sequencing
 The order of the bases along DNA contains the genetic inheritance.
 Determination of the sequence is based on chemical reactions rather than physical
analysis
 DNA is cleaved at specific sequences by restriction endonucleases
 For example, the restriction enzyme AluI cleaves between G and C in the four-base
sequence AG-CT Note that the sequence is identical to that of its complement, (3’)-TC-
GA-(5’)
 Other restriction enzymes produce other cuts permitting partially overlapping sequences
of small pieces to be produced for analysis

Analytical Methods

 The Maxam–Gilbert method uses organic chemistry to cleave phosphate linkages at

with specificity for the adjoining heterocycle
 The Sanger dideoxy method uses enzymatic reactions
 The Sanger method is now widely used and automated, even in the sequencing of
genomes

The Sanger Dideoxy Method

 The fragment to be sequenced is combined with:

 A small piece of DNA (primer), whose sequence is complementary to that on the 3’ end of
the restriction fragment
 The four 2’-deoxyribonucleoside triphosphates
(dNTPs)
The Dideoxy Nucleotides

 The solution also contains small amounts of the four 2’, 3’-dideoxyribonucleoside
triphosphates (ddNTPs)
 Each is modified with a different fluorescent dye molecule

The Dideoxy Method - Growing the and Stopping the Copied Chains

 DNA polymerase is added and a strand of DNA complementary to the restriction

fragment begins to grow from the end of the primer
 Whenever a dideoxyribonucleotide is incorporated, chain extension cannot continue

Dideoxy Method - Analysis

 The product is a mixture of dideoxy-terminated DNA fragments with fluorescent tags

 These are separated according to weight by electrophoresis and identified by their
specific fluorescence
 DNA Synthesis
 DNA synthesizers use a solid-phase method starting with an attached, protected
nucleotide
 Subsequent protected nucleotides are added and coupled
 After the final nucleotide has been added, the protecting groups are removed and the
synthetic DNA is cleaved from the solid support
 The bases are protected from reacting

DNA Synthesis: Attachment

 Attachment of a protected deoxynucleoside to a polymeric or silicate support as an ester

of the 3’—OH group of the deoxynucleoside
 The 5’—OH group on the sugar is protected as its p-dimethoxytrityl (DMT) ether

DNA Synthesis: DMT Removal

 Removal of the DMT protecting group by treatment with a moderately weak acid
DNA Synthesis: Coupling

 The polymer-bound (protected) deoxynucleoside reacts with a protected deoxynucleoside

containing a phosphoramidite group at its 3’ position, catalyzed by tetrazole, a reactive
heterocycle

DNA Synthesis: Oxidation and Cycling

*Phosphite is oxidized to phosphate by I

2
*The cycle is repeated until the sequence is complete

DNA Synthesis: Clean-up

 All protecting groups are removed and the product is released from the support by
treatment with aqueous NH3
 The Polymerase Chain Reaction (PCR)
 Copies DNA molecules by unwinding the double helix and copying each strand using
enzymes
 The new double helices are unwound and copied again
 The enzyme is selected to be fast, accurate and heat-stable (to survive the unwinding)
 Each cycle doubles the amount of material
 This is exponential template-driven organic synthesis

PCR: Heating and Reaction

 The subject DNA is heated (to separate strands) with

 Taq polymerase (enyzme) and Mg2+
 Deoxynucleotide triphosphates
 Two, oligonucleotide primers, each complementary to the sequence at the end of one
of the target DNA segments

PCR: Annealing and Growing

 Temperature is reduced to 37 to 50°C, allowing the primers to form H-bonds to their

complementary sequence at the end of each target strand

PCR: Taq Polymerase

 The temperature is then raised to 72°C, and Taq polymerase catalyzes the addition of
further nucleotides to the two primed DNA strands
PCR: Growing More Chains

 Repeating the denature–anneal–synthesize cycle a second time yields four DNA copies, a
third time yields eight copies, in an exponential series.
 PCR has been automated, and 30 or so cycles can be carried out in an hour