Chapter 26Chapter 26Nucleosides, Nucleotides,Nucleosides, Nucleotides,

and Nucleic Acidsand Nucleic Acids

26.126.1Pyrimidines and PurinesPyrimidines and Purines

Pyrimidines and Purines

In order to understand the structure and properties of DNA and RNA, we need to look at their structural components.

We begin with certain heterocyclic aromatic compounds called pyrimidines and purines.

Pyrimidines and Purines

Pyrimidine and purine are the names of the parent compounds of two types of nitrogen-containing heterocyclic aromatic compounds.

Pyrimidine Purine

N

N NH

N

NN 1

2

34

57

8

9

1

2

3

4

5

66

Pyrimidines and Purines

Amino-substituted derivatives of pyrimidine and purine have the structures expected from their names.

4-Aminopyrimidine 6-Aminopurine

H

N N

NN

HH

NH2

N

N

H

H

HH2N

Pyrimidines and Purines

But hydroxy-substituted pyrimidines and purines exist in keto, rather than enol, forms.

enol

N

N

H

H

HHO

N

N

H

H

HO

H

keto

Pyrimidines and Purines

But hydroxy-substituted pyrimidines and purines exist in keto, rather than enol, forms.

H

N N

NN

HH

OH

enol keto

H

N N

N

H

H

O

HN

Important Pyrimidines

Pyrimidines that occur in DNA are cytosine and thymine. Cytosine and uracil are the pyrimidines in RNA.

HN

NH

O

O

Uracil

HN

NH

O

O

CH3

Thymine

HN

NH

NH2

O

Cytosine

Important Purines

Adenine and guanine are the principal purines of both DNA and RNA.

Adenine

N

N

NH2

N

NH

Guanine

O

HN

NH

N

N

H2N

Caffeine and Theobromine

Caffeine (coffee) and theobromine (coffee and tea) are naturally occurring purines.

Caffeine

N

N

O

N

N

H3C

O

CH3

CH3

Theobromine

O

HN

NN

N

CH3

CH3

O

26.226.2NucleosidesNucleosides

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.

NH2

HO OH

OHOCH2

O

N

N

Table 26.2

Pyrimidine nucleosides

Cytidine occurs in RNA; its 2-deoxy analog occurs in DNA.

Cytidine

O

N

NH

O

HO

O

H3C

HOCH2

Table 26.2

Pyrimidine nucleosides

Thymidine occurs in DNA.

Thymidine

Table 26.2

Pyrimidine nucleosides

HOCH2

O

N

NH

O

OHHO

O

Uridine occurs in RNA.

Uridine

Table 26.2

Purine nucleosides

Adenosine

Adenosine occurs in RNA; its 2-deoxy analog occurs in DNA.

HOCH2 O

OHHO

N

N

N

NH2

N

Table 26.2

Purine nucleosides

Guanosine

Guanosine occurs in RNA; its 2-deoxy analog occurs in DNA.

HOCH2

N

NH

O

O

HO

N NH2

N

OH

26.326.3NucleotidesNucleotides

Nucleotides are phosphoric acid esters of Nucleotides are phosphoric acid esters of nucleosides.nucleosides.

Adenosine 5'-Monophosphate (AMP)

Adenosine 5'-monophosphate (AMP) is also called 5'-adenylic acid.

OCH2PHO

O

HO

O

OHHO

N

N

N

NH2

N

5'

1'

2'3'

4'

Adenosine Diphosphate (ADP)

OP

O

HO

HO

OCH2P

O

HOO

OHHO

N

N

N

NH2

N

Adenosine Triphosphate (ATP)

OP

O

HO

O

OCH2P

O

HO

O

OHHO

N

N

N

NH2

N

P

O

HO

HO

ATP is an important molecule in several biochemical processes including:

energy storage (Sections 26.4-26.5)phosphorylation

ATP and Phosphorylation

ATP +

hexokinaseThis is the first step in the metabolism of glucose.

ADP +

O

OHHO

HOHO(HO)2POCH2

O

HOCH2 O

OHHO

HOHO

cAMP and cGMP

Cyclic AMP and cyclic GMP are "second messengers" in many biological processes. Hormones (the "first messengers") stimulate the formation of cAMP and cGMP.

Cyclic adenosine monophosphate (cAMP)

CH2 O

OH

N

N

N

NH2

N

O

P

HO O

O

O

P

HO O

O

cAMP and cGMP

Cyclic AMP and cyclic GMP are "second messengers" in many biological processes. Hormones (the "first messengers") stimulate the formation of cAMP and cGMP.

Cyclic guanosine monophosphate (cGMP)

CH2

N

NH

O

O

N NH2

N

OH

26.426.4BioenergeticsBioenergetics

Bioenergetics

Bioenergetics is the thermodynamics of biological processes.

Emphasis is on free energy changes (G).

When G is negative, reaction is spontaneous in the direction written.

When G is 0, reaction is at equilibrium.

When G is positive, reaction is not spontaneous in direction written.

Standard Free Energy (G°)

Sign and magnitude of G depends on what the reactants and products are and their concentrations.

In order to focus on reactants and products, define a standard state.

The standard concentration is 1 M (for a reaction in homogeneous solution).

G in the standard state is called the standard free-energy change and given the symbol G°.

mA(aq) nB(aq)

Standard Free Energy (G°)

Exergonic: An exergonic reaction is one for which the sign of G° is negative.

Endergonic: An exergonic reaction is one for which the sign of G° is positive.

mA(aq) nB(aq)

Standard Free Energy (G°)

It is useful to define a special standard state for biological reactions.

This special standard state is one for which the pH = 7.

The free-energy change for a process under these conditions is symbolized as G°'.

mA(aq) nB(aq)

26.526.5ATP and BioenergeticsATP and Bioenergetics

Hydrolysis of ATP

G°' for hydrolysis of ATP to ADP is –31 kJ/mol.

Relative to ADP + HPO42–, ATP is a "high-

energy" compound.

When coupled to some other process, the conversion of ATP to ADP can provide the free energy to transform an endergonic process to an exergonic one.

ATP + H2O ADP + HPO42–

Glutamic Acid to Glutamine

+ NH4+–OCCH2CH2CHCO–

+NH3

O O

+ H2OH2NCCH2CH2CHCO–

+NH3

O O

G°' = +14 kJ Reaction is endergonic.

Glutamic Acid to Glutamine

+ NH4+–OCCH2CH2CHCO–

+NH3

O O

Reaction becomes exergonicwhen coupled to the hydrolysisof ATP.

+ ATP

+ HPO42–H2NCCH2CH2CHCO–

+NH3

O O

G°' = –17 kJ

+ ADP

Glutamic Acid to Glutamine

–OCCH2CH2CHCO–

+NH3

O O

Mechanism involvesphosphorylation of glutamic acid.

+ ATP

OCCH2CH2CHCO–

+NH3

O O

+ ADPP

O

–O

–O

Glutamic Acid to Glutamine

Followed by reaction of phosphorylated glutamic acid with ammonia.

H2NCCH2CH2CHCO–

+NH3

O O

+ HPO42–

OCCH2CH2CHCO–

+NH3

O O

+ NH3P

O

–O

–O

26.626.6Phosphodiesters, Phosphodiesters,

Oligonucleotides, and Oligonucleotides, and PolynucleotidesPolynucleotides

Phosphodiesters

A phosphodiester linkage between two nucleotides is analogous to a peptide bond between two amino acids.

Two nucleotides joined by a phosphodiester linkage gives a dinucleotide.

Three nucleotides joined by two phosphodiester linkages gives a trinucleotide, etc. (See next slide)

A polynucleotide of about 50 or fewer nucleotides is called an oligonucleotide.

3'

5'HOCH2

O

O

N

N

N

N

P OCH2O

HO

NH2

OCH2O

NH

N

N

N

HO

O

NH2HO

O P

H3C

O

O

OO

NH

N

Fig. 26.1The

trinucleotide ATG

phosphodiester linkages between 3' of one nucleotide and 5' of the next

A

T

G

free 5' end

free 3' end

26.726.7Nucleic AcidsNucleic Acids

Nucleic acids are polynucleotides.Nucleic acids are polynucleotides.

Nucleic Acids

Nucleic acids first isolated in 1869 (Johann Miescher).

Oswald Avery discovered (1945) that a substance which caused a change in the genetically transmitted characteristics of a bacterium was DNA.

Scientists revised their opinion of the function of DNA and began to suspect it was the major functional component of genes.

Composition of DNA

Erwin Chargaff (Columbia Univ.) studied DNAs from various sources and analyzed the distribution of purines and pyrimidines in them.

The distribution of the bases adenine (A), guanine (G), thymine (T), and cytosine (C) varied among species.

But the total purines (A and G) and the total pyrimidines (T and C) were always equal.

Moreover: %A = %T, and %G = %C

Composition of Human DNA

Adenine (A) 30.3% Thymine (T) 30.3%

Guanine (G) 19.5% Cytosine (C) 19.9%

Total purines: 49.8% Total pyrimidines: 50.1%

For example:

Purine Pyrimidine

Structure of DNA

James D. Watson and Francis H. C. Crick proposed a structure for DNA in 1953.

Watson and Crick's structure was based on:•Chargaff's observations•X-ray crystallographic data of Maurice Wilkins and Rosalind Franklin•Model building

26.826.8Secondary Structure of DNA:Secondary Structure of DNA:

The Double HelixThe Double Helix

Base Pairing

Watson and Crick proposed that A and T were present in equal amounts in DNA because of complementary hydrogen bonding.

2-deoxyribose

N

NN

N N

H

H

N

N

O CH3

O

H

2-deoxyriboseA T

Base Pairing

Watson and Crick proposed that A and T were present in equal amounts in DNA because of complementary hydrogen bonding.

Base Pairing

Likewise, the amounts of G and C in DNA were equal because of complementary hydrogen bonding.

N

NN

N O

NH

H

H

N

N

N

O

HH

2-deoxyribose

2-deoxyribose

G C

Base Pairing

Likewise, the amounts of G and C in DNA were equal because of complementary hydrogen bonding.

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.

•Gives proper Chargaff ratios (A=T and G=C)

•Because each pair contains one purine and one pyrimidine, the A---T and G---C distances between strands are approximately equal.

•Complementarity between strands suggests a mechanism for copying genetic information.

O

O

ĞO

ĞO

ĞO

O

O

O

O

OP

O

O

P O

OO

P O

OO

O

O

OĞ

OĞ

OĞ

O

O

O

O

O P

O

O

PO

OO

PO

OO

C G

AT

AT

CG

3'

5'

5'

5'

5' 5'

5'

5'

5'

3'

3'3'

3'3'

3'

3'

Fig. 26.4

Two antiparallel strands of DNA are paired by hydrogen bonds between purine and pyrimidine bases.

Fig. 26.5

Helical structure of DNA. The purine and pyrimidine bases are on the inside, sugars and phosphates on the outside.

26.926.9Tertiary Structure of DNA:Tertiary Structure of DNA:

SupercoilsSupercoils

DNA is coiled

A strand of DNA is too long (about 3 cm in length) to fit inside a cell unless it is coiled.

Random coiling would reduce accessibility to critical regions.

Efficient coiling of DNA is accomplished with the aid of proteins called histones.

Histones

Histones are proteins rich in basic amino acids such as lysine and arginine.

Histones are positively charged at biological pH.DNA is negatively charged.

DNA winds around histone proteins to form nucleosomes.

Histones

Each nucleosome contains one and three-quarters turns of coil = 146 base pairs.Linker contains about 50 base pairs.

Histones

Nucleosome = Histone proteins + Supercoiled DNA

26.1026.10Replication of DNAReplication of DNA

Fig. 26.8 DNA Replication

Fig. 26.8 DNA Replication

Fig. 26.8 DNA Replication

Fig. 26.8 DNA Replication

Elongation of the Growing DNA Chain

The free 3'-OH group of the growing DNA chain reacts with the 5'-triphosphate of the appropriate nucleotide.

Fig. 26.9: Chain Elongation

OH CH2

OO P

O

O–

O P

O

O–

O P

O

O–

O–Adenine,Guanine,

Cytosine, orThymine

••OH

CH2OPOO

Adenine,Guanine,

Cytosine, orThymine

O

O–

••

Poly-nucleotide

chain

Poly-nucleotide

chain

Fig. 26.9: Chain Elongation

OH CH2

OO P

O–

Adenine,Guanine,

Cytosine, orThymine

O CH2OPO

OAdenine,Guanine,

Cytosine, orThymine

O

O–

•• ••

Poly-nucleotide

chain

Poly-nucleotide

chain

O

O P

O

O–

O P

O

O–

O––

26.1126.11Ribonucleic AcidsRibonucleic Acids

DNA and Protein Biosynthesis

According to Crick, the "central dogma" of molecular biology is:

"DNA makes RNA makes protein."

Three kinds of RNA are involved.Messenger RNA (mRNA)Transfer RNA (tRNA)Ribosomal RNA (rRNA)

There are two main stages.TranscriptionTranslation

Transcription

In transcription, a strand of DNA acts as a template upon which a complementary RNA is biosynthesized.

This complementary RNA is messenger RNA (mRNA).

Mechanism of transcription resembles mechanism of DNA replication.

Transcription begins at the 5' end of DNA and is catalyzed by the enzyme RNA polymerase.

Fig. 26.10: Transcription

Only a section of about 10 base pairs in the DNAis unwound at a time. Nucleotides complementaryto the DNA are added to form mRNA.

The Genetic Code

The nucleotide sequence of mRNA codes for the different amino acids found in proteins.

There are three nucleotides per codon.

There are 64 possible combinations of A, U, G, and C.

The genetic code is redundant. Some proteins are coded for by more than one codon.

UUU Phe UCU Ser UAU Tyr UGU Cys U

UUC Phe UCC Ser UAC Tyr UGC Cys C

UUA Leu UCA Ser UAA Stop UGA Stop A

UUG Leu UCG Ser UAG Stop UCG Trp G

U

C

A

G

U

C

A

G

U

C

A

G

U C A G

U

C

A

G

First letter

Second letter

Third letter

Table 26.4

UUU Phe UCU Ser UAU Tyr UGU Cys U

UUC Phe UCC Ser UAC Tyr UGC Cys C

UUA Leu UCA Ser UAA Stop UGA Stop A

UUG Leu UCG Ser UAG Stop UCG Trp G

CUU Leu CCU Pro CAU His CGU Arg U

CUC Leu CCC Pro CAC His CGC Arg C

CUA Leu CCA Pro CAA Gln CGA Arg A

CUG Leu CCG Pro CAG Gln CCG Arg G

AUU Ile ACU Thr AAU Asn AGU Ser U

AUC Ile ACC Thr AAC Asn AGC Ser C

AUA Ile ACA Thr AAA Lys AGA Arg A

AUG Met ACG Thr AAG Lys ACG Arg G

GUU Val GCU Ala GAU Asp GGU Gly U

GUC Val GCC Ala GAC Asp GGC Gly C

GUA Val GCA Ala GAA Glu GGA Gly A

GUG Val GCG Ala GAG Glu GCG Gly G

U C A G

U

C

A

G

U

C

UAA Stop UGA Stop A

UAG Stop G

U

C

A

G

AUU Ile ACU Thr AAU Asn AGU Ser U

AUC Ile ACC Thr AAC Asn AGC Ser C

AUA Ile ACA Thr AAA Lys AGA Arg A

AUG Met ACG Thr AAG Lys ACG Arg G

U

C

A

G

U C A G

U

C

A

G

AUG is the "start" codon. Biosynthesis of allproteins begins with methionine as the first aminoacid. This methionine is eventually removed afterprotein synthesis is complete.

UAA, UGA, and UAG are "stop" codons thatsignal the end of the polypeptide chain.

Transfer tRNA

There are 20 different tRNAs, one for each amino acid.

Each tRNA is single stranded with a CCA triplet at its 3' end.

A particular amino acid is attached to the tRNA by an ester linkage involving the carboxyl group of the amino acid and the 3' oxygen of the tRNA.

Transfer RNA

Example—Phenylalanine transfer RNA

One of the mRNA codons for phenylalanine is:

UUC5' 3'

AAG3' 5'

The complementary sequence in tRNA is calledthe anticodon.

Fig. 26.11: Phenylalanine tRNA

Ribosomal RNA

Most of the RNA in a cell is ribosomal RNA.

Ribosomes are the site of protein synthesis. They are where translation of the mRNA sequence to an amino acid sequence occurs.

Ribosomes are about two-thirds RNA and one-third protein.

It is believed that the ribosomal RNA acts as a catalyst—a ribozyme.

26.1226.12Protein BiosynthesisProtein Biosynthesis

Protein Biosynthesis

During translation the protein is synthesized beginning at its N-terminus.

mRNA is read in its 5'-3' direction.Begins at the start codon AUGEnds at stop codon (UAA, UAG, or UGA)

Fig. 26.12: Translation

Reaction that occurs is nucleophilic acyl substitution. Ester is converted to amide.

Methionine at N-terminusis present as its N-formylderivative.

Fig. 26.12: Translation

Fig. 26.12: Translation

Ester at 3' end of alanine tRNA is Met-Ala.

Process continues along mRNA until stop codon is reached.

26.1326.13AIDSAIDS

AIDS

Acquired Immune Deficiency Syndrome

More than 22 million people have died from AIDS since disease discovered in 1980s.

Now fourth leading cause of death worldwide and leading cause of death in Africa (World Health Organization).

HIV

Virus responsible for AIDS in people is Human Immunodeficiency Virus (HIV).

Several strains of HIV designated HIV-1, HIV-2, etc.

HIV is a retrovirus. Genetic material is RNA, not DNA.

HIV

HIV inserts its own RNA and an enzyme (reverse transcriptase) in T4 lymphocyte cell of host.

Reverse transcriptase catalyzes the formation of DNA complementary to the HIV RNA.

HIV reproduces and eventually infects other T4 lympocytes.

Ability of T4 cells to reproduce decreases, interfering with bodies ability to fight infection.

AIDS Drugs

AZT and ddI are two drugs used against AIDS that delay onset of symptoms.

ON O

N3

O

H3C

HOCH2

O

NH

AZT

O

N

O

HOCH2

O

NH

ddI

N

N

H

H H

H

AIDS Drugs

Protease inhibitors are used in conjunction with other AIDS drugs.

Several HIV proteins are present in the same polypeptide chain and must be separated from each other in order to act.

Protease inhibitors prevent formation of HIV proteins by preventing hydrolysis of polypeptide that incorporates them.

26.1426.14DNA SequencingDNA Sequencing

DNA Sequencing

Restriction enzymes cleave the polynucleotide to smaller fragments.

These smaller fragments (100-200 base pairs) are sequenced.

The two strands are separated.

DNA Sequencing

Single stranded DNA divided in four portions.

Each tube contains adenosine, thymidine, guanosine, and cytidine plus the triphosphates of their 2'-deoxy analogs. POCH2

OH

OO

O

OH

P

O

OH

P

O

HO base

HHO

O

DNA Sequencing

The first tube also contains the 2,'3'-dideoxy analog of adenosine triphosphate (ddATP); the second tube the 2,'3'-dideoxy analog of thymidine triphosphate (ddTTP), the third contains ddGTP, and the fourth ddCTP. POCH2

OH

OO

O

OH

P

O

OH

P

O

HO base

HH

O

DNA Sequencing

Each tube also contains a "primer", a short section of the complementary DNA strand, labeled with radioactive phosphorus (32P).

DNA synthesis takes place, producing a complementary strand of the DNA strand used as a template.

DNA synthesis stops when a dideoxynucleotide is incorporated into the growing chain.

DNA Sequencing

The contents of each tube are separated by electrophoresis and analyzed by autoradiography.

There are four lanes on the electrophoresis gel.

Each DNA fragment will be one nucleotide longer than the previous one.

Figure 26.13

Figure 26.13

26.1526.15The Human Genome ProjectThe Human Genome Project

Human Genome Project

In 1988 National Research Council (NRC) recommended that the U.S. undertake the mapping and sequencing of the human genome.

International Human Genome Sequencing Consortium (led by U.S. NIH) and Celera Genomics undertook project. Originally competitors, they agreed to coordinate efforts and published draft sequences in 2001.

26.1626.16DNA ProfilingDNA Profiling

and theand thePolymerase Chain ReactionPolymerase Chain Reaction

DNA Profiling

DNA sequencing involves determining the nucleotide sequence in DNA.

The nucleotide sequence in regions of DNA that code for proteins varies little from one individual to another, because the proteins are the same.

Most of the nucleotides in DNA are in "noncoding" regions and vary significantly among individuals.

Enzymatic cleavage of DNA give a mixture of polynucleotides that can be separated by electrophoresis to give a "profile" characteristic of a single individual.

PCR

When a sample of DNA is too small to be sequenced or profiled, the polymerase chain reaction (PCR) is used to make copies ("amplify") portions of it.

PCR amplifies DNA by repetitive cycles of the following steps.

1. Denaturation2. Annealing ("priming")3. Synthesis ("extension" or "elongation")

Figure 26.14: (PCR)

Target region

(a) Consider double-stranded DNA containinga polynucleotide sequence (the target region)that you wish to amplify.

(b) Heating the DNA to about 95°C causes the strands to separate. This is the denaturation step.

(c) Cooling the sample to ~60°C causes oneprimer oligonucleotide to bind to one strand andthe other primer to the other strand. This is theannealing step.

Figure 26.14: (PCR)

(c) Cooling the sample to ~60°C causes oneprimer oligonucleotide to bind to one strand andthe other primer to the other strand. This is theannealing step.

(d) In the presence of four DNA nucleotides andthe enzyme DNA polymerase, the primer is extended in its 3' direction. This is the synthesisstep and is carried out at 72°C.

Figure 26.14: (PCR)

This completes one cycle of PCR.

(d) In the presence of four DNA nucleotides andthe enzyme DNA polymerase, the primer is extended in its 3' direction. This is the synthesisstep and is carried out at 72°C.

Figure 26.14: (PCR)

This completes one cycle of PCR.

(e) The next cycle begins with the denaturationof the two DNA molecules shown. Both arethen primed as before.

Figure 26.14: (PCR)

(f) Elongation of the primed fragments completesthe second PCR cycle.

Figure 26.14: (PCR)

(f) Elongation of the primed fragments completesthe second PCR cycle.

(g) Among the 8 DNAs formed in the secondcycle are two having the structure shown.

Figure 26.14: (PCR)

The two contain only the target region andand are the ones that increase disproportionately in subsequent cycles.

(g) Among the 8 DNAs formed in the secondcycle are two having the structure shown.

Figure 26.14: (PCR)

Table 26.5

Cycle Total DNAs Contain only target0 (start) 1 01 2 02 4 03 8 24 16 85 32 2210 1,024 1,00420 1,048,566 1,048,52630 1,073,741,824 1,073,741,764

26.1726.17Recombinant DNA TechnologyRecombinant DNA Technology