Dna,rna

Dr. Wolf's CHM 424 28- 1

Nucleic AcidsNucleic Acids


28.128.1Pyrimidines and PurinesPyrimidines and Purines


Pyrimidines and PurinesPyrimidines and Purines

In order to understand the structure and In order to understand the structure and properties of DNA and RNA, we need to look at properties of DNA and RNA, we need to look at their structural components.their structural components.We begin with certain heterocyclic aromatic We begin with certain heterocyclic aromatic compounds called pyrimidines and purines.compounds called pyrimidines and purines.



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

PyrimidinePyrimidine PurinePurine

NN

NN NNHH

NN

NNNN 11

2233

44

5577

88

99

11

22

33

44

55

6666



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

4-Aminopyrimidine4-Aminopyrimidine 6-Aminopurine6-Aminopurine

HH

NN NN

NNNN

HHHH

NNHH22

NN

NN

HH

HH

HHHH22NN



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

enolenol

NN

NN

HH

HH

HHHHOO

NN

NN

HH

HH

HHOO

HHketoketo



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

HH

NN NN

NNNN

HHHH

OOHH

enolenol ketoketo

HH

NN NN

NN

HHHH

OO

HHNN


Important PyrimidinesImportant Pyrimidines

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

HHNN

NNHH

OO

OO

UracilUracil

HHNN

NNHH

OO

OO

CHCH33

ThymineThymine

HHNN

NNHH

NNHH22

OO

CytosineCytosine


Important PurinesImportant Purines

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

AdenineAdenine

NN

NN

NNHH22

NN

NNHH

GuanineGuanine

OO

HHNN

NNHH

NN

NN

HH22NN


Caffeine and TheobromineCaffeine and Theobromine

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

CaffeineCaffeine

NN

NN

OO

NN

NN

HH33CC

OO

CHCH33

CHCH33

TheobromineTheobromine

OO

HHNN

NNNN

NN

CHCH33

CHCH33

OO


28.228.2NucleosidesNucleosides


NucleosidesNucleosides

The classical structural definition is that a The classical structural definition is that a nucleoside is a pyrimidine or purine N-glycoside nucleoside is a pyrimidine or purine N-glycoside of of DD-ribofuranose or 2-deoxy--ribofuranose or 2-deoxy-DD-ribofuranose.-ribofuranose.Informal use has extended this definition to Informal use has extended this definition to apply to purine or pyrimidine N-glycosides of apply to purine or pyrimidine N-glycosides of almost any carbohydrate.almost any carbohydrate.The purine or pyrimidine part of a nucleoside is The purine or pyrimidine part of a nucleoside is referred to as a referred to as a purine or pyrimidine basepurine or pyrimidine base..


NNHH22

HOHO OHOH

OOHOCHHOCH22

OO

NN

NN

Table 28.2Table 28.2

Pyrimidine nucleosidesPyrimidine nucleosides

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

Cytidine


OO

NN

NNHH

OO

HOHO

OO

HH33CC

HOCHHOCH22



Thymidine occurs in DNAThymidine occurs in DNA

Thymidine




HOCHHOCH22

OO

NN

NNHH

OO

OHOHHOHO

OO

Uridine occurs in RNAUridine occurs in RNA

Uridine



Purine nucleosidesPurine nucleosides

Adenosine

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

HOCHHOCH22 OO

OHOHHOHO

NN

NN

NN

NHNH22

NN



Purine nucleosidesPurine nucleosides

Guanosine

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

HOCHHOCH22

NN

NHNH

OO

OO

HOHO

NN NHNH22

NN

OHOH


28.328.3NucleotidesNucleotides

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


Adenosine 5'-Monophosphate (AMP)Adenosine 5'-Monophosphate (AMP)

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

OCHOCH22PPHOHO

OO

HOHOOO

OHOHHOHO

NN

NN

NN

NHNH22

NN

5'5'

1'1'

2'2'3'3'4'4'


Adenosine Diphosphate (ADP)Adenosine Diphosphate (ADP)

OOPP

OO

HOHO

HOHO

OCHOCH22PP

OO

HOHOOO

OHOHHOHO

NN

NN

NN

NHNH22

NN


Adenosine Triphosphate (ATP)Adenosine Triphosphate (ATP)

OOPP

OO

HOHO

OO

OCHOCH22PP

OO

HOHOOO

OHOHHOHO

NN

NN

NN

NHNH22

NN

PP

OO

HOHO

HOHO

ATP is an important molecule in several ATP is an important molecule in several biochemical processes including:biochemical processes including:

energy storage (Sections 28.4-28.5)energy storage (Sections 28.4-28.5)phosphorylationphosphorylation


cAMP and cGMPcAMP and cGMP

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

Cyclic adenosine monophosphate (cAMP)Cyclic adenosine monophosphate (cAMP)

CHCH22 OO

OHOH

NN

NN

NN

NHNH22

NN

OO

OOPPOO

HOHO


cAMP and cGMPcAMP and cGMP

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

OO

OOPPOO

HOHO

Cyclic guanosine monophosphate (cGMP)Cyclic guanosine monophosphate (cGMP)

CHCH22

NN

NHNH

OO

OO

NN NHNH22

NN

OHOH


28.628.6Phosphodiesters, Phosphodiesters,

Oligonucleotides, and Oligonucleotides, and PolynucleotidesPolynucleotides


PhosphodiestersPhosphodiesters

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

Two nucleotides joined by a phosphodiester Two nucleotides joined by a phosphodiester linkage gives a dinucleotide.linkage gives a dinucleotide.

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

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


3'

5'HOCH2

O

O

N

N

N

N

P OCH2O

HO

NH2

OCH2O

NH

N

N

N

HO

O

NH2HO

O P

H3CO

O

OO

NH

N

Fig. 28.1Fig. 28.1The The

trinucleotide trinucleotide ATGATG

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

AA

TT

GG

free 5' endfree 5' end

free 3' endfree 3' end


28.728.7Nucleic AcidsNucleic Acids

Nucleic acids are polynucleotides.Nucleic acids are polynucleotides.


Nucleic AcidsNucleic Acids

Nucleic acids first isolated in 1869 (Johann Nucleic acids first isolated in 1869 (Johann Miescher)Miescher)Oswald Avery discovered (1945) that a Oswald Avery discovered (1945) that a substance which caused a change in the substance which caused a change in the genetically transmitted characteristics of a genetically transmitted characteristics of a bacterium was DNA.bacterium was DNA.Scientists revised their opinion of the function of Scientists revised their opinion of the function of DNA and began to suspect it was the major DNA and began to suspect it was the major functional component of genes.functional component of genes.


Composition of DNAComposition of DNA

Erwin Chargaff (Columbia Univ.) studied DNAs Erwin Chargaff (Columbia Univ.) studied DNAs from various sources and analyzed the from various sources and analyzed the distribution of purines and pyrimidines in them.distribution of purines and pyrimidines in them.The distribution of the bases adenine (A), The distribution of the bases adenine (A), guanine (G), thymine (T), and cytosine (C) guanine (G), thymine (T), and cytosine (C) varied among species.varied among species.But the total purines (A and G) and the total But the total purines (A and G) and the total pyrimidines (T and C) were always equal.pyrimidines (T and C) were always equal.Moreover: %A = %T, and %G = %CMoreover: %A = %T, and %G = %C


Composition of Human DNAComposition of Human DNA

Adenine (A) 30.3%Adenine (A) 30.3% Thymine (T) 30.3%Thymine (T) 30.3%Guanine (G) 19.5%Guanine (G) 19.5% Cytosine (C) 19.9%Cytosine (C) 19.9%Total purines: 49.8%Total purines: 49.8% Total pyrimidines: 50.1%Total pyrimidines: 50.1%

For example:For example:

PurinePurine PyrimidinePyrimidine


Structure of DNAStructure of DNA

James D. Watson and Francis H. C. Crick James D. Watson and Francis H. C. Crick proposed a structure for DNA in 1953.proposed a structure for DNA in 1953.Watson and Crick's structure was based on:Watson and Crick's structure was based on:

•Chargaff's observations•Chargaff's observations•X-ray crystallographic data of Maurice •X-ray crystallographic data of Maurice Wilkins and Rosalind FranklinWilkins and Rosalind Franklin•Model building•Model building


28.828.8Secondary Structure of DNA:Secondary Structure of DNA:

The Double HelixThe Double Helix

FORMS OF DNAFORMS OF DNA



Base PairingBase Pairing

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

2-deoxyribose2-deoxyribose

N

NN

N N

H

H

NN

O CH3

O

H

2-deoxyribose2-deoxyriboseAA TT



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



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

NNN

N O

NH

H

HN

N

N

O

HH



GG CC



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


The DNA DuplexThe DNA Duplex

Watson and Crick proposed a double-stranded Watson and Crick proposed a double-stranded structure for DNA in which a purine or structure for DNA in which a purine or pyrimidine base in one chain is hydrogen pyrimidine base in one chain is hydrogen bonded to its complement in the other.bonded to its complement in the other.••Gives proper Chargaff ratios (A=T and G=C)Gives proper Chargaff ratios (A=T and G=C)••Because each pair contains one purine and Because each pair contains one purine and

one pyrimidine, the A---T and G---C distances one pyrimidine, the A---T and G---C distances between strands are approximately equal. between strands are approximately equal. ••Complementarity between strands suggests a Complementarity between strands suggests a

mechanism for copying genetic information.mechanism for copying genetic information.


O

O

ĞO

ĞO

ĞO

O

O

O

O

OPO

OP O

OO

P O

OO

O

O

OĞ

OĞ

OĞ

O

O

O

O

O PO

OPO

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. 28.4Fig. 28.4

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


Fig. 28.5Fig. 28.5

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


28.928.9Tertiary Structure of DNA:Tertiary Structure of DNA:

SupercoilsSupercoils


DNA is coiledDNA is coiled

A strand of DNA is too long (about 3 cm in A strand of DNA is too long (about 3 cm in length) to fit inside a cell unless it is coiled.length) to fit inside a cell unless it is coiled.Random coiling would reduce accessibility to Random coiling would reduce accessibility to critical regions.critical regions.Efficient coiling of DNA is accomplished with the Efficient coiling of DNA is accomplished with the aid of proteins called aid of proteins called histones.histones.


HistonesHistones

Histones are proteins rich in basic amino acids Histones are proteins rich in basic amino acids such as lysine and arginine.such as lysine and arginine.Histones are positively charged at biological pH.Histones are positively charged at biological pH.DNA is negatively charged.DNA is negatively charged.DNA winds around histone proteins to form DNA winds around histone proteins to form nucleosomes.nucleosomes.


Histones Histones

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


Histones Histones

NucleosomeNucleosome == Histone proteinsHistone proteins + Supercoiled DNA+ Supercoiled DNA


28.1028.10Replication of DNAReplication of DNA


Fig. 28.8 DNA Replication

A T C C G T A G G A T T A GC

5'3'

AT G CG A T C C T G A A T C

3'5'

The DNA to be copied is a double helix, shown here as flat for clarity.

The two strands begin to unwind. (next slide)


A T C C G T A G G A

T T A G

C

AT G CG AT C C T G A A T C

3'

5'

5'

3'


Each strand will become a template for construction of its complement.


A'

T'G'

C'

A T C C G T A G G AT T A G

C

5'

3'

AT G CG AT C C T G A A T C

5'

5' T' A'

3'

3'

leading strand

lagging strand

3'

5'

C'T'A'A'


Two new strands form as nucleotides that are complementary to those of the original strands are joined by phosphodiester linkages.

Polynucleotide chains grow in the 5'-3' direction—continuous in the leading strand, discontinuous in the lagging strand.


A' T' C' C' G' T' A' G' G' A' T' T' A' G'C'

5'3'

AT G CG A T C C T G A A T C

3'5'

A T C C G T A G G A T T A GC

5'3'

A'T' G' C'G' A' T' C' C' T' G' A' A' T' C'

3'5'

+


Two duplex DNAs result, each of which is identical to the original DNA.


Elongation of the growing DNA chainElongation of the growing DNA chain

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


Fig. 28.9 Chain elongationFig. 28.9 Chain elongation

OHOH CHCH22

OOOO PP

OO

O–O–

OO PP

OO

O–O–

OO PP

OO

O–O–

OO––Adenine,Guanine,

Cytosine, orThymine

••••OHOH

CHCH22OPOOPOOO

Adenine,Guanine,

Cytosine, orThymine

OO

O–O–

••••Poly-Poly-

nucleotidenucleotidechainchain


Fig. 28.9 Chain elongationFig. 28.9 Chain elongation

OHOH CHCH22

OOOO PP OO––

Adenine,Guanine,

Cytosine, orThymine

OO CHCH22OPOOPO

OOAdenine,Guanine,

Cytosine, orThymine

OO

O–O–

•••• ••••Poly-Poly-

nucleotidenucleotidechainchain

OO

OO PP

OO

O–O–

OO PP

OO

O–O–

OO––––


28.1128.11Ribonucleic AcidsRibonucleic Acids


DNA and Protein BiosynthesisDNA and Protein Biosynthesis

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

"DNA makes RNA makes protein.""DNA makes RNA makes protein."Three kinds of RNA are involved.Three kinds of RNA are involved.

messenger RNA (mRNA)messenger RNA (mRNA)transfer RNA (tRNA)transfer RNA (tRNA)ribosomal RNA (rRNA)ribosomal RNA (rRNA)

There are two main stages.There are two main stages.transcriptiontranscriptiontranslationtranslation


TranscriptionTranscription

In transcription, a strand of DNA acts as a In transcription, a strand of DNA acts as a template upon which a complementary RNA is template upon which a complementary RNA is biosynthesized. biosynthesized. This complementary RNA is This complementary RNA is messenger RNAmessenger RNA (mRNA).(mRNA).Mechanism of transcription resembles Mechanism of transcription resembles mechanism of DNA replication.mechanism of DNA replication. Transcription begins at the 5' end of DNA and is Transcription begins at the 5' end of DNA and is catalyzed by the enzyme catalyzed by the enzyme RNA polymeraseRNA polymerase..


Fig. 28.10 TranscriptionFig. 28.10 Transcription

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


The Genetic CodeThe Genetic Code

The nucleotide sequence of mRNA codes for The nucleotide sequence of mRNA codes for the different amino acids found in proteins.the different amino acids found in proteins.There are three nucleotides per codon.There are three nucleotides per codon.There are 64 possible combinations of A, U, G, There are 64 possible combinations of A, U, G, and C.and C.The genetic code is redundant. Some proteins The genetic code is redundant. Some proteins are coded for by more than one codon.are coded for by more than one codon.


UUUUUU PhePhe UCUUCU Ser Ser UAUUAU TyrTyr UGUUGU CysCys UUUUCUUC PhePhe UCCUCC SerSer UACUAC TyrTyr UGCUGC CysCys CCUUAUUA LeuLeu UCAUCA SerSer UAAUAA StopStop UGAUGA StopStop AAUUGUUG LeuLeu UCGUCG SerSer UAGUAG StopStop UCGUCG TrpTrp GG

UUCCAAGGUUCCAAGGUUCCAAGG

UU CC AA GG

UU

CC

AA

GG

First letterFirst letter

Second letterSecond letter

Third letterThird letter

Table 28.3 (p 1175)Table 28.3 (p 1175)


UUUUUU PhePhe UCUUCU Ser Ser UAUUAU TyrTyr UGUUGU CysCys UUUUCUUC PhePhe UCCUCC SerSer UACUAC TyrTyr UGCUGC CysCys CCUUAUUA LeuLeu UCAUCA SerSer UAAUAA StopStop UGAUGA StopStop AAUUGUUG LeuLeu UCGUCG SerSer UAGUAG StopStop UCGUCG TrpTrp GGCUUCUU LeuLeu CCUCCU Pro Pro CAUCAU HisHis CGUCGU ArgArg UUCUCCUC LeuLeu CCCCCC ProPro CACCAC HisHis CGCCGC ArgArg CCCUACUA LeuLeu CCACCA ProPro CAACAA GlnGln CGACGA ArgArg AACUGCUG LeuLeu CCGCCG ProPro CAGCAG GlnGln CCGCCG ArgArg GGAUUAUU IleIle ACUACU Thr Thr AAUAAU AsnAsn AGUAGU SerSer UUAUCAUC IleIle ACCACC ThrThr AACAAC AsnAsn AGCAGC SerSer CCAUAAUA IleIle ACAACA ThrThr AAAAAA LysLys AGAAGA ArgArg AAAUGAUG MetMet ACGACG ThrThr AAGAAG LysLys ACGACG ArgArg GGGUUGUU ValVal GCUGCU Ala Ala GAUGAU AspAsp GGUGGU GlyGly UUGUCGUC ValVal GCCGCC AlaAla GACGAC AspAsp GGCGGC GlyGly CCGUAGUA ValVal GCAGCA AlaAla GAAGAA GluGlu GGAGGA GlyGly AAGUGGUG ValVal GCGGCG AlaAla GAGGAG GluGlu GCGGCG GlyGly GG

UU CC AA GG

UU

CC

AA

GG


UUCC

UAAUAA StopStop UGAUGA StopStop AAUAGUAG StopStop GG

UUCC

AA GGAUUAUU IleIle ACUACU Thr Thr AAUAAU AsnAsn AGUAGU SerSer UUAUCAUC IleIle ACCACC ThrThr AACAAC AsnAsn AGCAGC SerSer CCAUAAUA IleIle ACAACA ThrThr AAAAAA LysLys AGAAGA ArgArg AAAUGAUG MetMet ACGACG ThrThr AAGAAG LysLys ACGACG ArgArg GG

UU CC AA GG

UU CC AA GG

UU

CC

AA

GG

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 tRNATransfer tRNA

There are 20 different tRNAs, one for each There are 20 different tRNAs, one for each amino acid.amino acid.Each tRNA is single stranded with a CCA triplet Each tRNA is single stranded with a CCA triplet at its 3' end.at its 3' end.A particular amino acid is attached to the tRNA A particular amino acid is attached to the tRNA by an ester linkage involving the carboxyl group by an ester linkage involving the carboxyl group of the amino acid and the 3' oxygen of the of the amino acid and the 3' oxygen of the tRNA.tRNA.


Transfer RNATransfer RNA

Example—Phenylalanine transfer RNAExample—Phenylalanine transfer RNA

One of the mRNA codons for phenylalanine is:One of the mRNA codons for phenylalanine is:

UUCUUC5'5' 3'3'

AAGAAG3'3' 5'5'

The complementary sequence in tRNA is calledThe complementary sequence in tRNA is calledthe the anticodonanticodon..


Fig. 28.11 Phenylalanine tRNAFig. 28.11 Phenylalanine tRNA

OCCHCHOCCHCH22CC66HH55

++NHNH33

OO

Anticodon

3'

5'3'

5'


Ribosomal RNARibosomal RNA

Most of the RNA in a cell is ribosomal RNAMost of the RNA in a cell is ribosomal RNARibosomes are the site of protein synthesis. Ribosomes are the site of protein synthesis. They are where They are where translationtranslation of the mRNA of the mRNA sequence to an amino acid sequence occurs.sequence to an amino acid sequence occurs.Ribosomes are about two-thirds RNA and one-Ribosomes are about two-thirds RNA and one-third protein.third protein.It is believed that the ribosomal RNA acts as a It is believed that the ribosomal RNA acts as a catalyst—a catalyst—a ribozyme.ribozyme.


28.1228.12Protein BiosynthesisProtein Biosynthesis


Protein BiosynthesisProtein Biosynthesis

During translation the protein is synthesized During translation the protein is synthesized beginning at its N-terminus.beginning at its N-terminus.mRNA is read in its 5'-3' directionmRNA is read in its 5'-3' direction

begins at the start codon AUGbegins at the start codon AUGends at stop codon (UAA, UAG, or UGA)ends at stop codon (UAA, UAG, or UGA)


Fig. 28.12 TranslationFig. 28.12 Translation

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

Methionine at N-terminusMethionine at N-terminusis present as its N-formylis present as its N-formylderivative.derivative.



Ester at 3' end of Ester at 3' end of alanine tRNA is Met-Ala.alanine tRNA is Met-Ala.Process continues Process continues along mRNA until stop along mRNA until stop codon is reached.codon is reached.


28.1328.13AIDSAIDS


AIDSAIDS

Acquired immune deficiency syndromeAcquired immune deficiency syndromeMore than 22 million people have died from More than 22 million people have died from AIDS since disease discovered in 1980sAIDS since disease discovered in 1980sNow fourth leading cause of death worldwide Now fourth leading cause of death worldwide and leading cause of death in Africa (World and leading cause of death in Africa (World Health Organization)Health Organization)


HIVHIV

Virus responsible for AIDS in people is HIV Virus responsible for AIDS in people is HIV (human immunodeficiency virus)(human immunodeficiency virus)Several strains of HIV designated HIV-1, HIV-2, Several strains of HIV designated HIV-1, HIV-2, etc.etc.HIV is a HIV is a retrovirusretrovirus. Genetic material is RNA, not . Genetic material is RNA, not DNA.DNA.


HIVHIV

HIV inserts its own RNA and an enzyme HIV inserts its own RNA and an enzyme ((reverse transcriptasereverse transcriptase) in T4 lymphocyte cell of ) in T4 lymphocyte cell of host.host.Reverse transcriptase catalyzes the formation of Reverse transcriptase catalyzes the formation of DNA complementary to the HIV RNA.DNA complementary to the HIV RNA.HIV reproduces and eventually infects other T4 HIV reproduces and eventually infects other T4 lympocytes.lympocytes.Ability of T4 cells to reproduce decreases, Ability of T4 cells to reproduce decreases, interfering with bodies ability to fight infection.interfering with bodies ability to fight infection.


AIDS DrugsAIDS Drugs

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

ONN OO

NN33

OOHH33CC

HOCHHOCH22

OO

NNHH

AZTAZT

O

NN

OO

HOCHHOCH22

OO

NNHH

ddIddI

NN

NN

HH

HH HH

HH


AIDS DrugsAIDS Drugs

Protease inhibitors are used in conjunction with Protease inhibitors are used in conjunction with other AIDS drugs.other AIDS drugs.Several HIV proteins are present in the same Several HIV proteins are present in the same polypeptide chain and must be separated from polypeptide chain and must be separated from each other in order to act.each other in order to act.Protease inhibitors prevent formation of HIV Protease inhibitors prevent formation of HIV proteins by preventing hydrolysis of polypeptide proteins by preventing hydrolysis of polypeptide that incorporates them.that incorporates them.


28.1428.14DNA SequencingDNA Sequencing


DNA SequencingDNA Sequencing

Restriction enzymes cleave the polynucleotide Restriction enzymes cleave the polynucleotide to smaller fragments.to smaller fragments.These smaller fragments (100-200 base pairs) These smaller fragments (100-200 base pairs) are sequenced.are sequenced.The two strands are separated.The two strands are separated.



Single stranded DNA divided in four portions.Single stranded DNA divided in four portions.Each tube contains adenosine, thymidine, Each tube contains adenosine, thymidine, guanosine, and cytidine plus the triphosphates guanosine, and cytidine plus the triphosphates of their 2'-deoxy analogs.of their 2'-deoxy analogs. POCHPOCH22

OHOH

OOOO

OO

OHOH

PP

OO

OHOH

PP

OO

HOHO basebase

HHHHOO

OO



The first tube also contains the 2,'3'-dideoxy analog of The first tube also contains the 2,'3'-dideoxy analog of adenosine triphosphate (ddATP); the second tube the adenosine triphosphate (ddATP); the second tube the 2,'3'-dideoxy analog of thymidine triphosphate (ddTTP), 2,'3'-dideoxy analog of thymidine triphosphate (ddTTP), the third contains ddGTP, and the fourth ddCTP.the third contains ddGTP, and the fourth ddCTP. POCHPOCH22

OHOH

OOOO

OO

OHOH

PP

OO

OHOH

PP

OO

HOHO basebase

HHHH

OO



Each tube also contains a "primer," a short Each tube also contains a "primer," a short section of the complementary DNA strand, section of the complementary DNA strand, labeled with radioactive phosphorus (labeled with radioactive phosphorus (3232P).P).DNA synthesis takes place, producing a DNA synthesis takes place, producing a complementary strand of the DNA strand used complementary strand of the DNA strand used as a template.as a template.DNA synthesis stops when a dideoxynucleotide DNA synthesis stops when a dideoxynucleotide is incorporated into the growing chain.is incorporated into the growing chain.



The contents of each tube are separated by The contents of each tube are separated by electrophoresis and analyzed by electrophoresis and analyzed by autoradiography.autoradiography.There are four lanes on the electrophoresis gel.There are four lanes on the electrophoresis gel.Each DNA fragment will be one nucleotide Each DNA fragment will be one nucleotide longer than the previous one.longer than the previous one.


Figure 27.29Figure 27.29

TT

TTGG

TTGGAA

TTGGAACC

TTGGAACCAA

TTGGAACCAATT

TTGGAACCAATTAA

TTGGAACCAATTAACC

TTGGAACCAATTAACCGG

TTGGAACCAATTAACCGGTT

ddAddA ddTddT ddGddG ddCddCSequence of Sequence of fragmentfragment


Figure 27.29Figure 27.29

TT

TTGG

TTGGAA

TTGGAACC

TTGGAACCAA

TTGGAACCAATT

TTGGAACCAATTAA

TTGGAACCAATTAACC

TTGGAACCAATTAACCGG

TTGGAACCAATTAACCGGTT

ddAddA ddTddT ddGddG ddCddC

AA

AACC

AACCTT

AACCTTGG

AACCTTGGTT

AACCTTGGTTAA

AACCTTGGTTAATT

AACCTTGGTTAATTGG

AACCTTGGTTAATTGGCC

AACCTTGGTTAATTGGCCAA

Sequence of Sequence of fragmentfragment

Sequence of Sequence of original DNAoriginal DNA


28.1528.15The Human Genome ProjectThe Human Genome Project


Human Genome ProjectHuman Genome Project

In 1988 National Research Council (NRC) In 1988 National Research Council (NRC) recommended that the U.S. undertake the recommended that the U.S. undertake the mapping and sequencing of the human mapping and sequencing of the human genome.genome.International Human Genome Sequencing International Human Genome Sequencing Consortium (led by U.S. NIH) and Celera Consortium (led by U.S. NIH) and Celera Genomics undertook project. Orginally Genomics undertook project. Orginally competitors, they agreed to coordinate efforts competitors, they agreed to coordinate efforts and published draft sequences in 2001.and published draft sequences in 2001.


28.1628.16DNA ProfilingDNA Profiling

and theand thePolymerase Chain ReactionPolymerase Chain Reaction


DNA ProfilingDNA Profiling

DNA sequencing involves determining the DNA sequencing involves determining the nucleotide sequence in DNA.nucleotide sequence in DNA.The nucleotide sequence in regions of DNA that The nucleotide sequence in regions of DNA that code for proteins varies little from one individual code for proteins varies little from one individual to another, because the proteins are the same.to another, because the proteins are the same.Most of the nucleotides in DNA are in Most of the nucleotides in DNA are in "noncoding" regions and vary significantly "noncoding" regions and vary significantly among individuals.among individuals.Enzymatic cleavage of DNA give a mixture of Enzymatic cleavage of DNA give a mixture of polynucleotides that can be separated by polynucleotides that can be separated by electrophoresis to give a "profile" characteristic electrophoresis to give a "profile" characteristic of a single individual. of a single individual.


PCRPCR

When a sample of DNA is too small to be When a sample of DNA is too small to be sequenced or profiled, the sequenced or profiled, the polymerase chain polymerase chain reactionreaction (PCR) is used to make copies (PCR) is used to make copies ("amplify") portions of it.("amplify") portions of it.PCR amplifies DNA by repetitive cycles of the PCR amplifies DNA by repetitive cycles of the following steps.following steps.

1. Denaturation1. Denaturation2. Annealing ("priming")2. Annealing ("priming")3. Synthesis ("extension" or "elongation")3. Synthesis ("extension" or "elongation")


Figure 28.14 (PCR)Figure 28.14 (PCR)

Target regionTarget region

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

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


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

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



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

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



This completes one cycle of PCR. This completes one cycle of PCR.

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



This completes one cycle of PCR. This completes one cycle of PCR.

((ee) The next cycle begins with the denaturation) The next cycle begins with the denaturationof the two DNA molecules shown. Both areof the two DNA molecules shown. Both arethen primed as before.then primed as before.



((ee) The next cycle begins with the denaturation) The next cycle begins with the denaturationof the two DNA molecules shown. Both areof the two DNA molecules shown. Both arethen primed as before.then primed as before.

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



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

((gg) Among the 8 DNAs formed in the second) Among the 8 DNAs formed in the secondcycle are two having the structure shown. cycle are two having the structure shown.



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

((gg) Among the 8 DNAs formed in the second) Among the 8 DNAs formed in the secondcycle are two having the structure shown. cycle are two having the structure shown.


Dr. Wolf's CHM 424 28- 100


CycleCycle Total DNAsTotal DNAs Contain only targetContain only target0 (start)0 (start) 11 0011 22 0022 44 0033 88 2244 1616 8855 3232 22221010 1,0241,024 1,0041,0042020 1,048,5661,048,566 1,048,5261,048,5263030 1,073,741,8241,073,741,824 1,073,741,7641,073,741,764

Dr. Wolf's CHM 424 28- 101

End of Chapter 28End of Chapter 28

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