dna,rna
TRANSCRIPT
Dr. Wolf's CHM 424 28- 1
Nucleic AcidsNucleic Acids
Dr. Wolf's CHM 424 28- 2
28.128.1Pyrimidines and PurinesPyrimidines and Purines
Dr. Wolf's CHM 424 28- 3
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.
Dr. Wolf's CHM 424 28- 4
Pyrimidines and PurinesPyrimidines 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
Dr. Wolf's CHM 424 28- 5
Pyrimidines and PurinesPyrimidines and Purines
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
Dr. Wolf's CHM 424 28- 6
Pyrimidines and PurinesPyrimidines and Purines
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
Dr. Wolf's CHM 424 28- 7
Pyrimidines and PurinesPyrimidines and Purines
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
Dr. Wolf's CHM 424 28- 8
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
Dr. Wolf's CHM 424 28- 9
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
Dr. Wolf's CHM 424 28- 10
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
Dr. Wolf's CHM 424 28- 11
28.228.2NucleosidesNucleosides
Dr. Wolf's CHM 424 28- 12
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..
Dr. Wolf's CHM 424 28- 13
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
Dr. Wolf's CHM 424 28- 14
OO
NN
NNHH
OO
HOHO
OO
HH33CC
HOCHHOCH22
Table 28.2Table 28.2
Pyrimidine nucleosidesPyrimidine nucleosides
Thymidine occurs in DNAThymidine occurs in DNA
Thymidine
Dr. Wolf's CHM 424 28- 15
Table 28.2Table 28.2
Pyrimidine nucleosidesPyrimidine nucleosides
HOCHHOCH22
OO
NN
NNHH
OO
OHOHHOHO
OO
Uridine occurs in RNAUridine occurs in RNA
Uridine
Dr. Wolf's CHM 424 28- 16
Table 28.2Table 28.2
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
Dr. Wolf's CHM 424 28- 17
Table 28.2Table 28.2
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
Dr. Wolf's CHM 424 28- 18
28.328.3NucleotidesNucleotides
Nucleotides are phosphoric acid esters of Nucleotides are phosphoric acid esters of nucleosides.nucleosides.
Dr. Wolf's CHM 424 28- 19
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'
Dr. Wolf's CHM 424 28- 20
Adenosine Diphosphate (ADP)Adenosine Diphosphate (ADP)
OOPP
OO
HOHO
HOHO
OCHOCH22PP
OO
HOHOOO
OHOHHOHO
NN
NN
NN
NHNH22
NN
Dr. Wolf's CHM 424 28- 21
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
Dr. Wolf's CHM 424 28- 22
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
Dr. Wolf's CHM 424 28- 23
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
Dr. Wolf's CHM 424 28- 24
28.628.6Phosphodiesters, Phosphodiesters,
Oligonucleotides, and Oligonucleotides, and PolynucleotidesPolynucleotides
Dr. Wolf's CHM 424 28- 25
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.
Dr. Wolf's CHM 424 28- 26
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
Dr. Wolf's CHM 424 28- 27
28.728.7Nucleic AcidsNucleic Acids
Nucleic acids are polynucleotides.Nucleic acids are polynucleotides.
Dr. Wolf's CHM 424 28- 28
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.
Dr. Wolf's CHM 424 28- 29
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
Dr. Wolf's CHM 424 28- 30
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
Dr. Wolf's CHM 424 28- 31
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
Dr. Wolf's CHM 424 28- 32
28.828.8Secondary Structure of DNA:Secondary Structure of DNA:
The Double HelixThe Double Helix
Dr. Wolf's CHM 424 28- 33
FORMS OF DNAFORMS OF DNA
Dr. Wolf's CHM 424 28- 34
Dr. Wolf's CHM 424 28- 35
Dr. Wolf's CHM 424 28- 36
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
Dr. Wolf's CHM 424 28- 37
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.
Dr. Wolf's CHM 424 28- 38
Base PairingBase Pairing
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
2-deoxyribose2-deoxyribose
2-deoxyribose2-deoxyribose
GG CC
Dr. Wolf's CHM 424 28- 39
Base PairingBase Pairing
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.
Dr. Wolf's CHM 424 28- 40
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.
Dr. Wolf's CHM 424 28- 41
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.
Dr. Wolf's CHM 424 28- 42
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.
Dr. Wolf's CHM 424 28- 43
28.928.9Tertiary Structure of DNA:Tertiary Structure of DNA:
SupercoilsSupercoils
Dr. Wolf's CHM 424 28- 44
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.
Dr. Wolf's CHM 424 28- 45
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.
Dr. Wolf's CHM 424 28- 46
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.
Dr. Wolf's CHM 424 28- 47
Histones Histones
NucleosomeNucleosome == Histone proteinsHistone proteins + Supercoiled DNA+ Supercoiled DNA
Dr. Wolf's CHM 424 28- 48
28.1028.10Replication of DNAReplication of DNA
Dr. Wolf's CHM 424 28- 49
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)
Dr. Wolf's CHM 424 28- 50
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'
Fig. 28.8 DNA Replication
Each strand will become a template for construction of its complement.
Dr. Wolf's CHM 424 28- 51
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'
Fig. 28.8 DNA Replication
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.
Dr. Wolf's CHM 424 28- 52
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'
+
Fig. 28.8 DNA Replication
Two duplex DNAs result, each of which is identical to the original DNA.
Dr. Wolf's CHM 424 28- 53
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.
Dr. Wolf's CHM 424 28- 54
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
Dr. Wolf's CHM 424 28- 55
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––––
Dr. Wolf's CHM 424 28- 56
28.1128.11Ribonucleic AcidsRibonucleic Acids
Dr. Wolf's CHM 424 28- 57
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
Dr. Wolf's CHM 424 28- 58
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..
Dr. Wolf's CHM 424 28- 59
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.
Dr. Wolf's CHM 424 28- 60
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.
Dr. Wolf's CHM 424 28- 61
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)
Dr. Wolf's CHM 424 28- 62
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
Dr. Wolf's CHM 424 28- 63
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.
Dr. Wolf's CHM 424 28- 64
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.
Dr. Wolf's CHM 424 28- 65
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..
Dr. Wolf's CHM 424 28- 66
Fig. 28.11 Phenylalanine tRNAFig. 28.11 Phenylalanine tRNA
OCCHCHOCCHCH22CC66HH55
++NHNH33
OO
Anticodon
3'
5'3'
5'
Dr. Wolf's CHM 424 28- 67
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.
Dr. Wolf's CHM 424 28- 68
28.1228.12Protein BiosynthesisProtein Biosynthesis
Dr. Wolf's CHM 424 28- 69
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)
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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.
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Fig. 28.12 TranslationFig. 28.12 Translation
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Fig. 28.12 TranslationFig. 28.12 Translation
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.
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28.1328.13AIDSAIDS
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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)
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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.
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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.
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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
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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.
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28.1428.14DNA SequencingDNA Sequencing
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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.
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DNA SequencingDNA Sequencing
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
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DNA SequencingDNA Sequencing
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
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DNA SequencingDNA Sequencing
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.
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DNA SequencingDNA Sequencing
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.
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Figure 27.29Figure 27.29
TT
TTGG
TTGGAA
TTGGAACC
TTGGAACCAA
TTGGAACCAATT
TTGGAACCAATTAA
TTGGAACCAATTAACC
TTGGAACCAATTAACCGG
TTGGAACCAATTAACCGGTT
ddAddA ddTddT ddGddG ddCddCSequence of Sequence of fragmentfragment
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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
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28.1528.15The Human Genome ProjectThe Human Genome Project
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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.
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28.1628.16DNA ProfilingDNA Profiling
and theand thePolymerase Chain ReactionPolymerase Chain Reaction
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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.
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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")
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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.
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((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.
Figure 28.14 (PCR)Figure 28.14 (PCR)
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((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.
Figure 28.14 (PCR)Figure 28.14 (PCR)
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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.
Figure 28.14 (PCR)Figure 28.14 (PCR)
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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.
Figure 28.14 (PCR)Figure 28.14 (PCR)
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((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.
Figure 28.14 (PCR)Figure 28.14 (PCR)
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((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.
Figure 28.14 (PCR)Figure 28.14 (PCR)
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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.
Figure 28.14 (PCR)Figure 28.14 (PCR)
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Table 28.4Table 28.4
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
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End of Chapter 28End of Chapter 28