dna. genetics & dna dna: picture 51 b-dna: the advent of modeling

DNADNA

Genetics & DNA

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DNA: Picture 51http://info.bio.cmu.edu/courses/03231/ProtStruc/ProtStruc.htm

B-DNA: The advent of modeling

http://molvis.sdsc.edu/pdb/dna_b_form.pdb

46 Å

12 base sequence

(1953-2003)

http://info.bio.cmu.edu/courses/03231/ProtStruc/ProtStruc.htm














DNA: Size, Shape & Self Assemblyhttp://www.umass.edu/microbio/chime/beta/pe_alpha/atlas/atlas.htm

Views & Algorithms

10.85 Å10.85 Å

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http://www.umass.edu/microbio/chime/beta/pe_alpha/atlas/atlas.htm






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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.

NN

NN

NN

NN

NN

NNHH

PyrimidinePyrimidine PurinePurine

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

NucleosidesNucleosides

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..

Uridine and AdenosineUridine and Adenosine

Uridine and adenosine are pyrimidine and purine Uridine and adenosine are pyrimidine and purine nucleosides respectively of nucleosides respectively of DD-ribofuranose.-ribofuranose.

UridineUridine(a pyrimidine nucleoside)(a pyrimidine nucleoside)

AdenosineAdenosine(a purine nucleoside)(a purine nucleoside)

OO

NN

HOCHHOCH22

HHNN

OO

OHOHHOHO

OO

NN NN

NN NNHOCHHOCH22 OO

OHOHHOHO

NNHH22

NucleotidesNucleotides

NucleotidesNucleotides

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

NN NN

NN NN

OO

OHOHHOHO

NNHH22

OCHOCH22PPHOHO

OO

HOHO

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

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

NN NN

NN NN

OO

OHOHHOHO

NNHH22

OCHOCH22PPHOHO

OO

HOHO1'1'

2'2'3'3'

4'4'5'5'

Adenosine Diphosphate (ADP)Adenosine Diphosphate (ADP)

NN NN

NN NN

OO

OHOHHOHO

NNHH22

OCHOCH22PPOO

OO

HOHO

PP

OO

HOHO

HOHO

Adenosine Triphosphate (ATP)Adenosine Triphosphate (ATP)

NN NN

NN NN

OO

OHOHHOHO

NNHH22

OCHOCH22PPOO

OO

HOHO

PP

OO

HOHO

OOPP

OO

HOHO

HOHO

ATP Stores EnergyATP Stores Energy

AMPAMP

ADPADP

ATPATPEach step is endothermic.Each step is endothermic.

Energy for each step comes Energy for each step comes from carbohydrate metabolism from carbohydrate metabolism (glycolysis).(glycolysis).

Reverse process is exothermic Reverse process is exothermic and is the source of biological and is the source of biological energy.energy.

G° for hydrolysis of ATP to G° for hydrolysis of ATP to ADP is –35 kJ/molADP is –35 kJ/mol

Adenosine 3'-5'-Cyclic Monophosphate Adenosine 3'-5'-Cyclic Monophosphate (cAMP)(cAMP)

Cyclic AMP is an important regulator of many Cyclic AMP is an important regulator of many biological processes.biological processes.

NN NN

NN NN

OO

OHOHOO

NNHH22

CHCH22

PPOO

HOHO

OO

Nucleic AcidsNucleic Acids

Nucleic AcidsNucleic Acids

Nucleic acids are polymeric nucleotides Nucleic acids are polymeric nucleotides ((polynucleotides).polynucleotides).

5' Oxygen of one nucleotide is linked to the 3' 5' Oxygen of one nucleotide is linked to the 3' oxygen of another.oxygen of another.

What are the bases?What are the bases?

O

NH2N

N N

N

OHO

OCH2

P OCH2–O

O O

NH2

N

N

O OH

O

P OCH2

O

–O

O OH

O N

NH

O

O

P–O

O

O

3'

3'

3'

5'

5'

5'

A section of a A section of a polynucleotide polynucleotide chain.chain.

Structure and Replication of DNA:Structure and Replication of DNA:The Double HelixThe Double Helix

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

Base PairingBase Pairing

Watson and Crick proposed that A and T were Watson and Crick proposed that A and T were equal because of complementary hydrogen equal because of complementary hydrogen bonding.bonding.

2-deoxyribose2-deoxyribose

N

NN

N N

H

H

N

N

O CH3

O

H

2-deoxyribose2-deoxyriboseAA TT

Base PairingBase Pairing

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

N

NN

N O

NH

H

H

N

N

N

O

HH



GG CC

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.

O

O

–O

–O

–O

O

O

O

O

OP

O

O

P O

OO

P O

OO

O

O

O–

O–

O–

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'

DNADNA

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.

DNADNA

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.

DNA Replication

C

G

T

AAs the double helix unwinds, each strand acts as a template upon which its complement is constructed.

DNA Replication

A

C

G

T

T'

A'

G'

C'

DNA-Directed Protein BiosynthesisDNA-Directed Protein Biosynthesis

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

Transcription is the formation of a strand of Transcription is the formation of a strand of mRNA using one of the DNA strands as a mRNA using one of the DNA strands as a template.template.

The nucleotide sequence of the mRNA is The nucleotide sequence of the mRNA is complementary to the nucleotide sequence of complementary to the nucleotide sequence of the DNA template.the DNA template.

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..


A G G T C A C T G

T C C A G T G A CA

TG

C TT

T

T

T

TC

C

C

C

C

A

A

AA

A

G

G

A

G

G

G

As double-stranded DNA unwinds, As double-stranded DNA unwinds, a complementary strand of mRNA a complementary strand of mRNA forms at the 5' end.forms at the 5' end.

5'5'

3'3'


A G G T C A C T G

T C C A G T G A CA

TG

C TT

T

T

T

TC

C

C

C

C

A

A

AA

A

G

G

A

G

G

G

A

AG

GU

C

5'5'

3'3'

Uracil is incorporated in RNA Uracil is incorporated in RNA instead of thymine.instead of thymine.

TranslationTranslation

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.

mRNA CodonsmRNA Codons

AlanineAlanine ArginineArginine AsparagineAsparagine Aspartic AcidAspartic Acid CysteineCysteine

GCUGCU GCAGCA CGUCGU CGA CGA AAUAAU GAU GAU UGUUGUGCCGCC GCGGCG AGAAGA CGC CGC AACAAC GACGAC UGC UGC

CGGCGG AGG AGG

Glutamic acidGlutamic acid GlutamineGlutamine GlycineGlycine HistidineHistidine IsoleucineIsoleucineGAAGAA CAACAA GGUGGU GGAGGA CAUCAU AUUAUU AUAAUAGAGGAG CAGCAG GGCGGC GGGGGG CACCAC AUCAUC

LeucineLeucine LysineLysine MethionineMethionine PhenylalaninePhenylalanine ProlineProlineUUAUUA CUUCUU AAAAAA AUGAUG UUUUUU CCUCCU CCA CCA CUACUA UUGUUG AAGAAG UUCUUC CCC CCC CGCGCUCCUC CUGCUG

SerineSerine ThreonineThreonine TryptophanTryptophan TyrosineTyrosine ValineValineUCUUCU UCA UCA ACUACU ACAACA UGGUGG UAUUAU GUUGUU GUAGUAAGUAGU UCC UCC ACCACC ACGACG UACUAC GUC GUC GUGGUGUCGUCG AGC AGC

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.

Phenylalanine tRNAPhenylalanine tRNA

Phenylalanine tRNAPhenylalanine tRNA OCCHCHOCCHCH22CC66HH55

NHNH33++

OO

A

CC

A A A

A

A

AA

A

AA

AA

A

A

A

A

AC CC

C

CC

CC

C C

CC

U

U

U U

U

UU

UU

UU

GG

G

G GG

GG

G

GG

G

UG G

G

GC

This AAA triplet is This AAA triplet is complementary to a complementary to a UUU tripletUUU tripletof mRNA; it is an of mRNA; it is an anticodonanticodon..

Protein SynthesisProtein Synthesis

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.

Gel ElectrophoresisGel Electrophoresis

The bloody glove?The bloody glove?

TT

TTGG

TTGGAA

TTGGAACC

TTGGAACCAA

TTGGAACCAATT

TTGGAACCAATTAA

TTGGAACCAATTAACC

TTGGAACCAATTAACCGG

TTGGAACCAATTAACCGGTT

ddAddA ddTddT ddGddG ddCddCSequence of Sequence of fragmentfragment

The bloody glove?The bloody glove?

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

Genetic Fingerprinting

OJ and the bloody glove!

ForensicsPaternityID-militaryFoodWineAnthropology

OTHER APPLICATIONS: Transgenic Crops

CLONING

Hello Dolly!

dna. genetics & dna dna: picture 51 b-dna: the advent of modeling

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