Basic Molecular Biology

January 2002

Macromolecules and their unit components

Macro molecules unit components

Nucleic acids nucleotides(DNA& RNA)

Proteins amino acids

Carbohydrates Sugars

Lipids Fatty acids√

√√

Amino acids

NC

CH

H

O

OH

H

R

aminegroup

carboxyl

side chain

NC

CH

H

O

O

H

H

R

-+

pH 6-7

Amino acids groups

Group Characteristics Names Example (-Rx)

non-polar hydrophobic Ala, Val, Leu,Ile, Pro, PheTrp, Met

polar hydrophilic Gly , Ser, Thr,(non-charged) Cys, Tyr, Asn

Gln

acidic negatively Asp, Glu charged

basic positively Lys, Arg, His charged

CH CH2

CH3

CH3 Leu

Thr

CH2C

O

O- Asp

CH2CH2CH2CH2NH3

+

Lys

CH

OH

CH3

Total = 20

peptides have different sequences

+ NH 3

CH

C

O

NH

CH

C

O

NH O -

CH

C

O

NH

CH

C

O

SH

CH 2

OH

CH

CH2

CH 2

OH

CH2

C

OO-

Tyr------Cys--------Asp-----------Ser = Y-C-D-S

Primary structureThe primary structure of a protein is defined as its sequence of amino acids.

Connexin26MDWGTLQSIL GGVNKHSTSI GKIWLTVLFI FRIMILVVAA KEVWGDEQAD FVCNTLQPGC KNVCYDHHFP ISHIRLWALQ LIMVSTPALL VAMHVAYRRH EKKRKFMKGE IKNEFKDIEE IKTQKVRIEG SLWWTYTTSI FFRVIFEAVF MYVFYIMYNG FFMQRLVKCN AWPCPNTVDC FISRPTEKTV FTVFMISVSG ICILLNITEL CYLFVRYCSG KSKRPV

Questions:

How is the information about protein sequences- stored in the cell ?- duplicated after cell division?

Answer:

...DNA...

BASE

TRIPHOSPHATE

RIBOSESUGAR

NUCLEOTIDE

N

NN

N

NH2

OCH2OP

O

OH

OP

O

OH

OH

OP

O

OH

OH

Nucleotides

Purines Pyrimidines

ATP

NH2

N

NN

N

OCH2OP

O

OH

OP

O

OH

OHOH

OP

O

OH

OH

O

OCH2OP

O

OH

OP

O

OH

OHOH

OP

O

OH

OH

N

N

O

O

H2N

GTP

NH2

CTP

CH3

TTP (UTP)

Nucleotide chain

OCH2OP

O

OH

OP

O

OH

OHO

OP

O

OH

OH

OCH2OP

O

OP

O

OH

HO

OHO

OP

O

OH

OH

N

O

OCH2OP

O

HO

OHOH

1’

2’3’

4’

5’

1’

2’3’

4’

5’

1’

2’3’

4’

5’

base

base

base

5’ end

3’ end

Nucleotide Pairing

N

NN

N

N

N

N

O

CH3

H HO

H

ThymidineAdenine

H- bonds

T A

Nucleotide Pairing

Guanine

N

NN

N

O

H

N

N

N

O

CH3

N

H

H

H

H

H- bonds

Cytosine

N

NN

N

N

N

N

O

CH3

H HO

H

ThymidineAdenine

H- bonds

T A

C G

The double helix

A

A

A

A A

T

T

TT

T

C

C

C

C

C GG

G

G

G

A T

CGA

T

5’ACGGGTACATGAC3’ |||||||||||||3’TGCCCATGTACTG5’

antiparallel complimentary strand

Types of Nucleic Acids

DNAnuclear DNA (linear)mitochondrial DNA (circular)plasmid DNA (circular

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

Differences between RNA and DNA

1) ribose sugar

OCH2

OH

RNA DNA

(ribonucleic acid) (deoxy ribonucleic acid)

OCH2

OHOH

Differences between RNA and DNA

1) ribose sugar

OCH2

OH

RNA DNA

(ribonucleic acid) (deoxy ribonucleic acid)

OCH2

OHOH

2 )T and U

N

N

O

N

N

O

CH3

uracyl thymidine

O

O

Differences between RNA and DNA

1) ribose sugar

OCH2

OH

RNA DNA

(ribonucleic acid) (deoxy ribonucleic acid)

OCH2

OHOH

2 )T and U

N

N

O

N

N

O

CH3

uracyl thymidine

3) strand single double

O

O

DNA replication

5’TGAC ||||3’ACTG

ATGGGTACA

TACCCATGT

CATGAC

GTACTG

GGG 3’

CCC 5’||||||||||||||||||

DNA replication

5’TGAC ||||3’ACTG

ATGGGTAC

A

TACCCATGT

CATGAC

GTACTG

GGG 3’

CCC 5’|||

A

G

DNA polymerase

DNA replication

5’TGAC ||||3’ACTG

ATGGGTAC

A

TACCCATGT

CATGAC

GTACTG

GGG 3’

CCC 5’|||A

GT

T

DNA replication

5’TGAC ||||3’ACTG

ATGGGTAC

A

TACCCATGT

CATGAC

GTACTG

GGG 3’

CCC 5’|||A

GT

TG

C

DNA replication

5’TGAC ||||3’ACTG

ATGGGTAC

A

TACCCATGT

CATGAC

GTACTG

GGG 3’

CCC 5’|||A

GT

TG

C

G

A

DNA replication

5’ACGGGTACATGAC |||||||||||||3’TGCCCATGTACTG

ACGGGTACA

TGCCCATGT

CATGAC

GTACTG

GGG 3’

CCC 5’|||5’ACGGGT3’

3’ACTG5’

5’ACGGGTACATGAC |||||||||||||3’TGCCCATGTACTG

ACGGGTAC 3’||||||||TGCCCATG 5’

5’ACGGGTACATGAC |||||||||||||3’TGCCCATGTACTG

ACGGGTAC 3’||||||||TGCCCATG 5’

semiconservative replication

old

new

new

old

DNA proof-reading

5’TGAC ||||3’ACTG

ATGGGTAC

A

TACCCATGT

CATGAC

GTACTG

GGG 3’

CCC 5’|||A

GT

TG

C

T

A

DNA polymerase has exonuclease activity: it can cut out unpaired bases to prevent mistakes(mutations) in DNA replication

DNA proof-reading

5’TGAC ||||3’ACTG

ATGGGTAC

A

TACCCATGT

CATGAC

GTACTG

GGG 3’

CCC 5’|||A

GT

TG

C

T

A

DNA polymerase has exonuclease activity: it can cut out unpaired bases to prevent mistakes(mutations) in DNA replication

DNA proof-reading

5’TGAC ||||3’ACTG

ATGGGTAC

A

TACCCATGT

CATGAC

GTACTG

GGG 3’

CCC 5’|||A

GT

TG

CT

A

DNA polymerase has exonuclease activity: it can cut out unpaired bases to prevent mistakes(mutations) in DNA replication

G

DNA(basic facts)• DNA is located in the cell nucleus

38 44Number ofchromosomes

46

• DNA subdivided in chromosomes whose number differ among species (46 in humans, 22+22+X +Y)

• DNA is associated with histones protein evenly distributed approximately every 200bp. (DNA+histones =chromatin)

From DNA to protein

A

A

A

A A

T

T

TT

T

C

C

C

C

C GG

G

G

GA T

CGAT

+ N H 3

C HR 1

C O

N H

CH R 2

C O

N H

O -

C HR 3

C O

N H

C H R 4

CO

Protein synthesisDNA

ACGTCTCAATGCAGAGTT

ACGUCUCAARNA

Transcription RNA polymerasenuclear factors

Protein

Translationribosomes (proteins+ rRNA)t RNAs

Thr-Ser-Gln

Transcription

5’TGAC ||||3’ACTG

ATGGGTACA

TACCCATGT

CATGAC

GTACTG

GGG 3’

CCC 5’||||||||||||||||||

Transcription

3’

3’

TGACATGGGTACACATGACGGG

ACTGTACCCATGTGTACTGCCC 5’

5’

U AUAU

RNA polymerase

G

NUCLEUS

CYTOSOL

Transcription

AAAAUGAUAU UGAUAUAAAAMESSANGER RNA (mRNA)

Translation(from mRNA to protein)

Triplet codon: three nucleotides code for one amino acid

AUG = Met ACA = ThrCAU = His UUU = Phe

Met

UAC

transfer RNA (tRNA)there are 20 different tRNA(each one carrying a specific amino acid)

anticodon

Translation

AUG ACG UCU CAA mRNA

Translation

AUG ACG UCU CAA

Met

UAC

mRNA

Thr

UGC

Translation

AUG ACG UCU CAA

Met

UAC

mRNA

Ser

AGA

Thr

UGC

Translation

AUG ACG UCU CAA

Met

mRNA

Thr

UGC

Ser

Translation

AUG ACG UCU CAA

Met

Gln

GUU

mRNA

Translation

AUGAUAGCCGAUU

Ribosome

mRNA

Translation

AUGAUAGCCGAUU

Ribosome

Translation

AUGAUAGCCGAUU

Ribosome

growing protein

N-terminus

C-terminus

Start and Stop codonsACCA-AUG-AUA-GCC-GAU-GGG-UGA-GGAG

The start codon is AUG and it also codes for Methionine

There are three stop codons UGA, UAA and UAG

Codon Degeneracy

-there are 20 amino acids but 64 codons -for some amino acid there is more than one codon-the last of the three bases is the least specific

UUU UUAUUGUUC

PheCAACAG Gln

CGUCGCCGG ArgCGAAGAAGG

AUG Met UGG Trp

Post-translational modification

After synthesis a protein can undergo one or more modifications. e.g.

• Tertiary structure : regulated by chaperons

• Glycosidation: addition of sugars

• Proteolitic cleavage

• Phosphorylation: addition of phosphate groups to Tyr, Thr and Ser.

• Other....

MUTATIONS• AGTFTHEDOGATETHECATANDTHEBUNENDAFAT

AGTF THE DOG ATE THE CAT AND THE BUN END AFAT

AGTF THE DOG ATE THH CAT AND THE BUN END AFAT

AGTF THE HOG ATE THE CAT AND THE BUN END AFAT

AGTF THE DOG ATE THE CAT END THEBUNENDAFAT

AGTF THE DOP GAT ETH ECA TAN DTH EBU NEN DAF AT

AGTF THE DOG ATE THC ATA NDT HEB UNE NDA FAT

AGTF THE DOG ATE CAT AND THE BUN END AFAT

E

THE

MUTATIONS• UGUAC AUG UAU ACG UCU CAA UGA UCCA

Met Tyr Ser Thr Gln STOP

POINT MUTATIONS• UGUAC AUG UAU ACG UCU CAG UGA UCCA Met Tyr Ser Thr Gln STOP

• UGUAC AUG UAU ACG CCU CAA UGA UCCA Met Tyr Ser Pro Gln STOP

• UGUAC AUG UAA ACG UCU CAA UGA UCCA Met STOP

MUTATIONS• UGUAC AUG UAU ACG UCU CAA UGA UCCA

Met Tyr Ser Thr Gln STOP

Deletions• UGUAC AUG UAU CGU CUC AAU GAU CCA Met Tyr Arg Leu Asn Asp Pro

• UGUAC AUG UAU UCU CAA UGA UCCAMet Tyr Thr Gln STOP

Insertion• UGUAC AUG UAU ACG AUC UCA AUG AUC

Met Tyr Ser Ile Ser Met Ile

A

A

ACG

Restriction enzymes

Enzymes found in bacteria that have the ability to cut DNA at specific sites

ACGTGCCATGAATTCGCATGATGCATGCTCGAGCATAGCTGCACGGTACTTAAGCGTACTACGTACGAGCTCGTATCG

EcoRI (GATTC)

AATTCGCATGATGCATGCTCGAGCATAGC GCGTACTACGTACGAGCTCGTATCGACGTGCCATG

TGCACGGTACTTAA

Restriction enzymes

ACGTGCCATGAATTCGCATGATGCATGCTCGAGCATAGCTGCACGGTACTTAAGCGTACTACGTACGAGCTCGTATCG

EcoRI (GATTC) XhoI (CTCGAG)

AATTCGCATGATGCATGCTCG GCGTACTACGTACGA AGCATAGC

GCTCGTATCGACGTGCCATGTGCACGGTACTTAA

recombination

ACGTGCCATGAATTCGCATCATGCGAATTCATAGCTGCACGGTACTTAAGCGTACTACGCTTAAGTATCG

TAGCATGAATTCGCATCGATCATCGTACTTAAGCGTAGCTAG

PLASMID

recombination

TAGCATG AATTCGCATCGATCATCGTACTTAA GCGTAGCTAG

PLASMID

ACGTGCCATG AATTCGCATCATGCG AATTCATAGCTGCACGGTACTTAA GCGTACTACGCTTAA GTATCG

recombination

TAGCATGAATTCGCATCATGCGAATTCGCATCGATCATCGTACTTAAGCGTACTACGCTTAAGCGTAGCTAG

PLASMID

ACGTGCCATG AATTCATAGCTGCACGGTACTTAA GTATCG

Exercise 1

5’ACGTGCCATGAATTCGCATCATGCGAATTCATAGC 3’3’TGCACGGTACTTAAGCGTACTACGCTTAAGTATCG 5’

5’ACGTGCCATGAATTCGCATCATGCGAATTCATAGC 3’

5’UGAAUUCGCAUCAUGCGAAUUCAUAGC 3’3’TGCACGGTACTTAAGCGTACTACGCTTAAGTATCG 5’

Transcribe the following DNA sequence into RNA starting from the base indicated by the arrow

Exercise 2Translate the following RNA sequence into a protein sequence starting from the first start codon

5’-ACCAUCCAGAGGACAAGAUGGAUUGGGGCACACUACAGAGCA UCCUCGGGUAAGGUGUCAACAA-3’

AUG GAU UGG GGC ACA CUA CAG AGC AUC CUC GGG UAA GGU GUC AAC AA

AUG GAU UGG GGC ACA CUA CAG AGC AUCMet Asp Trp Gly Thr Lys Gln Ser Iso M D W G T L Q S I

CUC GGG UAA GGUGUCAACAALeu Gly Stop L G

General plan• Lecture 1 Jan 9th (2hrs)

– DNA structure,replication,– Transcription translation– Techniques (to introduce practical)– Computer: retrieve sequence and digestion map

• Lecture 2 Jan 16th (3hrs)– Lab digest CX26 wt and mutant– Regulation of gene expression– Molecular biology of the ear– Run gel– Exercises- pseudo-hybridisation– (assign developmental gene)

• Lecture 3 development – Molecular biology of the ear: development– Papers on development– Develop map of ear development

plan

• Review of protein structure (primary)

• Structure of dna

• DNA replication (basics)

• Transcription

• Translation

• Exercise transcription translation

• Basic techniques: restriction enzymes Computer retrieve cx26 sequence perform restriction map