Basic Molecular Biology
January 2002
Macromolecules and their unit components
√
√
√
Macro molecules
unit components
Nucleic acids
(DNA& RNA)
nucleotides
Proteins
amino acids
Carbohydrates
Sugars
Lipids
Fatty acids
Amino acids
H
amine
group
H
O
H
C
N
C
OH
R
H +
H N
H
H
C
R
carboxyl
side chain
O
C
O
pH 6-7
Amino acids groups
Group
Characteristics
Names
Example (-Rx)
CH3
non-polar
polar
hydrophobic
hydrophilic
(non-charged)
Ala, Val, Leu,
Ile, Pro, Phe
Trp, Met
Gly , Ser, Thr,
Cys, Tyr, Asn
Gln
CH
CH3
Leu
OH
CH
CH3
acidic
negatively
charged
Asp, Glu
positively
charged
Thr
O
C CH2
O-
basic
CH2
Asp
Lys, Arg, His
NH3 + CH2 CH2 CH2 CH2
Total = 20
Lys
peptides have different sequences
CH2
SH
O
CH 2
C
CH
+ NH 3
NH
CH
CH 2
CH
O
C
CH
O
CH
O-
CH
C
NH
OH
NH
2
C
O
C
O-
O
OH
Tyr------Cys--------Asp-----------Ser
= Y-C-D-S
Primary structure
The primary structure of a protein is defined as its
sequence of amino acids.
Connexin26
MDWGTLQSIL
FRIMILVVAA
KNVCYDHHFP
VAMHVAYRRH
IKTQKVRIEG
MYVFYIMYNG
FISRPTEKTV
CYLFVRYCSG
GGVNKHSTSI
KEVWGDEQAD
ISHIRLWALQ
EKKRKFMKGE
SLWWTYTTSI
FFMQRLVKCN
FTVFMISVSG
KSKRPV
GKIWLTVLFI
FVCNTLQPGC
LIMVSTPALL
IKNEFKDIEE
FFRVIFEAVF
AWPCPNTVDC
ICILLNITEL
Questions:
How is the information about protein sequences
- stored in the cell ?
- duplicated after cell division?
Answer:
...DNA...
NUCLEOTIDE
NH2
N
N
BASE
N
N
OH
OH
P
O
OH
OH
O
P
O
O
P
O CH2
O
RIBOSE
SUGAR
O
TRIPHOSPHATE
OH
Nucleotides
Purines
ATP
Pyrimidines
GTP
TTP (UTP) CTP
O NH2
O NH2
N
N
H2 N
N
N
OH
OH
P
O
O
P
O
O
N
OH
OH
O
P
CH3
N
O CH2
O
OH
O
OH
P
O
OH
OH
OH
O
P
O
O
P
O
CH2
O
O
OH
OH
OH
Nucleotide chain
base
5’ end
OH
OH
P
O
O
OH
OH
P
P
O
O
O
5’
O
CH2
4’
O
1’ N
3’
O
OH
OH
P
O
2’
O
OH
O
P
O
O
HO
P
O
base
OH
5’
O
CH2
O
4’
1’
3’
HO
2’
O
OH
P
O
O
base
5’
CH2
4’
O
1’
3’
OH
2’
OH
3’ end
Nucleotide Pairing
H- bonds
CH3
O
H
H
N
N
H
N
N
N
O
Thymidine
N
Adenine
T A
N
Nucleotide Pairing
H- bonds
CH3
O
H
H
N
N
H
N
N
N
Thymidine
N
N H
N
O
Adenine
T A
H- bonds
H
CH3
O
N
H
N
N
N
O
Cytosine
H
N
N
N
H
C G
Guanine
AG
TC
The double helix
G
A AC
T T
C G T
A A T
A T
C G
C GA G
T
C
5’ACGGGTACATGAC3’
|||||||||||||
3’TGCCCATGTACTG5’
antiparallel complimentary strand
Types of Nucleic Acids
DNA
nuclear 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
RNA
CH2
DNA
CH2
O
O
1) ribose sugar
OH
OH
OH
(ribonucleic acid) (deoxy ribonucleic acid)
Differences between RNA and DNA
RNA
CH2
DNA
CH2
O
O
1) ribose sugar
OH
OH
OH
(ribonucleic acid) (deoxy ribonucleic acid)
O
O
2 )T and U
N
CH3
N
O
O
N
uracyl
N
thymidine
Differences between RNA and DNA
RNA
CH2
DNA
CH2
O
O
1) ribose sugar
OH
OH
OH
(ribonucleic acid) (deoxy ribonucleic acid)
O
O
2 )T and U
N
O
O
N
3) strand
CH3
N
N
uracyl
thymidine
single
double
DNA replication
GGG 3’
5’TGACATGGGTACA
||||||||||||||||||| |||
3’ACTG TACCCATGT GTACTG CCC 5’
DNA replication
5’TGAC
||||
3’ACTG
DNA polymerase
GGG 3’
|||
CCC 5’
DNA replication
5’TGAC
||||
3’ACTG
GGG 3’
|||
CCC 5’
DNA replication
5’TGAC
||||
3’ACTG
GGG 3’
|||
CCC 5’
DNA replication
5’TGAC
||||
3’ACTG
GGG 3’
|||
CCC 5’
DNA replication
5’ACGGGTACATGAC
|||||||||||||
3’TGCCCATGTACTG
semiconservative
replication
5’ACGGGTACATGACACGGGTAC 3’
|||||||||||||||||||||
3’TGCCCATGTACTGTGCCCATG 5’
5’ACGGGTACATGACACGGGTAC 3’
|||||||||||||||||||||
3’TGCCCATGTACTGTGCCCATG 5’
GGG 3’
|||
CCC 5’
old
new
new
old
DNA proof-reading
5’TGAC
||||
3’ACTG
GGG 3’
|||
CCC 5’
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
GGG 3’
|||
CCC 5’
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
GGG 3’
|||
CCC 5’
DNA polymerase has exonuclease activity: it can cut out unpaired
bases to prevent mistakes(mutations) in DNA replication
DNA(basic facts)
• DNA is located in the cell nucleus
• DNA is associated with histones protein
evenly distributed approximately every
200bp. (DNA+histones =chromatin)
• DNA subdivided in chromosomes whose
number differ among species (46 in
humans, 22+22+X +Y)
38
44
Number of
chromosomes
46
From DNA to protein
+N
AG
TC
R1
O
NH
CH
O
R2
C
NH
R3
A
C GT
C GA G
T
C
CH
C
G
A
A C
T T
C G T
A A T
H3
CH
O
C
NH
CH
O
C
O
-
R4
Protein synthesis
DNA
ACGTCTCAA
TGCAGAGTT
RNA polymerase
nuclear factors
Transcription
RNA
ACGUCUCAA
ribosomes (proteins+ rRNA)
t RNAs
Translation
Protein
Thr-Ser-Gln
Transcription
GGG 3’
5’TGACATGGGTACA
||||||||||||||||||| |||
3’ACTG TACCCATGT GTACTG CCC 5’
Transcription
5’ TGACATGGGTACACATGACGGG 3’
UG AUAU
3’ACTGTACCCATGTGTACTGCCC 5’
RNA polymerase
Transcription
CYTOSOL
NUCLEUS
UGAUAUAAAA
UGAUAUAAAA
MESSANGER RNA (mRNA)
Translation
(from mRNA to protein)
Triplet codon:
three nucleotides code for one amino acid
AUG = Met
CAU = His
ACA = Thr
UUU = Phe
anticodon UAC
transfer RNA (tRNA)
there are 20 different tRNA
(each one carrying a specific
amino acid)
Met
Translation
AUG ACG UCU CAA
mRNA
Translation
AUG ACG UCU CAA
UAC
Met
mRNA
Translation
AUG ACG UCU CAA
UAC
UGC
Met
Thr
mRNA
Translation
AUG ACG UCU CAA
UGC
Met
Thr
AGA
Ser
mRNA
Translation
AUG ACG UCU CAA
mRNA
UGC
GUU
Met
Ser
Thr
Gln
Translation
Ribosome
mRNA
AUGAUAGCCGAUU
Translation
Ribosome
AUGAUAGCCGAUU
Translation
Ribosome
AUGAUAGCCGAUU
N-terminus
growing protein
C-terminus
Start and Stop codons
ACCA-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
UUA
Phe
UUG
UUC
AUG Met
CAA
CAG
UGG Trp
Gln
CGU
CGC
CGG Arg
CGA
AGA
AGG
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
E
AGTF
THE DOG ATE CAT AND THE BUN END
THE
AFAT
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 A
• UGUAC AUG UAU CGU CUC
Met Tyr Arg Leu
ACG
• UGUAC AUG UAU UCU CAA
Met Tyr Thr Gln
Insertion A
• UGUAC AUG UAU ACG AUC
Met Tyr Ser Ile
AAU GAU CCA
Asn Asp Pro
UGA UCCA
STOP
UCA AUG AUC
Ser Met Ile
Restriction enzymes
Enzymes found in bacteria that have the ability to cut DNA at
specific sites
EcoRI (GATTC)
AATTCGCATGATGCATGCTCGAGCATAGC
ACGTGCCATGAATTCGCATGATGCATGCTCGAGCATAGC
GCGTACTACGTACGAGCTCGTATCG
ACGTGCCATG
TGCACGGTACTTAAGCGTACTACGTACGAGCTCGTATCG
TGCACGGTACTTAA
Restriction enzymes
EcoRI (GATTC)
XhoI (CTCGAG)
AATTCGCATGATGCATGCTCG
ACGTGCCATGAATTCGCATGATGCATGCTCGAGCATAGC
ACGTGCCATG
AGCATAGC
GCGTACTACGTACGA
TGCACGGTACTTAAGCGTACTACGTACGAGCTCGTATCG
TGCACGGTACTTAA
GCTCGTATCG
recombination
ACGTGCCATGAATTCGCATCATGCGAATTCATAGC
TGCACGGTACTTAAGCGTACTACGCTTAAGTATCG
TAGCATGAATTCGCATCGATC
ATCGTACTTAAGCGTAGCTAG
PLASMID
recombination
ACGTGCCATG
AATTCGCATCATGCG
AATTCATAGC
TGCACGGTACTTAA
GCGTACTACGCTTAA
GTATCG
TAGCATG
AATTCGCATCGATC
ATCGTACTTAA
GCGTAGCTAG
PLASMID
recombination
ACGTGCCATG
TGCACGGTACTTAA
AATTCATAGC
GTATCG
TAGCATGAATTCGCATCATGCGAATTCGCATCGATC
ATCGTACTTAAGCGTACTACGCTTAAGCGTAGCTAG
PLASMID
Exercise 1
Transcribe the following DNA sequence into RNA starting from
the base indicated by the arrow
5’ACGTGCCATGAATTCGCATCATGCGAATTCATAGC 3’
3’TGCACGGTACTTAAGCGTACTACGCTTAAGTATCG 5’
5’ACGTGCCATGAATTCGCATCATGCGAATTCATAGC 3’
5’UGAAUUCGCAUCAUGCGAAUUCAUAGC 3’
3’TGCACGGTACTTAAGCGTACTACGCTTAAGTATCG 5’
Exercise 2
Translate 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 AUC
Met Asp Trp Gly Thr Lys Gln Ser Iso
M
D
W
G
T
L
Q
S
I
CUC GGG UAA GGUGUCAACAA
Leu 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
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