basic molecular biology
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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
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