Protein Group

1
Molecular biology and biological chemistry
Bio 2304 Høst 2003
Molecular biology and biological chemistry
Protein Group Department of Molecular
Biotechnology Faculty of Medicine University of
Troms, Norway
Erik Hjerde E-mailerikh_at_fagmed.uit.no Telephone
47 77 64 47 06 Fax [phone]
2
Molecular biology and biological chemistry
Bio 2304 Høst 2003
Overview

• Basic chemistry
• DNA structure
• Gene structure
• Protein structure
• Laboratory techniques

Protein Group Department of Molecular
Biotechnology Faculty of Medicine University of
Troms, Norway
Erik Hjerde E-mailerikh_at_fagmed.uit.no Telephone
47 77 64 47 06 Fax [phone]
3
Molecular biology and biological chemistry
Bio 2304 Høst 2003
Basic chemistry
Electrons
Neutrons
Nucleus
Protons
Protein Group Department of Molecular
Biotechnology Faculty of Medicine University of
Troms, Norway
Erik Hjerde E-mailerikh_at_fagmed.uit.no Telephone
47 77 64 47 06 Fax [phone]
4
Molecular biology and biological chemistry
Bio 2304 Høst 2003
Basic chemistry
Orbitals describes the 3-D space of the electrons
The farther an electron is from the nucleus the
greater its energy
Only two electrons in each orbital 1s is filled
first
Quantum leap
Quantum
Protein Group Department of Molecular
Biotechnology Faculty of Medicine University of
Troms, Norway
Erik Hjerde E-mailerikh_at_fagmed.uit.no Telephone
47 77 64 47 06 Fax [phone]
5
Molecular biology and biological chemistry
Bio 2304 Høst 2003
Basic chemistry
Orbitals describes the 3-D space of the electrons
The farther an electron is from the nucleus the
greater its energy
Only two electrons in each orbital 1s is filled
first
Atoms are most stable when their outermost
orbitals are filled or emptied
Electrons are shared between atoms covalent bond
Orbital overlapp covalent bond
Protein Group Department of Molecular
Biotechnology Faculty of Medicine University of
Troms, Norway
Erik Hjerde E-mailerikh_at_fagmed.uit.no Telephone
47 77 64 47 06 Fax [phone]
6
Molecular biology and biological chemistry
Bio 2304 Høst 2003
Basic chemistry
Orbitals describes the 3-D space of the electrons
The farther an electron is from the nucleus the
greater its energy
Only two electrons in each orbital 1s is filled
first
Atoms are most stable when their outermost
orbitals are filled or emptied
Electrons are shared between atoms covalent bond
Only unpaired electrons valence electrons - can
take part in a covalent bond
Protein Group Department of Molecular
Biotechnology Faculty of Medicine University of
Troms, Norway
Erik Hjerde E-mailerikh_at_fagmed.uit.no Telephone
47 77 64 47 06 Fax [phone]
7
Molecular biology and biological chemistry
Bio 2304 Høst 2003
Basic chemistry
Electronegativity
Electronegativity atoms affinity for electrons
Polar bond electrons are shared unequally
Hydrogen bond can exist between molecules with
polar bonds
Weaker than covalent bonds, but important in
biomolecules
Slightly negative charge
H-bond donor
Polar bonds
Polar bond
H-bond acceptor
Slightly positive charge
Slightly positive charge
Polar bonds
Protein Group Department of Molecular
Biotechnology Faculty of Medicine University of
Troms, Norway
Erik Hjerde E-mailerikh_at_fagmed.uit.no Telephone
47 77 64 47 06 Fax [phone]
8
Molecular biology and biological chemistry
Bio 2304 Høst 2003
Basic chemistry
Hydrophobic -Water insoluble -No Hydrogen bonds
Hydrophilic -Water loving -Hydrogen bonds
Amphiphilic -Hydrophilic and hydrophobic parts
Protein Group Department of Molecular
Biotechnology Faculty of Medicine University of
Troms, Norway
Erik Hjerde E-mailerikh_at_fagmed.uit.no Telephone
47 77 64 47 06 Fax [phone]
9
Molecular biology and biological chemistry
Bio 2304 Høst 2003
Central dogma
Francis H. Crick
Transcription
Translation
DNA
RNA
Protein
Refers to the flow of genetic information from
DNA to RNA to protein
Protein Group Department of Molecular
Biotechnology Faculty of Medicine University of
Troms, Norway
Erik Hjerde E-mailerikh_at_fagmed.uit.no Telephone
47 77 64 47 06 Fax [phone]
10
Molecular biology and biological chemistry
Bio 2304 Høst 2003
DNA structure
Transcription
Translation
DNA
RNA
Protein
DeoxyriboNucleic Acid Stores genetic information
(genes)
Protein Group Department of Molecular
Biotechnology Faculty of Medicine University of
Troms, Norway
Erik Hjerde E-mailerikh_at_fagmed.uit.no Telephone
47 77 64 47 06 Fax [phone]
11
Molecular biology and biological chemistry
Bio 2304 Høst 2003
DNA structure
Protein Group Department of Molecular
Biotechnology Faculty of Medicine University of
Troms, Norway
Erik Hjerde E-mailerikh_at_fagmed.uit.no Telephone
47 77 64 47 06 Fax [phone]
12
Molecular biology and biological chemistry
Bio 2304 Høst 2003
DNA structure
Nucleotides are linked together by phosphodiester
bonds
Sequence direction from 5 to 3
Direction of growt
Polymerase
Phosphodiester backbone
Negatively charged
Protein Group Department of Molecular
Biotechnology Faculty of Medicine University of
Troms, Norway
Erik Hjerde E-mailerikh_at_fagmed.uit.no Telephone
47 77 64 47 06 Fax [phone]
13
Molecular biology and biological chemistry
Bio 2304 Høst 2003
DNA structure
DNA dobbel helical structure with antiparallel
strands
Strands held together by hydrogen bonds between
base pairs AT and CG
Sugar-phosphate backbone
Base bair
Base pair
Nitrogenous base
The thread of life
Hydrogen bonds
Protein Group Department of Molecular
Biotechnology Faculty of Medicine University of
Troms, Norway
Erik Hjerde E-mailerikh_at_fagmed.uit.no Telephone
47 77 64 47 06 Fax [phone]
14
Molecular biology and biological chemistry
Bio 2304 Høst 2003
Molecular biology and biological chemistry
Bio 2304 Høst 2003
DNA structure
DNA dobbel helical structure with antiparallel
strands
Strands held together by hydrogen bonds between
base pairs AT and CG
Sequence on one strand is complementary to the
other strand
5 AATTGGCC 3
3 TTAACCGG 5
5GGCCAATT 3
Complementary
Reverse complementary
Protein Group Department of Molecular
Biotechnology Faculty of Medicine University of
Troms, Norway
Erik Hjerde E-mailerikh_at_fagmed.uit.no Telephone
47 77 64 47 06 Fax [phone]
Protein Group Department of Molecular
Biotechnology Faculty of Medicine University of
Troms, Norway
Erik Hjerde E-mailerikh_at_fagmed.uit.no Telephone
47 77 64 47 06 Fax [phone]
15
Molecular biology and biological chemistry
Bio 2304 Høst 2003
Gene structure
Transcription
Translation
DNA
RNA
Protein
Decodes genetic information
Stores genetic information (genes)
Protein Group Department of Molecular
Biotechnology Faculty of Medicine University of
Troms, Norway
Erik Hjerde E-mailerikh_at_fagmed.uit.no Telephone
47 77 64 47 06 Fax [phone]
16
Molecular biology and biological chemistry
Bio 2304 Høst 2003
Gene structure
Prokaryot
Eukaryot
-No discontinuous genes (no introns) -mRNA is
translated during transcription
-Genes are often split, coding sequences are
interrupted by introns -mRNA is processed before
transport to the cytoplasma where it is translated
Protein Group Department of Molecular
Biotechnology Faculty of Medicine University of
Troms, Norway
Erik Hjerde E-mailerikh_at_fagmed.uit.no Telephone
47 77 64 47 06 Fax [phone]
17
Molecular biology and biological chemistry
Bio 2304 Høst 2003
Gene structure
Promotor - DNA sequence which determines the site
of transcription initiation for an RNA polymerase
Promotor sequence
Gene
DNA
Transcription start
Negative regulator
Inactive inhibitor
RNA polymerase
Active inhibitor
DNA
DNA
Genes are on
Genes are off
Positive regulation
Negative regulation
Protein Group Department of Molecular
Biotechnology Faculty of Medicine University of
Troms, Norway
Erik Hjerde E-mailerikh_at_fagmed.uit.no Telephone
47 77 64 47 06 Fax [phone]
18
Molecular biology and biological chemistry
Bio 2304 Høst 2003
Gene structure
Promotor - DNA sequence which determines the site
of transcription initiation for an RNA polymerase
Promotor sequence
Gene
DNA
Transcription start
Negative regulator
Inactive inhibitor
RNA polymerase
Active inhibitor
DNA
DNA
Genes are on
Genes are off
Normal regulation
Negative regulation
Protein Group Department of Molecular
Biotechnology Faculty of Medicine University of
Troms, Norway
Erik Hjerde E-mailerikh_at_fagmed.uit.no Telephone
47 77 64 47 06 Fax [phone]
19
Molecular biology and biological chemistry
Bio 2304 Høst 2003
Gene structure
Promotor - DNA sequence which determines the site
of transcription initiation for an RNA polymerase
Promotor sequence
Gene
DNA
Transcription start
Negative regulator
Inactive inhibitor
RNA polymerase
Active inhibitor
DNA
DNA
Genes are on
Genes are off
Normal regulation
Negative regulation
Protein Group Department of Molecular
Biotechnology Faculty of Medicine University of
Troms, Norway
Erik Hjerde E-mailerikh_at_fagmed.uit.no Telephone
47 77 64 47 06 Fax [phone]
20
Molecular biology and biological chemistry
Bio 2304 Høst 2003
Gene structure
Transcription
Translation
DNA
RNA
Protein
The genetic code

• Conversion of nucleic acid sequence to protein
  sequence
• Sequence of nucleotides in DNA and RNA specify
  sequence of amino acids in protein
• Three nucleotides (codon) encode one amino acid
  or a stop signal
• 64 codons (43) for 20 amino acids, more than one
  codon can specify the same amino acid (genetic
  code is degenerate)
• The third nucleotide in the codon is irrelevant
  for many amino acids

Protein Group Department of Molecular
Biotechnology Faculty of Medicine University of
Troms, Norway
Erik Hjerde E-mailerikh_at_fagmed.uit.no Telephone
47 77 64 47 06 Fax [phone]
21
Molecular biology and biological chemistry
Bio 2304 Høst 2003
Gene structure
Reading frame - DNA
DNA
Starts at a start codon ATG
? G C T G A C C T A G C G G
A C A ?
A T G
TAA/ATG/TGA
Terminates at a stop codon TAA/TAG/TGA
? G C T G A C C T A G C G G
A C A ?
Open reading frame long reading frames,
possible genes
One DNA strand 3 reading frames
? G C T G A C C T A G C G G
A C A ?
Protein Group Department of Molecular
Biotechnology Faculty of Medicine University of
Troms, Norway
Erik Hjerde E-mailerikh_at_fagmed.uit.no Telephone
47 77 64 47 06 Fax [phone]
22
Molecular biology and biological chemistry
Bio 2304 Høst 2003
Gene structure
Translation of reading frame
Starts at a start codon - AUG
AUG ? ? ? G C U G A C C U A G
C G G A C A ? ? UAA/AUG/UGA
Terminates at a stop codon UAA, UAG, UGA
Protein Group Department of Molecular
Biotechnology Faculty of Medicine University of
Troms, Norway
Erik Hjerde E-mailerikh_at_fagmed.uit.no Telephone
47 77 64 47 06 Fax [phone]
23
Molecular biology and biological chemistry
Bio 2304 Høst 2003
Gene structure
? G C U G A C C U A G C G G
A C A ?
Normal translation of reading frame
? Ala Asp Leu Ala Asp ?
Give a functional product
Protein Group Department of Molecular
Biotechnology Faculty of Medicine University of
Troms, Norway
Erik Hjerde E-mailerikh_at_fagmed.uit.no Telephone
47 77 64 47 06 Fax [phone]
24
Molecular biology and biological chemistry
Bio 2304 Høst 2003
Gene structure
? G C U G A C C U A G C G G
A C A ?
? Ala Asp Leu Ala Asp ?
Deletion of a A
Reading frame shift Deletion changes the reading
frame downstream of the deletion Creates a stop
codon and translation terminates
? G C U G C C U A G C G G A
C A G ?
? Ala Ala Stop
Give a non-functional product
Protein Group Department of Molecular
Biotechnology Faculty of Medicine University of
Troms, Norway
Erik Hjerde E-mailerikh_at_fagmed.uit.no Telephone
47 77 64 47 06 Fax [phone]
25
Molecular biology and biological chemistry
Bio 2304 Høst 2003
Gene structure
? G C U G A C C U A G C G G
A C A ?
? Ala Asp Leu Ala Asp ?
? G C U G C C U A G C G G A
C A G ?
? Ala Ala Stop
Mutation A ? U In this case no alteration in the
reading frame and amino acid sequence
? G C U G A C C U U G C G G
A C A ?
? Ala Asp Leu Ala Asp ?
Give a functional product. Silent mutation
Protein Group Department of Molecular
Biotechnology Faculty of Medicine University of
Troms, Norway
Erik Hjerde E-mailerikh_at_fagmed.uit.no Telephone
47 77 64 47 06 Fax [phone]
26
Molecular biology and biological chemistry
Bio 2304 Høst 2003
Protein structure
Transcription
Translation
DNA
RNA
Protein
Primary structure
The workhorses of the cell Maintain and reproduce
DNA Carrying out metabolic and structural
functions to keep cells alive
Secondary structure
Tertiary structure
Quartenary structure
Protein Group Department of Molecular
Biotechnology Faculty of Medicine University of
Troms, Norway
Erik Hjerde E-mailerikh_at_fagmed.uit.no Telephone
47 77 64 47 06 Fax [phone]
27
Molecular biology and biological chemistry
Bio 2304 Høst 2003
Protein structure
NH3
Primary structure -linear information -string of
amino acids
COO-
Side chain
R
Amino acid -building blocks -20 different amino
acids
H
C?
O
H
Amino group
Carboxyl group
N
C
H
O
Peptide bond -joins amino acids -covalent bond
Peptide bond
Protein Group Department of Molecular
Biotechnology Faculty of Medicine University of
Troms, Norway
Erik Hjerde E-mailerikh_at_fagmed.uit.no Telephone
47 77 64 47 06 Fax [phone]
28
Molecular biology and biological chemistry
Bio 2304 Høst 2003
Protein structure
Classification Hydrophilic hydrophobic Aromatic
aliphatic Charged uncharged Small large
Protein Group Department of Molecular
Biotechnology Faculty of Medicine University of
Troms, Norway
Erik Hjerde E-mailerikh_at_fagmed.uit.no Telephone
47 77 64 47 06 Fax [phone]
29
Molecular biology and biological chemistry
Bio 2304 Høst 2003
Protein structure
Planarity -due to double bond character
Torsion angles -Psi and phi rotation determines
the folding
Peptide torsion angles
Dipole -the peptide has a dipole -can make
hydrogen bonds
Protein Group Department of Molecular
Biotechnology Faculty of Medicine University of
Troms, Norway
Erik Hjerde E-mailerikh_at_fagmed.uit.no Telephone
47 77 64 47 06 Fax [phone]
30
Molecular biology and biological chemistry
Bio 2304 Høst 2003
Protein structure
Secondary structure -local arrangements of amino
acids -formed by intramolecular hydrogen bonds
Tertiary structure -folding of the total peptide
chain -secondary strucure elements linked by
loops and turns
Quarternary structure -association of two or more
polypeptide chains to form a native protein
structure
Protein Group Department of Molecular
Biotechnology Faculty of Medicine University of
Troms, Norway
Erik Hjerde E-mailerikh_at_fagmed.uit.no Telephone
47 77 64 47 06 Fax [phone]