Download Lecture 4 - Université d`Ottawa

Nucleic acids
• Informational macromolecule
• Deoxyribonucleic acid (DNA) is the genetic material
• Ribonucleic acid (RNA)
– Messenger RNA (mRNA) carries information from DNA to the
ribosomes
– Ribosomal RNA (rRNA) and transfer RNA (tRNA) are involved
in protein synthesis
– RNAs involved in regulation of gene expression and processing
and transport of RNAs and proteins
Nucleic acids
• DNA and RNA are polymers of
nucleotides
• Nucleotides consist of
– Purine and pyrimidine bases
• Purines: adenine (A) and guanine (G)
• Pyrimidines: cytosine (C) and
thymine(T)
• RNA has uracil (U) in place of thymine
– 5 C sugar (5’ phosphorylated)
• D-Ribose (RNA)
• D-2’deoxyrobose (DNA)
– Phosphate: 1-3 phosphate at 5’C of sugar
Nitrogenous bases
• Structures are meaningful
• Reactive centers?
Base pairing: Hydrogen bonding
Hydrogen bonding btw complementary bases is the basis for double stranded DNA structure
Backbone
• Sugar phosphodiester forms
the backbone
• Ribose for RNA
• 2’-deoxyribose for DNA
• Nucleoside=covalent bonding
of C1 of sugar and a base
• Naming: Guanosine,
Adenosine, cytidine and
Thymidine, Uridine
• Nucelotide=
Nuceloside+5’phosphate (1-3)
•
Naming:
– Adenosine monophosphate
(AMP)
– Adenosine diphosphate (ADP)
– Adenosine triphosphate (ATP)
• Can you name the others?
Adenosine
Adenosine monophosphate (AMP)
Adenosine diphosphate (ADP)
Adenosine triphosphate (ATP)
Phosphodiester bond formation
• DNA polymerases catalyze the rxn
• uses complementary dNTPs
• dehydration reaction between
• 3’-OH of new strand and
• 5’-phosphate of incoming dNTP
• synthesis is 5’3’
• covalent bond is called
phosphodiester
• there is always a 5’-phosphate and
a 3’-OH that gives the DNA its polar
sense (5’3’)
• complementary strands are antiparallel
Phosphodiester bond formation
• DNA polymerases catalyze the rxn
• uses complementary dNTPs
• dehydration reaction between
• 3’-OH of new strand and
• 5’-phosphate of incoming dNTP
• synthesis is 5’3’
• covalent bond is called
phosphodiester
• there is always a 5’-phosphate and
a 3’-OH that gives the DNA its polar
sense (5’3’)
• complementary strands are antiparallel
DNA is an antiparallel helix
• Geometry of bases and their
spacial arrangement to form Hbond cause helix structure of
dDNA
• In B-form right handed dDNA
• pairing bases stack in the centre
• backbone intertwined
• creates minor and major
grooves
• 0.34 nm (3.4 A) rise per base
pair
• one full helix turn houses 10
nucleotides
Major groove
34 A
20 A
Central dogma
• Complementary base pairing allows one strand of DNA to
act as a template for synthesis of a complementary DNA
or RNA strand
• DNA is transcribed to pass genetic information to RNA
• The information in RNA is present in a triplet code where
every three bases stands for one of the 20 amino acids
• Translation: mRNA codes for protein
• This flow of information from DNA to protein is called
“central dogma” in cell biology
Information flow: DNAmRNAProtein
Central dogma and mutations
GAGGUG
• The DNA contains the instructions for the sequence of amino acids in
each protein
• The order of amino acids in a protein determines its shape and function
• Errors or faults, ie mutations, in the DNA can change the amino acid
sequence and function of the encoded protein
• Sickle cell anaemia is due to one nucleotide change affecting
hemoglobin  reduced O2 carrying capacity
Proteins
• Proteins are the most diverse of all macromolecules
• Each cell contains several thousand different proteins
• Proteins direct virtually all activities of the cell
• Functions of proteins include:
 Enzymes
 Structural components (e.g. keratin, collagen)
 Motility (e.g. actin)
 Regulatory (e.g. transcription factors)
 Transport (e.g. Na+-K+-ATPase)
 Receptors (e.g. insulin receptors)
 Transport and storage of small molecules (e.g. O2)
 Transmit information between cells (protein hormones),
 Defense against infection (antibodies)
Amino acids
• Polymers of 20 different amino acids.
• Each amino acid consists of the α carbon bonded to a
carboxyl group (COO−), an amino group (NH3+), a
hydrogen, and a distinctive side chain (R)
Amino acids
• Amino acids are grouped based on
characteristics of the side chains:
– Nonpolar side chains
– Polar side chains
– Side chains with charged basic groups
– Acidic side chains terminating in carboxyl
groups
Nonpolar amino acides
•
•
•
•
10 aa have nonpolar R-groups (hydrophobic)
Simplest is glycine (R=H)
2 contain S and two have cyclic side chains
Nonpolar aa tend to be burried in the hydrophobic core of proteins
Polar amino acides
• 5 aa have polar R-groups; either –OH or NH2 (hydrophilic)
• Partial charge; H-bond formation with water
• Polar aa tend to appear on the surface of proteins
Charged amino acids
•
•
•
•
3 aa have positively charged NH2 groups (basic)
Full charge; H-bond and ionic bond
Like Polar aa tend to appear on the surface of proteins
Might take part in catalytic core of enzymes
Charged amino acids
• 2 aa have negatively charged –COO- group (acidic)
• Full charge; H-bond and ionic bond
• tend to appear on the surface of proteins or enzyme catalytic core
Peptide bond formation
• Polypeptides: chains of
amino acids joined by
peptide bonds
• Number of aa’s varied
• oxytocin – 9 aa,
• insulin – 51 aa,
titin (connectin)– 34,350
aa’s
• Average 400-500 aa
• One end of a polypeptide
terminates in an α amino
group (N terminus)
• other end is an α carboxyl
group (C terminus)
Protein structure
• Sequence of amino acids in a protein is
determined by the order of nucleotide
bases in a gene (Primary structure)
• One can deduce aa sequence from the
sequence of nucleotides in the gene (or
mRNA)
• 3-D conformation is critical to proteins
function
• What determines the 3-D structure of proteins?
Protein secondary structure
Christian B. Anfinsen (1957)
• 3-D structure is a result of interactions between the amino acids
• Christian Anfinsen denatured ribonuclease (RNase) by heat
treatment; breaks H-bonds
• If the treatment was mild, the proteins would return to their
normal shape at room temperature
• This would mean that the information for folding the protein is in
its primary sequence (how could he test?)
Protein secondary structure
• Secondary structure: regular arrangement of amino acids
within localized regions
• There are 2 types of secondary structure:
-
The polypeptide can coil in a spiral helix shape
The polypeptide can fold to form a β pleated sheet (parallel or
antiparallel)
• Both are held together by hydrogen bonds between the CO and NH
groups of peptide bonds
Protein Tertiary structure
Observation:
• Similarly disrupting the disulfide
bonds (S-S) using chemical
denaturing agents (eg. βmercaptoethanol) denatures
proteins (-SH forms)
• Incubation under oxygen
refolded the RNase back to its
functional conformation (ie enzyme
gained capacity to degrade RNA)
• indicates a higher level of structure
important for function that relies on
covalent S-S bridge (tertiary structure)
Protein Tertiary structure
• Tertiary structure: folding of
secondary structural elements to form
a 3-D arrangement
RNase
• 2° elements connected by loops and less
ordered aa’s
• interactions btw the side chains of
amino acids in different regions of
protein stabilizes the 3° structure
- Covalent bonds (S-S bridge)
- Hydrophobic and hydrophilic
interactions
• In most proteins this results in
domains,
the basic units of tertiary structure
Insulin
Protein Quaternary structure
• Quaternary structure
consists of interactions
between different
polypeptide chains
• In multi-subunit
enzymes
• Hemoglobin, for example,
is composed of four
polypeptide chains
Protein structure: Summary
Campbell & Reece, 2002
Can you meet these objectives?
• Distinguish among nucleosides, nucleotides
and nucleic acids?
• Explain the structure of DNA?
• List some functions of proteins in cells?
• Describe and distinguish between amino
acids?
• Discuss the levels of protein structure and
organization of proteins?