Welcome to
Introduction to Bioinformatics
Introduction to Molecular Biology
DNA to protein
Molecular biology forms part of a long intellectual tradition
• c. 350 BCE - Aristotle begins the Western tradition of natural philosophy
• 1664 - Robert Hook coins the term “cell” in his treatise Micrographia
• 1676 - Anton van Leeuwenhoek discovers bacteria, later spermatozoa
• 1735 - Carl Linnaeus’ Systema Naturae lays the foundations for taxonomy
• 1859 - Charles Darwin’s Origin of the Species
• 1860s - Louis Pasteur disproves abiogenesis and develops the “Germ theory”
• 1866 - Gregor Mendel describes the “inheritance of traits” in peas.
• 1868 - Ernst Haeckel postulates that the nucleus responsible for heredity
• 1869 - Friedrich Miescher isolates a crude nucleic acid preparation
• 1880s - Cytologists work out the details of mitosis, meiosis and fertilization
Molecular biology forms part of a long intellectual tradition
• 1903 - Walter Sutton proposes a chromosomal theory of heredity
• 1908 - Thomas Hunt Morgan discovers that genes can mutate
• 1909 – Archibald Garrod proposes the “gene-enzyme” hypothesis
• 1915 – Morgan and colleagues publish linkage maps of D. melanogaster
• 1927 – H.J. Muller and L.J. Stadler show that radiation can induce mutation
• 1928 - Fredrick Griffith demonstrates genetic transformation
• 1931 - Barbara McClintock proves genetic recombination – “crossing over”
• 1944 - Avery, MacLeod & McCarty show that DNA carries genetic information
• 1940s - The “modern synthesis” - E. Mayr, T. Dobzhansky, & J. Huxley
• 1949 – Erwin Chargaff formulates “Chargaff’s rules” of DNA composition
• 1952 - Hershey and Chase demonstrate that DNA is the genetic material
• 1953 - James Watson and Francis Crick describe the double helix of DNA
• 1957 – Vernon Ingram - genes determine the sequence of amino acids
• 1958 - Matt Meselson and Frank Stahl prove semiconservative replication
Molecular biology forms part of a long intellectual tradition
1958 - Arthur Kornberg discovers DNA polymerase
1960 – Sam Weiss and Jerard Hurwitz independently discover RNA polymerase
1960s - Genetic code cracked by Crick, Marshall Nirenberg, Har Gobind Khorana, etc.
1961 - Charles Yanofsky & Sydney Brenner show colinearity of DNA & protein
1965 - Holley and Zamir determine the structure of a tRNA
20xx – Your contribution!!
The “Central Dogma” of Molecular Biology
Term coined by Francis Crick in 1956 to describe the flow
of information in the cell
DNA
RNA
Protein
Transcription
Replication
Translation
Information flow is compartmentalized
DNA
Transcription
Pre-mRNA
Processing
mRNA
Export
mRNA
Decay
mRNA
Translation
Protein
Decay
protein
What is the nature of the Gene?
Frederick Griffiths demonstrates “Transformation” of a
heritable character in the bacteria Streptococcus pneumoniae
What is the nature of the Gene?
Oswald Avery, Colin MacLeod & Maclyn McCarty
first show that DNA is the “genetic principle”
Enzymes
used to
degrade
proteins
The Hershey-Chase experiment confirms
that DNA is the stuff of heredity
Erwin Chargaff and his rules
1. Ratio of nucleotides depends on species
2. A=T and G=C no matter the organism
What is the structure of DNA?
1952
Rosalind Franklin and Maurice
Wilkins produce X-ray
diffraction images of DNA
crystals that suggested that
DNA must have some helical
arrangement
What is the structure of DNA?
1953
Francis Crick and James
Watson put together all of
the clues and correctly
deduce that DNA is a
Double Helix
DNA base pairing occurs through hydrogen bonds
A:T pairs: 2 bonds
G:C pairs: 2 bonds
The double helix strongly suggested that DNA replication
might proceed by a “semiconservative” process
The Meselson-Stahl experiment argues
for strand separation during DNA replication
Genes control the amino acid
sequence of proteins
•1957 – Vernon Ingram shows
that sickle cell haemoglobin
varies from wild type by the
substitution of one amino acid
Genes control the amino acid
sequence of proteins
Alteration of amino acid sequence is also
observed in all other hereditary anaemias!
DNA cannot directly specify the sequence
of amino acids in proteins
• Protein synthesis in eukaryotic cells known to take place in the
cytoplasm
• There must therefore be a SECOND information containing molecule
that gets its specificity from DNA, but then moves to the cytoplasm
• Attention immediately focuses on RNA – was easy to imagine that it
could be produced from a DNA template
•Torborn Caspersson and Jean Brachet demonstrated that RNA was
mostly in the cytoplasm
Jean Brachet
(1909-1998)
Discovery of mRNA
• T2 is a bacteriophage that infects
E. coli
• Completely shuts down normal
cellular transcription. Only viral
protein is made
• T2 RNA always has the same
composition as T2 DNA
• T2 carries none of its own RNA
• 32P is incorporated into RNA made
after T2 infection
• Only about 3-5% of cellular RNA
is messenger RNA!
The case for RNA
Missing methyl
group in uracil
relative to
thymine
Hydroxyl group
Chemically very similar to DNA
Easy to imagine RNA being produced
from a DNA template
There must be a molecular machine
that makes RNA from a DNA template
• Jerard Hurwitz and Sam Weiss independently
discover an enzyme that will only make RNA in the
presence of DNA.
• The enzyme uses ATP, GTP, CTP, and UTP as
precursors
POOR GUYS!
1959 Nobel prize was already awarded to Severo Ochoa
for what turned out later to be the WRONG ENZYME!
RNA Polymerase is a molecular
machine that carries out transcription
RNA is synthesised in the nucleus
but travels to the cytoplasm
Cells pulse-labelled with 3H coupled cytidine
T = 15 minutes
T = ~90 minutes
D.M. Prescott
Ribosomes are the site of protein synthesis
ribosomes studding the endoplastic reticulum
Shown using radio labelled amino acids in conjunction with ultracentrifugation to isolate
Different cell fractions. Where does the radioactivity end up at various times?
Ribosomes and associated rRNAs
are the factories for protein synthesis
Crick’s adaptor hypothesis
• Can folded RNA act directly as the template for protein synthesis?
• Seems unlikely:
• the nucleosides chemically want to react with water soluble groups
• but many amino acids are polar
• no clear way to discriminate chemically similar amino acids
Crick proposes that an adaptor molecule must fit between RNA
and the incoming amino acids, but its nature is unknown
Incoming amino acid
Adaptor molecule
RNA
Crick’s adaptors (tRNAs) are themselves
RNA molecules
• Self-folding by complementary
base pairs gives a structure with
several functional domains
• Account for ~10% of cellular RNA
abundance
• Typically includes several modified,
non-standard bases.
Zamecnick and Hoagland discover
aminoacyl synthetases
Enzymes that added an adaptor (that we now know to be
tRNA) to amino acids prior to their incorporation in proteins
It turns out these tRNAs are Crick’s proposed adaptors
Mahlon Hoagland
Paul Zamecnik
Translation proceeds through
a tRNA intermediate
Nature of the genetic code
• Obvious early on most likely a triplet code in order to code 20
amino acids:
• 4 x 4 nucleotides can specify 42 = 16 amino acids
• 4 x 4 x 4 nucleotides can specify 43 = 64 amino acids
• Code must be redundant
• Not overlapping – Sydney Brenner’s thought experiment
• Marshall Nirenberg and Heinrich Matthaei showed that a
homopolymer (UUUUUU…. etc. ) produced a poly-phenylalanine
protein
Khorana's synthetic RNA approach
to cracking the genetic code
Example RNA with two repeating units
RNAs with two repeating units:
(UCUCUCU → UCU CUC UCU) produced a polypetide consisting of
alternating Serine and CUC codes for Leucine
RNAs with three repeating units:
(UACUACUA → UAC UAC UAC, or ACU ACU ACU, or CUA CUA CUA)
produced three different strings of amino acids
RNAs with four repeating units including UAG, UAA, or UGA, produced
only dipiptides and tripeptides thus revealing that UAG, UAA and UGA are
stop codons.
The genetic code is (almost)
universal
Amino acids fall into five functional categories
Study Question 11
Degeneracy and frequency of amino acids
Most common
Leu Gly Ser
Least common
Trp Met His
Study Question 12
Single mutation from AGA
Silent: |
Hydrophilic/
Hydrophilic: |
Study Question 12
Single mutation from AGA
Silent: |
Conservative: |
Hydrophilic/
Hydrophilic: ||
Hydrophilic/
Hydrophobic: |
Study Question 12
Single mutation from AGA
Silent: ||
Conservative: |
Hydrophilic/
Hydrophilic: |||
Hydrophilic/
Hydrophobic: |
Other: |
Proteins have four levels of structure
The primary structure of proteins is determined by
peptide bonds between amino acids
Formation of this bond is associated with a small +ve DG
Protein synthesis depends on coupled reactions!
Secondary structure - the alpha helix
H bonds
Alpha helical conformation is stabilised by hydrogen bonding
Secondary structure – beta sheets
Parallel configuration
Antiparallel configuration
Secondary structures combine
to determine tertiary structure
The enzyme acetylcholinesterase bound to acetylcholine
Proteins combine to form quaternary structures
Collagen
Haemoglobin
Allosteric interactions
+ve
-ve
Interactions (usually with a small molecule) can
alter the shape and activity of an enzyme
Enzymes lower the activation energies
associated with biochemical reactions
DG
Typical energy of activation is 20-30 kcal/mol
Eukaryotic mRNA must often must be spliced
in order to produce a mature transcript
Exons often correspond to functional protein domains
and alternative splicing can give rise to variant proteins