Download Lecture 1 - Doolittle Lab

Spring, 2016
Osher Five Lecture Series
Russell Doolittle
Lecture 1: Avogadro’s Number
Lecture 2: The Discovery of X-Rays
Lecture 3: Nature of the Atomic Nucleus
Lecture 4: Sickle Cell Anemia: a Molecular Disease
Lecture 5: Unraveling the Genetic Code
Osher Lecture 5
Unraveling the Genetic Code
Russell Doolittle
June 1, 2016
Watson and Crick, 1953
DNA double helix model
E. Coli DNA
Micrograph by
Ruth Kavenoff
The 1953 model of DNA explained how information is replicated
generation to generation (A vs T, G vs C).
1955 The Central Dogma widely accepted.
DNA makes RNA makes protein
It was assumed that RNA was made in a directly complementary
fashion the same as DNA is replicated (A vs U, G vs C).
What wasn’t at all obvious was how the information in RNA
was translated into protein.
4 bases (A,G, C, T)
4 bases (A, G, C, U)
?
20 amino acids
DNA sequence
(4 letters)
protein sequence (20 letters)
Crick theorized that there must be some kind of
intermediate matching device between the RNA and the
protein.
It was called the “adapter hypothesis”.
By this time it was realized there were several kinds of RNA.
A “big” RNA particle that could be sedimented in an
ultracentrifuge (“ribosomal RNA”).
A small molecular weight soluble RNA
A variable sized RNA that was presumed to be the template
for various proteins, which are also variable sized.
DNA makes RNA makes protein
4 bases (in DNA and RNA) vs 20 amino acids (in proteins)
can’t be “singlet code” (1 base for each amino acid)
can’t be “doublet code” (2 bases for each amino acid (42 = 16)
must be “triplet code” (3 bases for each amino acid (43 = 64)
ribosome
large subunit
small subunit
The Main Players
Francis Crick (Nobel, 1962)
James Watson (Nobel, 1962)
Sydney Brenner (Nobel, 2002)
Severo Ochoa (Nobel, 1959)
Marshall Nirenberg (Nobel, 1968)
Robert Holly (Nobel, 1968)
Gobind Khorana (Nobel, 1968)
And with special tribute to the “RNA-Tie Club” and “the Yellow Berets”
The RNA Tie Club (founded 1954)
ALA (George Gamow)
ARG (Alex Rich)
ASP (Paul Doty)
ASN (Bob Ledley)
CYS (M. Ycas)
GLU (Robley Williams)
GLN (Alex Dounce)
GLY (Richard Feynman)
HIS (Melvin Calvin)
ISO (now ILE) (Norman Simons)
LEU (Edward Teller)
LYS (Erwin Chargaff)
MET (N. Metropolis)
PHE (Gunther Stent)
PRO (Jim Watson)
SER (Harold Gordon)
THR (Leslie Orgel)
TRY (now TRP) (Max Delbruck)
TYR (Francis Crick)
VAL (Sydney Brenner)
The Locales
Cambridge, England
New York City
Cold Spring Harbor, Long Island
Woods Hole, Massachusetts
Madison, Wisconsin
Bethesda, Maryland
The triplet code is comma-less.
The triplet code is non-overlapping.
The triplet code is degenerate.
The triplet code is universal (almost!).
Watch out for language changes.
“Soluble RNA” becomes “adaptor RNA” becomes “transfer RNA” (tRNA)
“template RNA” becomes “messenger RNA” (mRNA)
Energetically unfavorable in water.
Protein (peptide) hydrolysis is favored in water.
In the 1950’s several laboratories were trying to figure out how
proteins were made from a biochemical standpoint.
The standard biochemical strategy is to purify components
and then re-assemble them in the test tube (“in vitro”) to
see if they will react to give the expected product.
In this case the essential components were ribosomal RNA,
soluble RNA, activation enzymes, amino acids and ATP and GTP.
amino acid + ATP ->
amino acyl-AMP + tRNA
amino acyl-AMP + PP
-> amino acyl-tRNA + AMP
the adapter
Later it would be found that a family of 20 enzymes (one for each
amino acid) catalyzes both these steps.
Radioactivity was essential for the experiments to be described today.
C14 amino acids didn’t become available until the mid-1950’s.
In the mid-1950’s a bacterial enzyme was discovered that could make
RNA from individual dinucleotides (UDP, GDP, ADP, CDP).
It was known that DNA synthesis uses trinuclotides (TTP, GTP, ATP, CTP).
Ochoa thought this was how RNA was made in the cell, but his postdoc
(Marianne Grunberg-Manago) wisely convinced him the enzyme was mainly for
degrading RNA.
RNA (X = U,G,A,C)
nucleotide diphosphates
phosphate
nP + (XMP)n

nXDP
In the presence of simple phosphate, the enzyme polynucleotide phosphorylase
catalyzes the conversion of RNA into nucleotide diphosphates.
Importantly, the reaction can be run backwards in the test tube
to generate RNA polymers. If only one nucleotide diphosphate
is provides (e.g., GPP), the product is poly-G.
The 1961 International Congress of Biochemistry was held in August in Moscow.
Only a relatively few intrepid American biochemists went behind the Iron Curtain.
But upon their return an important finding reported at that meeting was
quickly circulated by telephone.
.“..poly-U makes poly-Phe..”.
(phone message, September, 1961)
Proc.Natl. Acad. Sci, USA, Oct. 15, 1961
The Nirenberg lab had used a poly-U oligomer as a substitute for
messenger RNA (aka template RNA).
The only amino acid (18 tried) to be incorporated into
“protein” was phenylalanine (Phe).
The poly-U had been made in vitro with the enzyme
polynucleotide phosphorylase as described by Grunberg-Manago
and Ochoa (although this was not mentioned).
Poly-U
In a “Note Added in Proof” the Nirenberg group reported they had
now tried poly-C as the artificial messenger and the only amino acid
incorporated into “protein” was proline (Pro).
PNAS Dec 15, 1961
The Ochoa lab immediately responded that they had (“also”
omitted) shown that poly-U makes poly-Phe.
The race was on!
Selected articles from
Nirenberg lab, 1961-63.
PNAS articles from
Ochoa lab, 1961-62.
Given that the codon for phenylalanine must be UUU and the
codon for proline must be CCC, efforts were immediately
undertaken to deduce the other codons on the basis of known
mutations in real proteins.
Variant human hemoglobins figured prominently in such reckonings.
For example, if Phe changed to Ser, then the codon for Ser must be
UUX, UXU or XUU, it was thought, X being one of the other
three bases (A, G or T).
PNAS, April, 1961
PNAS, June, 1962
Most of these predictions ended up being in error, although often
compositionally correct (but wrong order).
In 1964 the Nirenberg lab introduced a new strategy that gave
definitive answers. They used synthetic trinucelotides corresponding
to the various codons in combination with amino acyl-tRNAs.
Instead of looking for incorporation into “protein”, they
simply looked to see which combinations stuck to ribosomes,
The Nirenberg lab was making their synthetic triplets by
relatively crude enzymatic procedures. Not all 64 possible
were made.
By 1965 H. Gobind Khorana had made all 64 possible
triplets by chemical synthesis.
In 1964 Robert Holley completed the sequence of the 77-nucleotide
alanine tRNA.
Its general structure could be predicted on the basis of base
pairing (A:U and G:C).
The anti-codon for alanine (Ala) was at the expected position.
Ala = GCC, anti codon = GGC.
In the end, the code was found to be degenerate and universal.
Today’s slides (Osher5.pdf) available on dogfish website
(http://dogfish.ucsd.edu) under Lectures tab.
Proc.Natl. Acad. Sci, USA, Oct. 15, 1961
PNAS Dec. 15, 1962