Who was the mysterious
and possibly dangerous man we call ..........
........ Robert
W. Holley
(1922-1993) ?
Born on January
28, 1922 in Urbana, Illinois.
1942-
Recieved B.A. in Chemistry from
the University of Illinois.
1944-1946-
Spent
these two years with Professor Vincent du Vigneaud at Cornell
University where he was involved in
the first chemical synthesis of
penicillin.
1947-
Received a Ph.D. in organic
chemistry from Cornell University.
1948-
Became a professor of Organic
Chemistry at the Geneva Experiment Station at Cornell.
1955-56-
Took a sabbatical leave from Cornell and studied protein
synthesis. Towards the end of this year he carried out experiments on
the acceptor of activated amino
acids.
1968-
*Shared NOBEL
PRIZE
for Physiology/Medicine.
What was known at this point (1956) :
- Amino acids
are activated enzymatically to form amino acyl-adenylates
(Hoagland, Kellerl and Zanecnik, DeMoss, Genuth and
Novelli, and Berg and Newtons).
- The acceptor
of the activated amino acids was a low molecular weight RNA (Hoagland
et al., Otata and Nohara).
- The activated
amino acids became attached to a terminal adenosine residue on the tRNA
and that different tRNA’s were serving as acceptors for different amino
acids (Zachau, Acs, and Lipmann).
- The Craig
Countercurrent Distribution Technique- after a four year
wait- was finally developed.
- J.R. Penswick
found that a brief treatment with ribonuclease T1 at 0C splits the
molecule in two. Subsequent digestion with the same enzyme will give
fragments of the "left" and "right" parts of the tRNA.
- And of
course... Watson and Crick’s base-pairing scheme of A with U and G
and C.
- Also, very
relevant, in 1956 the "Flat-Top" is the choice of
hairstyle for men.
Holley and his crew
used a mix of ingenious logic, persistent work
ethic, and a little common sense to construct what we truly believe to
be:
THE BEST EXPERIMENT OF THE CENTURY:
it is to
genetics, what the Lion King was to Disney Animation.
Isolation of tRNA sample (at Cornell
University, 1958)
For reliable structural analysis, a highly purified
tRNA is required (i.e. for one amino acid)
Need RNA in large, readily available
quantities... therefore... use YEAST RNA!
Fractional Technique
- Craig
Countercurrent distribution technique.
- Distribution
of bulk yeast tRNA.
- Eventually
Alanine tRNA was selected.
IT WAS A
GAMBLE!
- they had no real assurance it was a pure sample!
- could have had other tRNA's, or molecules that
accepted tRNA's.
- so they gambled 4 years of development
- "If
starting material was impure,
we could expect that attempts
at structural analysis would
lead to hopeless confusion.
Fortunately this was not the
outcome"
~ Robert W. Holley, 1968
* Note: Amount of purified alanine
tRNA was very limited.
The amount available for experiments =
tens
of mg!
Total amount over 3 years work
=
1g of highly purified material!
(from over 300 lbs. of yeast)
Cleavage
of
Polynucleotide Chain: The
"real" experiment.
- Alanine tRNA is single chain, 80 nucleotide
residues.
- Full sequence needs to be identified for strutural
analysis.
Sentence/Word
Analogy:
Sentence composed of words totally 80 letters.
1) Break
sentence into words
1) Cleavage of
chain (into small fragments)
2) Identify words
vs. 2) Identify sequence of
fragments
3) reconstruct
sentence with correct order of words
3) reconstruct original sequence
by determining order
of
fragments.
LOGIC EXPERIMENT
Need
2 sets of fragments...
therefore use two different enzymes:
a) Pancreatic ribonuclease
(next to pyrimidine
nucleotides)
b) Takadiastase ribonuclease T1
(next
to guanylic acid and inosine residues)
These fragments isolated by ion-exchange
chromatography
- DEAE-cellulose column
was a newly developed technology, had the required
resolution for such a study.
- If necessary, also paper
electrophoresis/rechromatography.
The separated
fragments are hydrolyzed with alkali, such that the mononucleotides
they
were made up of could be identified (based on physical properties and
spectra)
*only useful for
dinucleotides* (because
position of attack is known)
For the larger
oligonucleotides they needed a more comprehensive
sequence determination method.
This involved
1) digestion with snake venom
phosphodiesterase => mix of
degradation products
2) chromatrography of products
3) alkaline hydolysis yields
3'-terminal nucleosides
4) analyze successive peaks to
determine sequence
List of all the fragments (Table 1, 2)
-> to do this sequencing
took 2.5 years!
Putting it all
together...
Couple of things Holley considered:
- End groups are unique!
3' -hydroxyl group ("right end")
free 5'-phosphate group ("left
end")
- There were several other
"unusual" nucleotides and sequences. e.g. I-
*So far, they
have compared two sets of smaller fragments to determine
the set of larger fragments they come from.
*To complete the
sequencing of the entire molecule, they had to compare
these larger fragments to
even larger fragments to determine
the entire chain
sequence!!
- e.g. They "bisected"
the RNA using RNaseT1 at 0oC in presence of Mg2++,
digestion of these half molecules indicated which of the fragments they
already
identified came from which half of the molecule (i.e. digestion of the
larger molecule would yield chromatographic
peaks
that were known, because of the
previous work) and so on...!!
Structural
Proof
- The analysis
of the large fragments gave sufficient information to
establish the sequences of the two halves of the tRNA.
- The terminal
sequences of the tRNA were already known, which only
made it possible to join them together in one way. The joining sequence
was -I-G-C- , which is the alanine tRNA’s anticodon.
Implications
- The first
known nucleotide sequence for a nucleic acid.
- The first
nucleotide sequence of a gene (this would be the sequence
of the gene that determines the structure of the alanine transfer RNA
in yeast.
* Also interesting, in that it paved the way for sequencing of
future structures, for example, the sequence
G-T-*-C-G at the time was
believed to be found the same 20 nucleotide
distance form the AA-acceptor end of the
molecule in ALL tRNA's, just
as in the Ala-tRNA.
- And it only took 9 years!
If that wasn’t
enough...
Three
dimensional structure:
- Initially
concerned with the interaction of the tRNA with the mRNA.
-
"Speculation suggests that the three-dimensional structure of a
transfer RNA, in the presence of the magnesium ion under conditions
suitable for protein synthesis, should have the codon triplet of
nucleotides, the anticodon, exposed in a way that will permit it to
interact with a triplet of nucleotides, the codon, in the messenger
RNA." Robert Holley
- E.B.
Kelly and J.R. Penswick suggested that there was a "cloverleaf"
arrangement with the -I-C-G- sequence exposed.
- This
meant a "Watson-and-Crick-type" pairing of A to U and G to C in
the double stranded regions. The unpaired regions would form loops.
- The
anticodon is always found on the middle loop.
All this jibba
jabba eventually looks like THIS
"That then is our story of the
alanine transfer RNA. It all followed
quite naturally from taking a
sabbatical leave. I strongly recommend
sabbatical leaves."
~
Robert W.
Holley, 1968
Useful Links:
Dr. Carr's Advanced
Genetics Site
Nobel
e-Museum (1968)
Robert
W. Holley's Nobel Lecture
Agricultural
Research Service: Essentially, a summary of Holley's work
a wicked presentaion on 50's
fashion, home of the Flat Top
