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:
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.

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.


- 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