29th April, 1965.

Dr. H. Gobind Khorana,
University of Wisconsin,
fnst, for Enzyme Research,
1702 University Avenue,
Madison 6r Wisconsin,
U.S,A,

Dear Gobind, '

',"

Very pieased to get your letter and to have the details
especially about poly AG.
results with aspartic,   I am however very puzzled by your'
             I think it is most important to see

whether any aspartic is incorporated.  My general impression,
both from your results and from Wrenberg%, is that binding
studies by themselves, can be very misleading.  The only safe
way is to test each trinucleotide against all 20 amino acids.
It is often found that a triplet binds one amino acid fairly
well, but others less well; the latter are usually artefacts,
These errors are increased by high Mg and low temperature.
,Noreover, there are triplets which we believe to be authentic,
which bind very poorly,  Thus in your table I: shall disregard
all ~assignrrhntti'based on binding alone,                        '. .,

  After all, there is no reason why the binding test using
trinucleotides should work at all, since it does not exactly
correspond to what occursin protein synthesis.  For this
reason Z attach much more importance to incorporation.  However,
I am not clear why binding to the polymer should be misleading,
and very puzzled that arginine S-RRA binds so poorly, and why
aspartic S-RNA binds at all, to poly AAG.  This is why f think
it important to be sure no aspartic is incorporated,

.

  X am still very pussled about the poly AAG results.  If it
were not for your results with poly AG I would not accept AGA
for arginine.  Incidentally are you certain that poly AG
incorporates no aspartic?


Dr. H, Gobfnd Khorana      -2-      29th April, 1965..



  ft*s always worth remembering that you ma have a strain
                -6
with a suppressor in it, and,that eventually   e result
should be checked using a different strain.  Xf you had a
93uppressoP which was misreading Sn the third place it would
explain your results.  However such a strain might incorporate
some aspN with poly A,  Could you check this? Xt might also
incorporate some ser using poly BAG.

   On other matters.  f'have heard from Jacques Fresco. His
results are more or less what one would expect; only a small_
amount of tryp is inoorporated and this may well be because the
repeating sequence is not exact.  His results suggest that AUA
ia Ileu,  However this could be a mistaken sequence, suoh as
Mu.  It could be checked by taking a rigorously repeating
poly AU and reacting it (when melted) to turn some A into I,
not 90% as Fresco did* but only, say9 3% - just enou& to
destroy the secondary structure.  Then if Ileu were incorpora-
ted we should know that AUA was Ileu, Could you try th.is?

  Row as to tri- and tetra-sequences, especially in relation
to ochres and amber&
            + ,, .: :.;-- : ,* ':'     We should very much like ,to have
                                                                           $Y   : .,,
                                                                           ,,
                      . .
           poly UAA
        and    poJy UAG  (or poly UAI)

'(R~'%~??&&se poly UAA is near enough the same as poly AAU
or poly AUA *I, 9: won"t repeat this remark in what follows)

because we aould use them to test the mechanism of suppression.
I have written out all possible repeating tri- and tetra-
sequences, and enclose copies of my notes (please check for
slips)*  The best tetra's would appear to bet
               poly UAAA
               poly UAAC
              poly UAAG
               poly UAM
               poly UAGU


                                                                           '
Dr. H,. Gobind Khorana    -3-,  29th April, 1965.'
                                                              

               ~                                  ! `,.                            ,,
However I 'have grave doubts .-that you will be able to-make
accurate repeating ,tetra"s by your present method,s, and      L
suggest you try poly UAA'and poly UAG first; ')I    ' .'

                                                                      

  Ther,e,have been no further,developments here about
suppressors, except that they have shown that YanofskyQa         '
strain'of E,':coli has an amber suppressor in t$ which puts in

,Tyr.


                     

                 

   Aboutthe'Gordon Conference,. Many thanks for the
promise of the money,'  I enclose a copy of a letter to Jim
and Marshall, which explains itself,  I do,hope that Yanofsky
can'come to the Gordon Conference.  In addition you should ',
know that Streisinger and'his colleagues have the following
results for phage lysosyme,

Wild type: ) ' L'

  Double mutant: 5
            


Using the latest version of the'code (copy' enclosed - it
incorporatesTtheir results) you will easily ,.be, able to show .'
$aFot$ro is only one solution, and that reading is from        '

       `as .Ochoa claims,  Their result also givesAw 5 new
codons; ADO ,y'ou think that Streisinger, or one,of his people,.
could b& squeezed into'the~ Gordon Conference?
  Finally' 3: should mention that Zachau now has for the serine
S-RNA (from yeast),the sequence%  4;. pUpUpIpGpApUp ,.e.
which is the antLcodon I'prsdioted for the pair-of codons        ,,
UCCJ and UCC.`  f thus believe that the anti-codon forphe is
,, IAA (or GAA),- not AAA - and for tyr it is IUA (or GUA)

2, H, d., Crick