Second, a reaction mixture containing formyl-35Smcthionyl-
tRNAMetF was chromatographed on a column
of ‘Sephadex G-75’. Virtually all of the incorporated
radioactivity chromatographed identically to the intact
a 2~ 2 haemoglobin, measured by absorbance at 563 nm,
present in the reticulocyte extract (Fig. 4a). This experiment
suggests that formylmethionine is incorporated into
intact, functional a and ~ chains and we confirmed this
by the experiment in Fig. 4b. Globin chains were prepared
from a similar in vitro reaction, and separated on a column
of carboxymethyl (CM) cellulose. Two peaks of radioactivity
were found; they eluted from the column slightly
before the authentic a and~ chain markers. OM-cellulose
separates largely according to protein charge, so that this
result is accounted for by incorporation of the formyl-
35S-mcthionine radioactivity into a and ~ chains with a
reduced positive charge, because of the blocked N-tcrminal
amino group.
Finally, when digested with trypsin, tho formyl-35Smethionine-
labelled a and ~ chains each yiPlded a single
radioactive peptide (Fig. 5). These have the expected
ionophoretic mobility of the amino-tf’rminal tryptic
peptides of a and ~ chains containing an N-terminal
formylmethionine. When those peptides were treated
with dilute HCl to remove the formyl residue, thEy had an
ionophoretic mobility consistent with the acquisition of a
Eingle positive charge (Fig. 6, top panel). Further characterization
of these peptides is in progress, but we suggest
that a formylmethionine residue can be incorporated at
the amino terminus of both a and ~ chains, and that no
other polypeptide beginning with formylmcthioninc is
produced in our syst0m.
Incorporation by Met-tRNAMetF
The low level of methionine incorporation from ass.
Met-tRNAMetF (Fig. l) could be explained by any of the
following hypotheses: (l) Mct-tRNAMetF, like fMcttRNAJIIetl<‘,
is actually used for the initiation of haemoglobin
synthesis, but the amino terminal methionine is
rapidly removed from the nascent chain; (2) MettRNAMetF
is used for initiation of a protein synthesized
in relatively small amounts, and (3) Met-tRNAMetF inserts
methionine into the internal positions of haemoglobin,
but only poorly.
Evidence that both Met-tRNAMetF and fMet-tRNAMetF
incorporate, respectively, methionine and formylmethionine
into the same position of the polypeptide chain is
derived from the experiment in Fig. 6. Protein synthe- The experiment described in Table 2 also strongly
indicates that Met-tRNAMetF and fMet-tRNAMetF compete
for the same function-the initiation of haemoglobin
synthesis. Thus (1) addition of Met-tRNAMetF inhibits
incorporation by formyl-358-Met-tRNA.MetF and conversely
non-radioactive fMet-tRNAMetF inhibits incorporation
by 35S-Met-tRNAMetF; and (2) the inhibition was
specific, because the net level of haemoglobin synthesismeasured
by incorporation of 14C-lysine (Table 2) or
358-Met-tRNAMetl\1 (unpublished)-was not appreciably
affected by either tRNA.
Furthermore, the low level of incorporation observed
with ass.Met-tRNAMetF as compared with formyl-358-MettRNAMetF
suggests that ass-methionine incorporated from
ass.Met-tRNAMetF is removed from the nascent chain,
whereas the formyl- 35S-methionyl product is stable. A
direct prediction of this is the conversion of a large amount
of radioactivity from ass.Met-tRN.AMetFto free methionine.To test this, a small amount of 35S-Met-tRNAMetl!’ was
added to a reticulocyte cell-free systp,m (Fig. 7). Results
show, first, that within 3 min more than 60 per cent of
the radioactivity was converted to acid soluble form.
Paper ionophoresis at pH 1·9 revealed that all of the 35S
radioactivity solubilized is indeed 35S-methionine. Second,
this acid solubilization requires protein synthesis on
cytoplasmic 80S ribosomes, for it is inhibited by cycloheximide
or aurintricarboxylate, but not by chloramphenicol.
Third, this acid solubilization is specific, for in
similar experiments with 35S-Met-tRNAMetM and formyl-
35S-Met-tRNAMetl<’ there was less than 15 per cent formation
of acid soluble radioactivity. Table 3 shows that acetyl-35S-methionyl tRNAMetF
is also incorporated efficiently into protein, whereas
acetyl- 35S-methionyl-tRNAMetM is not incorporated,
so that the ability of formyl or acetyl Met-tRNAMetF to
initiate protein synthesis is not simply to be attributed
to a blocked amino group, but rather to an intrinsic
property of tRNAMetF.