(Received for publication, April 26, 1995; and in revised form, June 30, 1995)
From the
Uracil, uridine, and pseudouridine were acetylated by refluxing
in acetic anhydride, and the products of acetylation were incubated
with a synthetic
peptide(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21) that
corresponds to the N-terminal 21 amino acid residues of human myelin
basic protein. Peptide bond formation, at the N terminus in peptide 1-21, was obtained with acetyluracil
and acetylpseudouridine, but not with acetyluridine. Transfer of an
acetyl group from acetyluracil and acetylpseudouridine depended on
acetylation in the N-heterocycle. X-ray crystallographic
analysis definitively established N-1 as the site of acetylation in
acetyluracil. Mass spectrometry of the acetylation products showed that
one acetyl group was transferred to peptide 1-21, in water, by
either acetyluracil or acetylpseudouridine at pH
6. Release of the
acetyl group by acylaminopeptidase regenerated peptide 1-21 (mass
spectrometry) and automated sequencing (for five cycles) of the
regenerated (deacetylated) peptide demonstrated that the N terminus was
intact. The findings are discussed in the context of a possible role
for pseudouridine in ribosome-catalyzed peptidyltransfer, with
particular reference being made to similarities between the possible
mechanism of acyl transfer by acetyluracil/pseudouridine and the
mechanism of carboxyl transfer by carboxylbiotin in acetyl CoA
carboxylase. The possibility that idiosyncratic appearance of a wide
range of acyl substituents in myelin basic protein could be related to
a peculiar involvement of ribosomal pseudouridine is mentioned.
The high group-transfer potential of aminoacyl substituents at the 2`,3`-hydroxyl termini in tRNAs (see (1) ) suggested that subsequent ribosome-catalyzed transfer of aminoacyl groups, from tRNAs into peptide linkages, might involve, as intermediate carriers of similar transfer potential, vicinal 2`,3`-hydroxyl termini in rRNA molecules. After it was clear that rRNA molecules had vicinal 2`,3`-hydroxyl termini but that they were unlikely to be implicated in peptidyl transfer (see ``Discussion''), it was of interest, in the context of a direct role for RNA in peptidyl transfer(2) , to evaluate evidence for other possible sites of high group-transfer potential in rRNA.
Almost 40 years ago, Spector and Keller (3) reported that the N-1 position in uracil could transfer acyl groups into peptide linkage(3) , but the discovery was made at a time when RNA was thought to contain only the four classical nucleosides. The authors noted that the N-1 position of uracil is blocked in the corresponding classical nucleoside (uridine) of RNA and concluded that the property was of chemical, not of biochemical interest. Ironically, a ``fifth nucleoside'' had been isolated from RNA (4) a year before the Spector and Keller report, but 3 years passed before the fifth nucleoside, the first modified nucleoside found in RNA, was identified as a uridine isomer, pseudouridine(5, 6, 7) .
The possible
relevance of the Spector and Keller finding for a prominent role of
pseudouridine during peptidyltransfer in the ribosome was only
articulated for publication during the past few
years(8, 9) . In this brief time, the Ofengand
laboratory has shown that all pseudouridine residues in the RNA of
large ribosomal subunits from Escherichia coli (9 (
)residues) through human (54
residues) are present
in the peptidyl transfer region of the
ribosome(10, 11, 12, 13) . Where it
was once possible to envision a network of transfer sites in the
ribosome, from tRNA termini via rRNA termini into peptide linkage, it
is now possible to envision a network of
sites.
The
sites are not evolutionarily conserved in the primary and secondary
structure of rRNA but could possibly constitute a network of transfer
sites within the stereostructure of the ribosome. Two residues,
including
(E. coli numbering), found in
most but not all (13) prokaryotic and mitochondrial large
subunit RNAs so far analyzed, and
, in the
``universal'' Um-Gm-
sequence of all
eukaryotic/cytosolic large subunit rRNAs so far studied, are
quasi-conserved, and as with many other large subunit RNA pseudouridine
residues, they are likely proximal to the 2`,3` termini of ribosomal
P-site tRNAs.
Correlation between the amount (14) and
sequence occurrence (Um-Gm-) (15) of
and
2`-O-methylnucleosides in eukaryotic (wheat) rRNA was first
noted more than 30 years ago. Parallel increments (
10-fold) in the
two types of modification between the rRNAs of bacteria and higher
eukaryotes (16, 17) was also noted, as was a potential
biosynthetic relation between them(18, 19) .
Interrelatedness of pseudouridylation and sugar-methylation in rRNA has
recently been discussed in the context of function and stereostructural
modeling at the peptidyltransfer center in the
ribosome(9, 12) . This report focuses on the more
specific consideration: a direct role of
in aminoacyltransfer.
From the outset, it was clear that the acyl group-transfer potential
of uracil (3) is greater than that observed for the vicinal
2`,3`-hydroxyl termini in tRNAs(1) , but it was equally clear
that, if it had group-transfer properties at all, the properties of
might differ from those of uracil. The most acidic ionizations
in uracil, uridine, and pseudouridine are 9.50, 9.25(20) , and
8.97(21) , respectively, and the very high group-transfer
potential of the -CO-NH- dissociation in uracil, which contains the N-1
(O-2) positions (22) , was revealed by experiment(3) .
As in uracil, the most acidic -CO-NH- ionization in pseudouridine is
allied with a very strong bathochromic shift (20 mµ) of the UV
maximum; however, the most acidic (-CO-NH-) ionization in uridine is
not allied with a bathochromic shift of its UV maximum, and it is
located between two carbonyl groups (see (22) ). It was
therefore posited (8) that N-1 (O-2) in pseudouridine, but not
N-3 (O-4) in uridine, might share the high acyl transfer potential
invested in N-1 (O-2) of uracil(3) . An analogous situation
obtains in urate/uric acid. For example, in urate, N-3 (analogous to
N-1 in uracil) is the most acidic site in the N-heterocycle,
whereas N-1 (analogous to N-3 in uracil) is flanked by carbonyl groups
and as with N-3 in uracil, is a relatively weak dissociation in the
purine base(23) .
It could only be determined by experiment
whether shares the anticipated (8) acyl group-transfer
properties of uracil. It was first necessary to establish whether
pseudouridine, like uracil, can accept acyl groups in its N-heterocycle and then whether pseudouridine can mediate
transfer of the acyl substituent into peptide linkage. For this
purpose, a model system involving the N-terminal sequence of human
myelin basic protein was developed, and the positive findings are
described in this report.
Figure 1: Fast atom bombardment mass spectrum of acetyluracil.
Figure 2: Molecular structure and bond lengths (AU) of acetyluracil.
Figure 3: Diagram illustrating the HPLC separation of acetylpseudouridine from other components of the reaction mixture in which acetylpseudouridine was used for peptide-bond synthesis. The product of peptide-bond synthesis eluted at 21 min and acetylated pseudouridine eluted at 31-33 min.
Figure 4: API mass spectrum, unacetylated peptide at m/z = 769.4 and acetylated peptide at m/z = 783.4.
Using the procedure described by Spector and Keller(3) , we have confirmed that monoacetyl uracil is made during reflux in acetic anhydride, and we have characterized the product by mass spectrometry. Most significantly, x-ray crystallography has established that acetylation occurs at N-1 as predicted by Spector and Keller(3) . Acetylation of uridine under the same conditions yields a product that contains three acetyl substituents, and acetylation of pseudouridine yields a product that contains four acetyl substituents. We have assumed, as previously shown for acetyluridine(27) , that in acetylpseudouridine, three of the acetyl substituents are at the 2`-, 3`-, and 5`-hydroxyl functions in the ribosyl substituent of the nucleoside and, as demonstrated here for uracil (Fig. 2), that the fourth acetyl substituent is at the N-1 position of uracil.
Because acetyluracil, but not acetyluridine
can transfer an acetyl group to the N terminus of
peptide 1-21 of human myelin basic protein, it is assumed that
N-1 acetylation in the N-heterocycle of acetylpseudouridine
accounts for its similar ability to transfer an acetyl group to the N
terminus in peptide 1-21 of MBP. This strengthens speculation (8) that pseudouridine in the ribosome could serve to
transfer acyl groups of high group transfer potential during protein
biosynthesis, possibly in addition to, or in concert with His-229 of
protein L2(30, 31) .
As recently noted(9) ,
the acyl-transfer site(N-1) in pseudouridine might participate in other
ways in the catalysis of ribosomal peptidyltransfer, e.g. by
proton abstraction. Whatever the case, all evidence adduced (10, 11, 12, 13) during the 3 years
since publication of the hypothesis (8) has only
amplified the notion that pseudouridine, in one capacity (8) or
another(9) , has a central, significant, and previously
unheralded role in the activity of the peptidyl transfer center of the
ribosome.
By contrast, the possibility that a circuit of vicinal hydroxyl groups might be engaged in transferring activated aminoacyl residues from the 2`,3` termini of tRNA molecules into peptide linkage, which had once seemed plausible when 5, 5.8, 18, and 26 S rRNAs were shown to have 2`,3`-hydroxyl termini(15, 32, 33, 34, 35) , declined steadily over the years. Accumulated information gradually revealed that the 2`,3` termini of rRNA molecules are not located in the peptidyltransfer center of the E. coli ribosome. In the first instance, it was shown that one of the Shine-Dalgarno hypotheses (36) was correct in placing the 3` terminus of E. coli 16 S rRNA in the immediate vicinity of the (mRNA) decoding site(37) , not the peptidyltransfer site of the ribosome.
Although an explanation of the reactivity of the N-1 position in
uracil is lacking, the ureido structure assumes first importance, as it
does in all putative functions for pseudouridine(9) . We
suggest that a mechanism similar to that proposed for transfer of
carboxyl groups of high group-transfer potential by biotin may be
operative, also, in the transfer of acyl groups of high group-transfer
potential by pseudouridine. The ureido group in biotin is the
catalytically active part of the acetyl-CoA carboxylase molecule to
which CO of high group-transfer potential has been
added(38, 39) .
Crystallographic studies suggest
that the reactivity of N-1` in biotin results in a partial enolate at
O-2`, which is in turn responsible for deprotonation of N-1`, rendering
this site nucleophilic and able to accept CO of high
group-transfer potential, i.e. N-1` can thereby accept the
active CO
that is generated in an ATP-dependent reaction.
It seems plausible that reactivity of the N-1 position in uracil
(pseudouridine) is likewise the result of partial enolate formation at
the exocyclic O-2 (see (22) ). Another similarity, this time
with respect to the subsequent mechanism of transfer, is noteworthy.
Carboxyl transfer (via biotin) between donor and acceptor in acetyl-CoA
carboxylase occurs by a gauche-trans rotation about the 2-6 and
6-7 bonds in the valeryl side chain of biotin(38) , and
this is reminiscent of the notion that increased rotational flexibility
about the C-C glycosyl bond in pseudouridine, relative to an
N-C glycosyl bond in uridine, may be a significant consideration
in transfer of aminoacyl groups (via pseudouridine) between P and A
sites in the ribosome (see (9) ).
The(1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21) N-terminal
peptide of MBP was chosen as a model peptide for these studies because
its acylation by a wide variety of fatty acids (40) may be
indicative of an idiosyncratic pathway of N-acetylation in MBP. It seems that the
ribosome itself, via pseudouridine residues in the peptidyl transfer
region, might serve to transfer acyl groups to N
termini in MBP. For instance, N
acetylation
occurs after about 25 amino acids are added to the growing polypeptide
(see (41) ), and a recent study of the routing of N
termini through the E. coli ribosome shows the exit
channel is wide enough to allow a nascent polypeptide to fold; residue
25 in a nascent polypeptide can cross-link to residue 2585 in 23 S
RNA(42) , which neighbours
in the
peptidyl transfer region.
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