© 7990 Oxford University Press
Nucleic Acids Research, Vol. 18, No. 16 4677
The role of modified purine 64 in initiator/elongator
discrimination of tRNA^61 from yeast and wheat germ
Stefan Kiesewetter, Giinther Ott and Mathias Sprinzl*
Laboratorium fur Biochemie, Universitat Bayreuth, Universitats-straBe 30, Postfach 10 12 51, D-8580
Bayreuth, FRG
Received June 18, 1990; Accepted July 17, 1990
ABSTRACT
The role of 2'-ribosylated adenosine 64 in tRNA,Mel
from yeast in initiation/elongation discrimination was
investigated. As measured by in vitro translation in
rabbit reticulocyte lysate, the specific removal of the
2'-ribosylphosphate at adenosine 64 via periodate
oxidation allows tRNAj"et to read internal AUG codons
of the globine messenger RNA. Yeast Met-tRNA"61
lacking the modification of nucleoside 64 forms ternary
complexes with GTP and elongation factor Tu from
Escherichia coli. The lack of modification at position
64 does not prevent tRNA"6' from participating in the
initiation process of in vitro protein synthesis. Wheat
germ tRNAfet has a 2'-ribosylated guanosine at
position 64. Removal of this modification from the
wheat germ tRNAfet enables it to read internal AUG
codons of globine and tobacco mosaic virus messenger
RNA in reticulocyte and wheat germ translation
systems, respectively.
INTRODUCTION
The first step in translating genetic information into a functional
protein is the recognition of the starting codon on the mRNA.
Although the genetic code is degenerate for most amino acids,
there is only one codon used for methionine in all cytoplasmic
translation systems. In order to ensure the correct initiation site
of translation, the protein synthetizing apparatus of the cell must
be able to distinguish between the AUG start codon and AUG
tripletts in the internal coding region of the mRNA. Thus, a highly
specialized methionine accepting tRNA is needed to recognize
exclusively the start codon. In procaryotes this tRNA is termed
tRNAfMcl, indicating the formylation of the attached methionine
residue; in eucaryotes the initiator tRNA"el is not formylated.
Efforts have been made to elaborate the structural features
which determine the specificity of initiator tRNAs. The crystal
structure of both procaryotic tRNAfMet [1] and eucaryotic
py
y
f
j ^ ' [2] have been determined. However, the reported
refinements are too rough to give detailed insight into the unique
role of these tRNAs during protein synthesis.
Mutants of E.coli tRNA"el have also been constructed [3-6]
* To whom correspondence should be addressed
to elucidate which features of the primary structure contributes
to its specific function. In the case of tRNA^et, the first base
pair of the aminoacyl stem (base pair 1-72 according to [7])
plays a discriminating role in determining the initiator function
of this tRNA. All tRNAsfMct from eubacteria and chloroplasts
contain a C r A 7 2 mismatch at this position. A mutant of this
tRNA, containing a regular C r G 7 2 base pair, forms EFTu-GTP-Met-tRNAfMeI ternary complexes [3-5] and is able to
act as an elongator tRNA in vivo [4].
The binding of bacterial IF-2GTPfMet-tRNAfMeI to the
ribosomal P-site is made possible by a stack of three G-C base
pairs in the last three positions of the anticodon stem of the tRNA.
Mutants of tRNAfMet having only one or two G-C base pairs in
this region of the molecule have a significantly diminished
initiation capability in in vitro translation [5].
Little is known, thus far, about the features which determine
the specificity of eucaryotic tRNA[*el. We have focused our
attention on a modification found recently in tRNAJ^" from
yeast [8] (figure 1), located near the junction of the T-stem and
the aminoacyl stem of the tRNA tertiary structure. This part of
the molecule is thought to be important for the interaction of
elongator aminoacyl-tRNAs with EF-la • GTP and EF-Tu • GTP
in eucaryotes and procaryotes, respectively [9]. It is possible that
the hydrophilic modification at adenosine 64 could prevent the
binding of the elongation factor to the aminoacylated initiator
tRNA, thus excluding it from the elongation process. We tested
this possibility by measuring the binding capacity of yeast initiator
tRNA^" to bacterial elongation factor Tu, which is homologous
in structure and function to yeast EF-la. Furthermore we
measured the modification-64-dependent activity of initiator
tRNAs from yeast and wheat germ during protein elongation in
vitro.
MATERIALS AND METHODS
tRNA"el from yeast was isolated from yeast bulk tRNA
(Boehringer, Mannheim, FRG) by BD cellulose chromatography
[10] and rechromatography on Sepharose 6B according to [11].
The isolated species could be aminoacylated by partially purified
E.coli Met-tRNA synthetase [12] to 1460 pmoles/A260 unit.
4678 Nucleic Acids Research, Vol. 18, No. 16
tRNAfa from wheat germ was a genereous gift of Dr.P. Sigler,
Yale University, New Haven, and was aminoacylatable with
partially purified E.coli Met-tRNA synthetase to 1321
pmoles/A260 units. Aminoacylations were performed in a buffer
containing 150 mM Tris/HCl pH 7.6; 60 mM KC1; 10 mM
MgCl2 and 5 mM ATP in the presence of 100 units/ ml
ATP(CTP):tRNA-nucleotidyltransferase from yeast (Dr. H.
Sternbach, Gottingen, FRG) at 37 CC for 20 min and using either
[ I4 C]methionine 2.15 GBq/mol or [35S]methionine 20.0
OH OH
Fig.l: The modification found in tRNA*1" from yeast: 2'-l"-j3-(5"-phosphoryl)ribosyl-adenosine [8].
0.200
GBq/mol (Amersham Buchler, Braunschweig, FRG). Wheat
germ and rabbit reticulocyte translation kits were obtained from
Promega, Atlanta, USA and Amersham Buchler, Braunschweig,
FRG, respectively.
Reverse phase HPLC of nucleosides was performed on a
Supelcosil LC-18 column (250x4.6 mm, Supelco, Bellefonte,
USA) using Beckman HPLC system gold, pump module 128 and
multiwavelength UV detector 167. Sample preparation and HPLC
conditions were as described [13].
Oxidative elimination of the phosphoribosyl residues from N-64
of yeast and wheat germ tRNAjMel, leading to tRNA,Me'(ox)
species, was carried out in a buffer containing 50 mM sodium
acetate, pH 6.5; 150 fM tRNAf1" and 10 mM sodium periodate
in a total volume of 100 /d. The mixture was incubated for 20
min at room temperature. After adding glucose to a final
concentration of 12 mM, the resulting mixture was allowed to
stand for a further 10 min [14]. The pH of the solution was then
changed by adding 1 M sodium acetate pH 4.5 up to a final
concentration of 200 mM. After ethanol precipitation the pellet
was washed twice with 70% ethanol/water (v/v) and dried in
vacuo. The pellet was dissolved in 300 /d 500 mM lysine, pH
9.0, and the resulting solution was incubated for 30 min at 37°C.
Subsequently, 0.5 units of alkaline phosphatase from E.coli
(Sigma, St. Louis, USA) were added and the mixture was allowed
to stand for a further 30 min at 37°C. One M sodium acetate,
pH 4.5, was then added to a final concentration of 200 mM.
tRNA|Mel(ox) was isolated by ethanol precipitation and
subsequent batch-purification on DEAE-Sephadex A 25
(Pharmacia, Uppsala, Sweden), followed by desalting on a Biogel
P6 (BioRad, Richmond, USA) column as described [14].
a
C
c
A
0.100
ij
m A
7
m G
m C -A
3
CM
i
m§G
ApG
III'6*
0.000
retention time [min]
Fig.2: Reversed phase HPLC nucleoside analysis of yeast tRNA^" (a), tRNAjMeI(ox) (b), wheat germ tRNAJ*" (c) and tRNA;Mcl(ox) (d).
Nucleic Acids Research, Vol. 18, No. 16 4679
tRNA|Met(ox) was methionylated in the presence of
ATP(CTP): tRNA-nucleotidyltransferase as described above, in
order to reattach the 3'-adenylate residue removed by periodate
treatment [14]. The methionine acceptances of yeast
tRNA^el(ox) and wheat germ tRNA,Mct(ox) were 1296 and 1145
pmoles/A26o units, respectively.
In vitro translation in rabbit reticulocyte lysate was performed
according to Wagner et al. [15]. The reaction mixture (100 /tl)
contained 60 /tl of rabbit reticulocyte lysate (not treated with
RNase, not depleted of amino acid), 75 mM KC1, 2 mM
MgCl2, 0.5 mM ATP, 0.2 mM GTP, 15 mM creatine
phosphate, 45 units/ml of creatine kinase (Boehringer,
Mannheim, FRG), 50 /tM tRNAbulk from calf liver, 50 /tM
methionine, 25 /tM hemin and 0.5 /tM [35S]Met-tRNA"et or
[35S]Met-tRNA^el(ox), respectively, and was incubated at 34°C.
Eighteen /tl aliquots of the reaction mixture were diluted into 200
/tl 0.1 M NaOH at the indicated times. The incorporation of
[35S]methionine into a//3 globine was determined by
hydrochloric acid/acetone and trichloroacetic acid precipitation
as described [16].
In vitro translation in wheat germ extract was performed as
described by Roberts und Paterson [17]. The reaction mixture
(60 /tl) consisting of 30 /tl wheat germ extract (amino acid
dependend, message dependend), 100 mM potassium acetate,
66.7 /*M of each biogene amino acid, 1 /tl TMV-RNA
(Amersham Buchler, Braunschweig, FRG) and 0.5 /iM tRNA
was incubated at 25 °C. Aliquots of 10 /tl were removed from
the mixture at the indicated times, diluted into 500 /tl 1 M NaOH
and heated to 37 °C for 10 min to ensure aminoacyl-tRNA
hydrolysis. The incorporation of [35S]methionine into the
formed polypeptides was determined after trichloroacetic acid
precipitation on nitrocellulose disks (46 /tm, Schleicher und
Schuell, Dassel, FRG).
For monocyclic Edman degradation in vitro protein
biosynthesis was performed in the absence of methionine. The
reaction was stopped after 20 min by adding 200 /tl of 0.1 M
NaOH. The resulting mixture was incubated for 10 min at 37°C,
then neutralized by adding 200 /tl 0.1 M HC1 and chilled on ice.
The formed polypeptides were isolated by acetone precipitation.
The precipitate was resuspended twice in 10% trichloroacetic
acid, then thoroughly washed with ice-cold acetone and
subsequently dried in vacuo. The colourless pellet was dissolved
in 400 /tl 50% pyridine/water (v/v). The monocyclic Edman
degradation was performed using a manual procedure developed
by Chang et al. [18]. All reactions were carried out under
nitrogen. A solution of 7.5 mM 4-dimethylaminoazo-
benzene-4'-isothiocyanate in pyridine (40 /tl) was added to 80
lil of the protein solution. The mixture was incubated for 30 min
at 55°C. Ten /tl of phenylisothiocyanate was then added to the
solution and the resulting mixture was allowed to stand for a
further 20 min at 55°C. After extraction of the solution with
n-heptane/ethylacetate 2:1 (v/v) the aqueous layer was dried and
subsequently dissolved in 50 /tl trifluoroacetic acid. The solution
was incubated for 10 min at 55°C. After removal of the
trifluoroacetic acid in vacuo the resulting pellet was dissolved
in 50 /tl H2O. The solution was extracted three times with
200 /tl butylacetate. Both the organic and the aqueous layers were
evaporated to dryness and the residues dissolved in 200 /tl formic
acid. The amount of [35S]methionine in each fraction was
determined by liquid scintillation counting.
The determination of equilibrium dissociation constants of the
ternary complex aa-tRNA • EF-Tu • GTP using EF-Tu from E. coli
was performed with a fluorescence-titration method as described
elsewhere [19].
RESULTS
Modification in yeast and wheat germ tRNAf16* at position
64
The ribose moiety in the 2'-position of adenosine 64 of yeast
tRNA"CI prevents enzymatic digestion of the phosphodiester
bond between A^ and G65 by nuclease PI. These nucleosides
therefore elute as a dimer during reverse phase HPLC [8,13]
of a PI—and phosphatase—treated digest of yeast tRNAf"
(figure 2). Oxidation of the tRNA with periodate [14] enables
acidic hydrolysis of the 2'-l"-O-glycosidic bond, leading to
specific removal of the additional ribose at adenosine 64. This
makes the phosphodiester bond between A^ and Gg5 accessible
for nuclease PI. After oxidation, the A*MpG65 dimer disappears
from the HPLC elution profile of the digested tRNA. Integration
of the elution peaks reveals an increase by one guanosine residue,
while the amount of adenosine is unchanged as compared with
the analysis of the unoxidized tRNA"et (figure 2). This indicates
the loss of the 3'-adenosine during the outlined procedure.
Terminal adenosine was again added to the tRNA via phosphatase
treatment and incorporation of AMP by an ATP(CTP): tRNA
nucleotidyltransferase catalyzed reaction [14]. This treatment
yields an initiator tRNA^^ox) which only lacks modification at
adenosine 64.
Initiator tRNAf161 from wheat germ contains a yet unidentified
modification at guanosine 64 [7]. Reversed phase HPLC
Table 1: Monocyclic Edman degradation of the translation products from in vitro translation either
in rabbit reticulocyte lysate, or wheat germ extract. The amount of the thiohydantoin derivative
of [35S]methionine indicates aminoterminally attached [35S]methionine. The quantity of
[35S]methionine in the remaining polypeptide indicates internally bound [35S]methionine. Data are
given in fmol.
tRNA
rabbit reticulocyte lysate
internal
aminoterminal
[35S]Met
[35S]Met
wheat germ extract*
internal
aminoterminal
[35S]Met
[35S]Met
tRNA"cl(yeast)
tRNA^oxXyeast)
A"ete t(wheat g e r m)
tRNA"
tRNA,Me'(ox)(wheat germ)
730
4450
525
2195
n.d.
n.d.
651
1463
945
1155
520
675
* Translation was performed in the presence of 65 pM methionine
n.d.
n.d.
993
946
4680 Nucleic Acids Research, Vol. 18, No. 16
nucleoside analysis of this tRNA shows an unknown elution peak
having a typical guanosine UV-absorption spectrum X^,, = 227
11111
\nax = 254 run (figure 2). As in the case with yeast, this
elution peak disappears after periodate treatment of wheat germ
tRNAj"el. Therefore, similar to A*^ of yeast tRNA|"e\ G*^ in
wheat germ tRNAf1" appears to carry a 2'-ribosyl residue. Like
the A*^ modification of yeast tRNAfet, the modification of
G*64 also contains a phosphate group. This conclusion is based
on experiments in which wheat germ tRNA[*et was treated with
alkaline phosphatase and subsequently digested by a mixture of
RNase Tl and RNase A to yield nucleoside-3'-phosphates.
Comparison of the nucleotide composition, as determined by ion
exchange HPLC [20] of native and phosphatase-treated wheat
germ tRNA,Mel, indicates the presence of an additional phosphate
group on an elution peak corresponding to G*M. This fraction
was identified as a guanosine derivative by UV-spectroscopy [data
not shown]. These results point to a structure of the modified
guanosine 64 which is closely related to the modification of
adenosine 64 in yeast tRNAfet [8]. Nucleoside and nucleotide
analyses are compatible with a 5-phosphorylated ribose moiety
which is attached to the 2'-hydroxyl group of guanosine 64 via
an O-glycosidic bond.
6000
Activity of yeast and wheat germ tRNA^16' in rabbit
reticulocyte translation system
The in vitro translation of globin mRNA in rabbit reticulocyte
lysate in the presence of yeast [35S]Met-tRNAiMet(ox), which
lacks the adenosine-64 phosphoribosylation was compared to
that with native yeast [35S]Met-tRNAiMct. The results show a
significantly enhanced incorporation of [35S]methionine into the
formed polypeptides for the translation system with the oxidized
analogue [Fig. 3]. To exclude the possibility that the observed
enhanced incorporation of [35S]methionine is due to deacylation
of [35S]Met-tRNAf4ct(ox) and reattachment of [35S]methionine to
elongator tRNAmMct, the reaction was performed in the presence
of a 100-fold molar excess of free unlabeled methionine over
[35S]Met-tRNA[*et.
In order to distinguish between an enhanced amino-terminal
insertion of [35S]methionine by tRNA;Met(ox) and incorporation
of methionine into internal methionine positions of the globine,
the translation products were isolated and submitted to monocyclic
Edman degradation. The results, shown in table 1, indicate that
internal AUG codons are read by eucaryotic initiator tRNA if
the modification at position 64 has been chemically removed by
oxidative elimination of the ribose moiety. However, the oxidative
removal of the ribose from position 64 does not interfere with
initiation, suggesting that the modification is not required for this
process.
The outlined experiments suggest that the modification at
adenosine 64 in yeast tRNA|"et prevents the binding of MettRNAf1" to one essential component of the elongation apparatus.
In order to investigate the effect of the adenosine-64 modification
in more detail, we selectively removed the 5"-phosphate group
of A*^. This was performed by treatment of yeast tRNA,Mel
with alkaline phosphatase, yielding tRNA,MeI(phos), which could
be aminoacylated up to 1457 pmol [35S]methionine per A260
unit. In vitro translation of globin mRNA in rabbit reticulocyte
Table 2: Equilibrium dissociation constants of EF-Tu • GTP • aa-tRNA ternary
complexes, determined as described [18] using EF-TuGTP from E.coli.
incubation time [mln]
Fig.3: a) In vitro translation in rabbit reticulocyte lysate in the presence of either
0.5 fiM [35S]Met-tRNAiMe' ( - A - ) , [35S]Met-tRNA,Mel(ox) ( - • - ) or
["SlMet-tRNA^phos) ( - • - ) .
tRNA
KDxl0"8M
tRNAjMeir- ••
Met
tRNA^'yeast
tRNAVal£.co//
tRNA^CI(ox)yeast
11
60
0.5
1.1
3000
Table 3: Nucleosides occupying position 64 in different initiator tRNAsMcl. Data
taken from [7].
10
16
20
86
Incubation time [mln]
Rg.4: In vitro translation in wheat germ extract with mRNA from tobacco mosaic
virus in the presence of either 0.5 pM [35S]-Met-tRNAflel ( - • - ) or 0.5 pM
[35S]Met-tRNA^"(ox) ( - • - ) from wheat germ.
tRNA
species
amount
of sequences
position 64
occupied by
modification
eubacteria
archaebact.
14
6
C
G
no
no
13
U,C
no
11
g
G,A
G
no
no
10
C,U
no
13
G,A
yes
mitochondria:
chordata and
achordata
plants and
fungi
chloroplasts
cytoplasma:
chordata and
achordata
plants and
fungi
Nucleic Acids Research, Vol. 18, No. 16 4681
lysate in the presence of [35S]Met-tRNAfe'(phos) has only a
slight effect on [35S]methionine incorporation into the translation
products (figure 3).
The activities of tRNAf" and tRNAf "(ox) from wheat germ
in the same translation assay are shown in Table 1. If translation
is performed in the presence of [35S]Met-tRNA;Mel(ox) the
insertion of [35S]methionine into internal positions of a/f3
globine was observed. Wheat germ [35S]Met-tRNAfel in the
presence of native. Only little incorporation of [35S]methionine
into the translation products occured.
Activity of wheat germ tRNAfel in wheat germ translation
system
The activities of [35S]Met-tRNA,MeI and [35S]Met-tRNAf"(ox)
were tested in the homologous wheat germ in vitro translation
system using RNA from tobacco mosaic virus as mRNA [17].
The insertion of [35S]methionine into the formed polypeptides
increases by approximately 100% if in vitro translation is carried
out in the presence of [35S]Met-tRNAf el(ox) rather than native
[35S] Met-tRNAfet (figure 4). Monocyclic Edman degradation
confirmed the increased methionine incorporation into internal
positions of the polypeptide (table 1), indicating a reading of
internal AUG codons by wheat germ tRNAf" which lacks the
modification at guanosine 64.
Formation of EF-Tu'GTP*aa-tRNA ternary complexes of
Met-tRNAf" and Met-tRNAf et(ox) from yeast
To explore the possibility that the modified adenosine 64 in yeast
tRNAf" acts as a steric rejection signal to prevent EF-la GTP
from binding to Met-tRNAf", the equilibrium dissociation
constants of the bacterial EF-Tu-GTP interaction with MettRNAf" and Met-tRNAf el(ox) were determined. Elongation
factor Tu from E. coli was used in these studies since no method
for the determination of dissociation constants of ternary
complexes using the eucaryotic elongation factors exists.
However, both factors have analogous function in protein
biosynthesis [21], and all aminoacyl-tRNAs tested thus far form
ternary complexes with bacterial EF-Tu. The mechanism of
binding aminoacyl-tRNA to eucaryotic EF-la-GTP and to
procaryotic EF-Tu-GTP may therefore be similar.
The results of the measurements are summarized in table 2.
Yeast Met-tRNAf "(ox) binds with a relatively high affinity to
the procaryotic elongation factor Tu, whereas the native MettRNAf1" from yeast with modified adenosine 64 is considerably
less efficient in this interaction. This is similar to the situation
in E.coli where the unformylated Met-tRNAfMet binds
approximately 50 fold more efficiently to procaryotic EFTu-GTP than the formylated species [22]. However, the
dissociation constant for yeast Met-tRNAf" (ox) (1.1X 10~8 M)
is in the same order of magnitude as for Val-tRNAVal
(0.5 x 10~8 M), a weakly binding E.coli elongator tRNA [23].
DISCUSSION
This work provides evidence for the enhanced participation of
initiator Met-tRNAsf" from yeast and wheat germ in the in
vitro protein elongation reaction after the excission of the
2'-phosphoribosylation on purine-64 of these tRNAs. Since the
molecular basis for the discrimination of initiator Met-tRNAf"
from the elongation process in eucaryotic protein biosynthesis
is not yet understood, it is reasonable to ask whether the unusual
bulky modification at purine-64 of these tRNAs has some function
in this process.
The base pair 50-64 is located near the junction of the
aminoacyl- and T-stems in the tRNA tertiary structure and is
important for the formation of the continuous stack between these
stems. This 'aminoacyl domain' is a structural element recognized
by some tRNA-interacting proteins. Alanine-tRNA synthetase,
for example, aminoacylates a minihelix^3 consisting of the
'aminoacyl domain' but missing the D-,variable- and anticodonstems and loops of E.coli tRNA^11 [24]. RNase P requires this
minimal structure for recognition and processing of the 5'-end
of tRNA precursors [25]. The aminoacylated 'aminoacyl domain'
of tRNA is also the minimal structure required for interaction
with the bacterial elongation factor Tu [9]. The
phosphoribosylation of residue 64 adds a bulky hydrophilic group
to the surface of this part of the tRNA.
In this work we use an E.coli EF-Tu as a model for EF-la
for two reasons. First, eucaryotic and procaryotic protein
elongation factors l a and Tu, respectively, show strong stuctural
and functional similarities [22]; secondly, an assay for aa-tRNA
interaction with EF-la is not available. We could demonstrate
that the phosphoribosylation at A*^ of yeast tRNAf" strongly
affects the interaction with bacterial EF-Tu. This observation,
however, must be verified for the eucaryotic EF-la. It is likely
that, in analogy with the procaryotic system, the removal of the
phosphoribosyl residue from A*^ will lead to enhanced MettRNAf". EF-la-GTP ternary complex formation and, in turn,
to a participation of initiator Met-tRNAf" in the protein
elongation cycle. The results of our in vitro translation
experiments offer evidence to support this hypothesis. Regardless
whether we use a heterologous (yeast tRNAf", globine mRNA,
reticulocyte lysate) or homologuos system (wheat germ
tRNAfet, TMV-RNA, wheat germ lysate), the chemical scission
of the phosphoribosyl residue from the initiator tRNA leads to
a markedly increased incorporation of methionine into internal
positions of the polypeptide. Thus the phosphoribosylation of
nucleoside 64 seems to prevent the yeast and wheat germ initiator
Met-tRNAs from participating in the elongation cycle.
The 2'-phosphoribosylation is probably a general feature of
cytoplasmic tRNAsf" in plants and fungi (table 3) and we
suggest that it may control the discrimination between their
initiator versus elongator function. A similar role was identified
for the unpaired residues 1 and 72 in bacterial initiator tRNAs
[3—5]. Whether this modification of initiator tRNAs in the
cytoplasm of plants and fungi plays a regulatory role during
translation is presently being studied in our laboratory.
ACKNOWLEDGEMENT
We thank Prof. P.Sigler for helpful discussions and suggestions.
This work was supported by the Deutsche Forschungsgemeinschaft, SFB 213, D5 and a NATO grant for scientific exchange
No. 870485.
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