Proc. Natl. Acad. Sci. USA
Vol. 96, pp. 875–880, February 1999
Biochemistry
Induced fit of a peptide loop of methionyl-tRNA
formyltransferase triggered by the initiator tRNA substrate
(RNA–protein interactions兾initiator tRNA formylation兾RNA footprinting兾protein conformational change)
VAIDYANATHAN RAMESH*, CHRISTINE MAYER*, MICHAEL R. DYSON, SADANAND GITE,
AND UTTAM L. RAJBHANDARY†
Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139
Communicated by Alexander Rich, Massachusetts Institute of Technology, Cambridge, MA, December 1, 1998 (received for review
November 2, 1998)
The crystal structure of E. coli MTF shows that the enzyme
consists of two domains connected by a linker region (refs.
13–16; Fig. 1). Cross-linking studies, mutational analysis, and
binding studies support the notion that the C-terminal domain,
although essential, is involved mostly in nonspecific interactions with the tRNA (refs. 13 and 17; S.G., Y. Li, V.R., and
U.L.R., unpublished results). The N-terminal domain is strikingly homologous in structure to E. coli glycinamide ribonucleotide formyltransferase (GARF), which, like MTF, also
uses N10-formyltetrahydrofolate as a formyl donor in formylation (13–15). A notable difference, however, is that the
N-terminal domain of MTF contains a highly conserved 16-aa
insertion sequence (residues 34–49 in the E. coli enzyme) in
the loop region between the second ␤-strand and second
␣-helix (Fig. 1). Suppressor mutants (18) in the E. coli MTF,
which compensate for a formylation-defective mutant initiator
tRNA fall within this insertion loop. Mutational analysis of the
conserved amino acid residues within the insertion sequence
indicates an important role for an invariant arginine, Arg-42,
in both substrate binding and catalysis (19). Further characterization of the arginine mutants suggested interactions between the invariant arginine and the determinants for formylation in the acceptor stem of initiator tRNA (19). These
results of suppressor and mutational analysis suggest strongly
that the 16-aa insertion sequence in MTF acts as a recognition
module for specific interactions with the determinants for
formylation in the acceptor stem of the initiator tRNA.
A segment of the insertion loop, 40AGRGKK45, spanning
the critical arginine (13), is unstructured in the crystal structure of the native enzyme and is uniquely susceptible to
proteolysis in MTF. Here we have used two types of experiments—protection of MTF against proteases and protection of
tRNA against nucleases—to study the specific interactions
between MTF and the initiator Met-tRNA.‡ The initiator
Met-tRNA specifically protects the enzyme against proteolytic
cleavage within the insertion loop. By using Arg-42 mutant
enzymes and mutant initiator tRNAs in protection experiments we show that the formation of a functional MTF䡠MettRNA (enzyme–substrate) complex is necessary for this protection. These results support the idea that a segment of the
recognition module in MTF, which is unstructured and accessible to proteases in the free enzyme, adopts a defined
conformation in the enzyme–substrate complex. Footprint
experiments (20) using RNase V1, T2, and T1 also suggest that
the recognition module protects mostly the acceptor stem of
the initiator Met-tRNA, which contains the critical determi-
ABSTRACT
A 16-aa insertion loop present in eubacterial
methionyl-tRNA formyltransferases (MTF) is critical for specific recognition of the initiator tRNA in Escherichia coli. We
have studied the interactions between this region of the E. coli
enzyme and initiator methionyl-tRNA (Met-tRNA) by using
two complementary protection experiments: protection of
MTF against proteolytic cleavage by tRNA and protection of
tRNA against nucleolytic cleavage by MTF. The insertion loop
in MTF is uniquely sensitive to cleavage by trypsin. We show
that the substrate initiator Met-tRNA protects MTF against
trypsin cleavage, whereas a formylation-defective mutant
initiator Met-tRNA, which binds to MTF with approximately
the same affinity, does not. Also, mutants of MTF within the
insertion loop (which are defective in formylation) are not
protected by the initiator Met-tRNA. Thus, a functional
enzyme–substrate complex is necessary for protection of MTF
against trypsin cleavage. Along with other data, these results
strongly suggest that a segment of the insertion loop, which is
exposed and unstructured in MTF, undergoes an induced fit
in the functional MTF䡠Met-tRNA complex but not in the
nonfunctional one. Footprinting experiments show that MTF
specifically protects the acceptor stem and the 3ⴕ-end region
of the initiator Met-tRNA against cleavage by double and
single strand-specific nucleases. This protection also depends
on formation of a functional MTF䡠Met-tRNA complex. Thus,
the insertion loop interacts mostly with the acceptor stem of
the initiator Met-tRNA, which contains the critical determinants for formylation.
Formylation of the initiator Met-tRNA (Met-tRNAfMet) by the
enzyme methionyl-tRNA formyltransferase (MTF) is important for the initiation of protein synthesis in eubacteria,
mitochondria, and chloroplasts (1–3). Escherichia coli initiator
tRNA mutants defective in formylation are essentially inactive
in initiation (4). Similarly, a strain of E. coli having a disruption
in the formylase gene grows extremely slowly (5). The formyl
group on the initiator methionyl-tRNA facilitates its binding to
the initiation factor IF2, which is necessary for the binding of
the tRNA to the ribosomal P site during initiation of protein
synthesis (6). MTF is highly specific in that it formylates only
the initiator Met-tRNA and no other aminoacyl-tRNA including the elongator methionyl-tRNA (7, 8). The most important
sequence and structural elements for formylation are clustered
in the acceptor stem of E. coli initiator tRNA (9–12). The
absence of a Watson-Crick base pair between positions 1 and
72, a unique feature of all eubacterial initiator tRNAs, is one
of the critical determinants.
Abbreviations: MTF, methionyl-tRNA formyltransferases; GARF,
glycinamide ribonucleotide formyltransferase.
*V.R. and C.M. contributed equally to this work.
†To whom reprint requests should be addressed. e-mail: bhandary@
wccf.mit.edu.
‡The initiator tRNAfMet species was used throughout this work.
2
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PNAS is available online at www.pnas.org.
875
876
Biochemistry: Ramesh et al.
FIG. 1. Schematic alignment of GARF and MTF sequences from
various sources. The amino acid numbering of MTF begins with serine
found at the N terminus of the E. coli protein. The amino acids Asn,
His, Ser/Thr, and Asp [thought to be involved in catalysis in GARF
(14–16) and also found in MTF] are indicated. Arrows indicate sites
of insertion in MTF compared with GARF.
nants for formylation, in the functional, but not in the nonfunctional, complex.
MATERIALS AND METHODS
Strains, Plasmids, and Enzymes. The cloning and expression of wild-type and mutant MTFs and MetRS as C-terminal
6⫻His fusion proteins has been described (18, 19). The
purified enzymes were stored in 20 mM Imidazole䡠HCl (pH
7.6), 150 mM NaCl, 10 mM 2-mercaptoethanol, and 50%
glycerol. E. coli UT481 containing the plasmid pEC7 and
overexpressing tRNA nucleotidyl transferase was provided by
M. P. Deutscher (University of Miami School of Medicine).
The enzyme was purified as described in ref. 21. RNase V1
(Cobra venom, 0.72 units兾␮l) was from Pharmacia Biotech and
was stored in 50% glycerol at ⫺20°C. RNases T2 and T1
(Sankyo, 10 units兾␮l) were from Calbiochem and were stored
at 4°C.
Preparation of Initiator tRNA Containing 3ⴕ-Amino-3ⴕDeoxyA (tRNAN) at the 3ⴕ End. The cloned wild-type and G72
mutant initiator tRNAs were purified from E. coli B by
electrophoresis of total tRNA on a 15% native polyacrylamide
gel (22). The purity of tRNAs, assessed by aminoacylation
assays, was ⬎95%. The 3⬘-terminal A of tRNA was exchanged
for 3⬘-amino-3⬘-deoxyA by using tRNA nucleotidyl transferase. The incubation mixture (20 ␮l) for this contained 50
mM Tris䡠HCl (pH 8.0), 10 mM MgCl2, 10 mM reduced
glutathione, 0.5 mM CTP, 4.8 mM 3⬘-amino-3⬘-deoxyATP
(Fluka), 1 mM sodium pyrophosphate, 1 A260 unit of initiator
tRNA, and 2 units of tRNA nucleotidyl transferase. Incubation
was at 37°C for 2 hr. After the incubation, the tRNA was
separated from the excess of 3⬘-amino-3⬘-deoxyATP by
DEAE-cellulose chromatography and recovered by ethanol
precipitation. The purified initiator tRNA analog (tRNAN)
could be quantitatively aminoacylated to yield [35S]MettRNAN in which the [35S]methionine is linked to the tRNA by
a base-stable peptide linkage. When using nonradioactive
methionine, the extent of aminoacylation of the initiator
tRNAN was monitored by silver staining of 15% polyacrylamide gels. The aminoacyl-tRNAN migrated slower than
tRNAN, and aminoacylation was essentially complete.
Preparative Aminoacylation of Initiator tRNAN with [35S]LMethionine. tRNAN (1.2 ␮M) was incubated in the aminoacylation buffer containing 250 ng of MetRS and 10 ␮M [35S]Lmethionine (1 ⫻ 106 cpm per pmol) at 37°C for 25 min. The
[35S]Met-tRNAN was purified by electrophoresis on a 12%
polyacrylamide–8M urea gel, located by autoradiography, and
eluted into a buffer containing 0.3 M sodium acetate (pH 5.5)
and 1 mM EDTA for 2–5 hr at 20°C. The tRNA was recovered
by ethanol precipitation and dissolved in distilled water to
approximately 30,000 cpm per microliter.
Proc. Natl. Acad. Sci. USA 96 (1999)
Expression and Purification of [35S]Methionine-Labeled
MTFs. Cultures of E. coli JM109兾pQE16FMTp (or the appropriate MTF mutant) were grown to mid-logarithmic phase
in 100 ml of 2⫻YT (1% yeast extract兾1.6% tryptone兾0.5%
NaCl) medium. The cells were collected, washed with 0.1 M
KH2PO4 (pH 7.4), and suspended in 50 ml of Dulbecco’s
Modified Eagle Medium without glutamine, methionine, and
cystine (ICN). The culture was then supplemented with glutamine (1 mM), ampicillin (100 ␮g兾ml), [35S]L-methionine [1
mCi (1 Ci ⫽ 37 GBq); 1,175 Ci兾mmole, NEN] and isopropyl
␤-D-thiogalactoside (IPTG) (1 mM) and grown for 3 hr at 37°C
(23). The [35S]methionine-labeled MTF was purified by using
Talon Sepharose affinity chromatography and was diluted with
the corresponding nonradioactive MTF or mutant MTF to a
specific activity of 86 ⫻ 103 cpm per microgram.
Proteolysis of MTF. Protease digestions were performed in
the standard buffer used for aminoacylation and formylation
containing 20 mM imidazole䡠HCl (pH 7.6), 150 mM NH4Cl, 2
mM ATP, 0.1 mM Na2EDTA, and 10 mM MgCl2 at 37°C with
trypsin (Promega; modified trypsin, 21, 500 units兾mg). The
reaction (10 ␮l) consisting of [35S]methionine-labeled MTF
(0.5 ␮M), wild-type or the G72 mutant initiator tRNAN
(1.25–80 ␮M), and MetRS (200 ng) was incubated in the
presence or absence of 1 mM methionine in the aminoacylation buffer for 15 min at 37°C. Trypsin (1:3.3 wt兾wt ratio of
trypsin to protein) was added and the mixture was incubated
for 10 min at 37°C. The reaction was stopped by the addition
of SDS buffer (24) and freezing in dry ice. The samples were
analyzed on 12% SDS兾PAGE gels, and the gels were dried and
subjected to autoradiography. The radioactivity was quantitated by phosphorimaging (Molecular Dynamics).
The G34C36 mutant initiator tRNA was purified and aminoacylated with valine by using a ValRS-enriched extract (25).
The Val-tRNA (G34C36) was recovered by using phenol
extraction and ethanol precipitation and was used for protease
digestion as described above.
Gel Mobility Shift Analysis of MTF䡠Met-tRNAN Complexes.
Binding reactions (10 ␮ l) were performed in 20 mM
imidazole䡠HCl (pH 7.6), 5 mM MgCl2, 150 mM NaCl, and 5%
glycerol. The mixture was incubated at room temperature for
15 min, and the complexes were resolved on a 6% native
polyacrylamide gel at 150 V for 2 hr. The gels were fixed and
dried, and the radioactivity was quantitated by phosphorimaging.
RNase V1, T2, and T1 Footprinting. The initiator MettRNAN, labeled either at the 5⬘ end with [32P] or at the 3⬘ end
with [35S]Met, (0.02 ␮M, ⬇40,000 cpm) was preincubated in a
buffer containing 20 mM imidazole䡠HCl, (pH 7.6), 150 mM
NH4Cl, and 5 mM MgCl2 for 10 min at 55°C and slowly cooled
to 20°C. MTF (0.09–18 or 27 ␮M) was added and the mixture
(10 ␮l) was left at 20°C for 10 min; 0.1 ␮g of yeast tRNAPhe and
either RNase V1 (0.005 units), RNase T2 (0.002 units), or
RNase T1 (0.01 units) was added, and the mixture was
incubated at 20°C. After 10 min, 0.3 M sodium acetate (pH 5.5;
90 ␮l) and total E. coli tRNA (2 ␮g) was added. The mixture
was extracted with an equal volume of phenol兾chloroform, an
additional 2 ␮g of carrier tRNA was added to the aqueous
layer, and tRNA fragments were recovered by ethanol precipitation on dry ice. The pellet was washed once in 80%
ethanol and dissolved in 6 ␮l of 8 M urea containing 0.03%
(wt兾vol) bromophenol blue and xylene cyanole and analyzed
on 18% polyacrylamide兾7 M urea sequencing gels. Products of
partial cleavage with alkali and RNase T1 were used as size
markers to identify the tRNA bands (26). The gel was fixed,
dried on a DEAE-cellulose paper, and subjected to autoradiography. Radioactivity in the bands on each lane was quantitated by phosphorimaging (Molecular Dynamics).
RESULTS
Susceptibility of the Insertion Loop in MTF to Proteolytic
Cleavage. Several lines of evidence indicate that the sequence
40AGRGKK45 within the insertion loop of MTF is uniquely
Biochemistry: Ramesh et al.
sensitive to proteases. First, attempts to purify MTF overproduced in E. coli by factor Xa cleavage of a maltose-binding
protein fused to MTF yielded a truncated MTF protein that
had been cleaved after Arg-42 within the insertion loop (D.
Mangroo, S.G., and U.L.R., unpublished data). This truncated
MTF is inactive in formylation. Second, suppressor mutants of
MTF with Gly-41 changed to Arg-41 or Lys-41 were found to
be partially cleaved in E. coli after Arg-41 or Lys-41 (18). Third,
the crystal structure of MTF (13) shows that amino acids
40–45 are disordered. Fourth, limited treatment of MTF with
trypsin (13) or with clostripain, an arginine-specific protease
(data not shown), resulted in a single cleavage after Arg-42.
Interestingly, the R42L mutant is not cleaved by clostripain
(data not shown), suggesting that Arg-38, the other arginine in
the insertion loop, is structured (13) and is not accessible to
cleavage. The R42L mutant is, however, susceptible to cleavage with trypsin after Lys-44 and Lys-45 (data not shown),
suggesting that these residues are, as indicated in the crystal
structure (13), also unstructured and therefore accessible to
cleavage by trypsin.
Protection of MTF Against Trypsin Cleavage by tRNA. We
showed previously that the R42L mutant of MTF was much
less active in formylation. Interestingly, the G41R兾R42L double mutant also was equally inactive, suggesting that the
requirements for arginine at position 42 cannot be fulfilled by
an arginine at position 41 (19). This result suggested that
amino acids 40–45 in the insertion loop, normally unstructured
and flexible, adopt a defined conformation upon binding to the
tRNA. Here, we have used protection against cleavage with
trypsin to probe for conformational changes in the insertion
loop on binding of the enzyme to the initiator Met-tRNA.
Because of the chemical instability of the ester linkage between methionine and tRNA in Met-tRNA, an initiator tRNA
analog, tRNAN, in which the 3⬘-terminal A residue is replaced
by 3⬘-amino-3⬘-deoxyA, was used (27). Upon aminoacylation
of tRNAN with MetRS, the methionine, which is initially
attached to the 2⬘-OH group through an ester linkage, migrates to the 3⬘ position and forms a base stable peptide
linkage (28). MTF labeled in vivo with [35S]Met was used to
follow the extent of cleavage of MTF with trypsin.
The extent of cleavage of MTF was monitored at a fixed
concentration of MTF (0.5 ␮M) and increasing concentrations
of tRNAN or Met-tRNAN (Fig. 2A). Increasing amounts of
tRNAN up to a 16-fold molar excess over MTF had little effect
on the extent of cleavage (Fig. 2 A, lanes 2, 4, 6, and 8) over
that, 48.7%, seen in the absence of any added tRNA (Fig. 2 A,
lane 10 and Right). In contrast, addition of Met-tRNAN
protected MTF against cleavage by trypsin in a concentrationdependent manner (Fig. 2 A, lanes 3, 5, 7, and 9 and Right). At
the highest concentration of Met-tRNAN used here (8 ␮M),
there was ⬎80% protection of MTF against trypsin cleavage
compared with the cleavage seen in presence of the uncharged
tRNAN (Fig. 2 A, lane 9 and Right).
In contrast to the protection against trypsin cleavage of MTF
by Met-tRNAN, there was virtually no protection (Fig. 2B) by
the G72 mutant initiator tRNAN (G72) or Met-tRNAN (G72).
The G72 mutant initiator tRNA, which contains a C1:G72 base
pair at the end of the acceptor stem, is a very poor substrate
for MTF both in vitro (9–11) and in vivo (12). Protection
against cleavage was marginal at best even at the highest
concentration (80 ␮M) of Met-tRNAN (G72), corresponding
to a 160-fold molar excess of tRNA over MTF (Fig. 2B, Right).
The G72 mutant Met-tRNAN, however, forms a complex with
MTF with about the same affinity as wild-type Met-tRNAN
(Table 1). These results suggest that formation of a complex
alone between MTF and the G72 mutant Met-tRNAN is not
sufficient for tRNA-dependent protection of MTF against
cleavage within the insertion loop by trypsin. A functional
MTF䡠Met-tRNAN complex is necessary for the protection.
Proc. Natl. Acad. Sci. USA 96 (1999)
877
FIG. 2. Effect of tRNA, uncharged (䊐) or aminoacylated (■), on
partial trypsin digestion of 35S-labeled MTF. Left panels show SDS-gel
electrophoretic analysis of the incubation mixture in the presence of
increasing amounts of initiator tRNAN or Met-tRNAN. (A) Wild-type
initiator tRNAN. (B) The G72 mutant initiator tRNAN. ⴱ indicates the
position of the long C-terminal MTF fragment, the radioactive band
at the bottom of the gel is the corresponding N-terminal fragment.
Percent cleavage in each lane was determined by adding the radioactivity in the fragments divided by the total radioactivity in that lane
based on phosphorimager analysis. (Right) Results of quantitation.
Effect of tRNAN and Met-tRNAN on Trypsin Cleavage of
Mutant MTFs. In view of the results above with the formylation-defective G72 mutant initiator tRNA, we examined
whether MTF mutants that are defective in enzyme function
would be protected against trypsin cleavage by wild-type
initiator Met-tRNA. The results summarized in Fig. 3 show
that the R42K mutant is protected to a small extent by
Met-tRNAN but only at very high concentrations (20 ␮M and
40 ␮M), whereas there was virtually no protection of the
Table 1. Apparent K ds of wild-type and mutant
MTF䡠Met-tRNAN complexes
Apparent Kd (␮M)
MTF
Wild-type tRNA
G72 mutant tRNA
Wild type
.45†
.32†
.4
4.16
0.5
—
R42K
R42L
Data are based on results of gel mobility shift analyses (29). The
binding reactions contained either the wild-type MTF (2.5 ␮M), the
R42K mutant (2.5 ␮M), or the R42L mutant (5 ␮M) and increasing
amounts of Met-tRNAN, wild-type, or the G72 mutant (0.25 ␮M to 1.5
␮M). The apparent K d values were calculated from the slope of a
double reciprocal plot of 1/r, where r is the fraction of MTF bound to
Met-tRNAN against 1/Met-tRNAN free using the equation 1/r ⫽ K d
(1/Met-tRNAN free) ⫹ 1 (30, 31). The apparent K d measured here for
the His-tagged wild-type MTF䡠Met-tRNAN complex using gel mobility
shift experiments is about the same as the K d reported for MTF䡠MettRNA complex based on quenching of tryptophan fluorescence of
MTF (32).
†Results of separate set of experiments.
878
Biochemistry: Ramesh et al.
FIG. 3. Effect of tRNA, uncharged (䊐) or aminoacylated (■), on
partial trypsin digestion of [35S]Met-labeled mutant MTFs. (A) The
R42K mutant MTF. (B) The G41R兾R42L mutant MTF. Percent
cleavage was calculated as described in the Legend of Fig. 2.
G41R兾R42L mutant even at the highest concentration (40
␮M) of Met-tRNAN. The R42K mutant of MTF forms a
complex with Met-tRNAN with about the same affinity as
wild-type MTF (Table 1). These results further support the
conclusion above that the formation of a functional MTF䡠MettRNAN complex is necessary for protection of MTF against
trypsin cleavage.
Effect of Mutant Initiator tRNA Aminoacylated with Valine
on Protection Against Trypsin Cleavage. The amino acid
attached to the initiator tRNA is also important for formylation of tRNA by MTF (25, 33–35). Previous studies have shown
that E. coli initiator tRNA that had been ‘‘misaminoacylated’’
with valine is a poor substrate for formylation, Vmax down
57-fold and Km up 2.2-fold (33). Therefore, we have investigated whether the G34C36 anticodon mutant of E. coli initiator tRNA, which is aminoacylated with valine (25), protects
MTF against trypsin cleavage. Because the ester linkage in
Val-tRNA is stable under the conditions of incubation (36, 37),
the G34C36 mutant initiator Val-tRNA was used as such
without exchange of the 3⬘-terminal A for 3⬘-deoxy-3⬘-amino
A. There was no protection of MTF against trypsin cleavage
even at the highest concentration (40 ␮M) of the G34C36
mutant Val-tRNA (data not shown).
Protection of Met-tRNAN by MTF Against RNases V1, T2,
and T1 Cleavages. The most important determinants for
formylation in the initiator tRNA are clustered toward the end
of the acceptor stem (10–12). A minor determinant is the
Proc. Natl. Acad. Sci. USA 96 (1999)
A11:U24 base pair in the D stem (10, 38). It was, therefore, of
interest to see whether the interaction of MTF with the
initiator tRNA is localized to these regions or extends to the
rest of the tRNA. This was studied by analyzing regions of the
tRNA protected by MTF against cleavages by the double
strand-specific RNase V1 and the single strand-specific
RNases T2 and T1 (20). Besides Met-tRNAN and the nuclease,
the incubation mixture contained either no MTF or increasing
amounts of MTF from 0.09 ␮M to 18 or 27 ␮M. To ensure that
the cleavages being monitored were caused by primary hits, the
Met-tRNAN was labeled either at the 3⬘ end with [35S]Met or
at the 5⬘ end with 32P. The results obtained with either labeled
tRNA were essentially the same. Fig. 4A (Left) shows the
results of a gel electrophoretic analysis of limited digests of
[35S]Met-tRNAN with RNase V1. The cleavage sites are numbered according to the phosphate, with the 5⬘-terminal phosphate being number 1. It can be seen that MTF protects the 3⬘
end of the acceptor stem at phosphates 67–71 in a concentration-dependent manner. At 0.09 ␮M MTF, there was already
50% protection of cleavage at phosphate 68 (Fig. 4A Left),
whereas at 18 ␮M MTF, there was ⬎90% protection of
cleavage at phosphates 67–71 (Fig. 4B Upper). Some other sites
showed enhanced cleavage at phosphates 30, 43, and 44 in the
anticodon stem and at phosphate 53 in the T␺C stem whereas
others showed slightly reduced cleavage at phosphates 29 in the
anticodon stem and phosphate 52 in the T␺C stem. These are
most likely the result of structural changes in the tRNA
induced by MTF. Similar enhancements of RNase V1 cleavage
in the anticodon stem have been noted before in complexes of
EF-Tu with aminoacyl-tRNA and IF2 with fMet-tRNA
(39, 40).
In contrast to protection against RNase V1 cleavage of
Met-tRNAN by MTF, there was only very weak protection of
the G72 mutant initiator tRNA, Met-tRNAN (G72), at phosphates 67 and 68 and none at phosphates 69, 70, and 71 in the
acceptor stem. (Fig. 4A Right and Fig. 4B Lower). Because the
G72 mutant Met-tRNAN forms a complex with MTF with
about the same affinity as wild-type Met-tRNAN (Table 1), this
result suggests that MTF dependent protection of the initiator
Met-tRNAN against RNase V1 cleavage also requires the
formation of a functional MTF䡠Met-tRNAN complex.
RNase T2 cleaved Met-tRNAN predominantly at the end of
the acceptor stem at phosphates 73–76 (Fig. 4C) and to some
FIG. 4. (A) Electrophoretic analysis of partial RNase V1 cleavage of 3⬘-end [35S]Met-labeled initiator Met-tRNAN and Met-tRNAN (G72) on
polyacrylamide gels. Met-tRNAN (lanes 4–10) and the G72 mutant Met-tRNAN (lanes 11–17) were incubated with increasing amounts of MTF
(0, 0.09, 0.3, 0.9, 3, 9, and 18 ␮M) and subjected to partial digestion with RNase V1. Lane 1, OH, alkaline ladder; lane 2, T1, RNase T1 digest
under denaturing conditions; lane 3, C, control without RNase VI treatment. (B) Quantitation of RNase VI cleavages in A by phosphorimager
analysis. The amount of tRNA cut at a given position (67–71) is plotted as a percentage of total tRNA loaded per lane. Open bars, cleavage of
Met-tRNAN, hatched bars, cleavage in the presence of 18 ␮M MTF. (C) Upper, electrophoretic analysis (on a 15% polyacrylamide gel) of a partial
RNase T2 digest of 5⬘-end 32P-labeled Met-tRNAN. The tRNA was bound to increasing amounts of MTF (0.09–9 ␮M), and the complexes were
analyzed by RNase T2 digestion; Lower, quantitation of RNase T2 footprinting by phosphorimager analysis.
Biochemistry: Ramesh et al.
extent in the D loop, the anticodon loop, and the variable loop
(data not shown). Because of the very short size of the
3⬘-terminal fragment(s) resulting from cleavages at phosphates
73–76, only 5⬘ 32P-labeled Met-tRNAN was used in the RNase
T2 cleavage experiments. MTF protected the cleavage of
tRNA by RNase T2 in the acceptor stem (Fig. 4C) but not in
the D loop, the anticodon loop, or the variable loop (data not
shown).
RNase T1 cleaved Met-tRNAN in the D loop and in the
variable loop. Neither of these regions were protected against
RNase T1 cleavage by MTF (data not shown). Thus, the
combined results on the effect of MTF on cleavage of MettRNAN by RNases V1, T2, and T1 suggest that MTF binds
primarily to the acceptor stem of Met-tRNAN (Fig. 5). There
are no major contacts between MTF and other regions of the
tRNA. We cannot exclude contacts between MTF and the
tRNA in the D stem, because the enzyme probes that we have
used do not cleave within the D stem.
Use of Mutant MTF Proteins in RNase V1 Footprinting of
Met-tRNAN. Three mutant proteins all carrying alterations in
Arg-42, MTF (R42K), MTF (R42L), and MTF (G41R兾R42L)
were also used for RNase V1 footprint analysis of the
MTF䡠Met-tRNA complex. The difference among the mutants
was marginal, although MTF (R42K) was somewhat better
than either MTF (R42L) or MTF (G41R兾R42L) mutants.
Both the MTF (R42K) and MTF (R42L) mutants required at
least 100 times more protein than wild-type MTF (⬎27 ␮M for
the mutants as compared with 0.09 ␮M for the wild-type MTF)
to provide 50% protection of cleavage at phosphate 68 (data
not shown). Although the binding affinity of the R42L mutant
MTF for Met-tRNAN is about 13-fold lower than that of the
wild-type MTF, the R42K enzyme forms a complex with
Met-tRNAN with about the same affinity as wild-type MTF
(Table 1). These data, therefore, support the conclusion that
the interaction of MTF with Met-tRNAN is strongly affected
by the mutation of Arg-42 and that a specific interaction
between the MTF and the acceptor stem of the tRNA is lost
in the mutants (19).
Use of Mutant Initiator tRNA Aminoacylated with Valine in
RNase V1 Footprinting. As mentioned above, initiator tRNA
aminoacylated with valine is a poor substrate for MTF (33).
Therefore, we have performed RNase V1 footprinting of
FIG. 5. Cloverleaf structure of E. coli initiator Met-tRNAN. Nucleotides playing a major role in formylation are boxed by solid lines,
whereas those playing a relatively minor role are boxed by dotted lines.
RNase cleavages that are decreased in the presence of MTF are
indicated with 䊞 whereas those that are increased are indicated with
䊝.
, RNase V1 cleavage;
, RNase T2 cleavage.
Proc. Natl. Acad. Sci. USA 96 (1999)
879
Val-tRNA (G34C36) in the presence of increasing amounts of
MTF. There was no protection against RNase V1 cleavages in
the acceptor stem except perhaps at the highest concentration
(9 ␮M) of MTF (data not shown).
DISCUSSION
Our results on protection of MTF against trypsin cleavage in
the insertion loop by the substrate Met-tRNAN strongly suggest that amino acids 40–45 in this region of MTF (which are
unstructured and flexible in the free enzyme) adopt a defined
structure in a functional (but not in an inactive) MTF䡠MettRNAN complex. The protection requires the formation of a
functional MTF䡠Met-tRNAN complex because the G72 mutant
initiator tRNA, which is a poor substrate for formylation, and
the G34C36 mutant initiator tRNA, which is aminoacylated
with valine, are both ineffective in protection of MTF against
trypsin cleavage (Figs. 2 and 4). Also, MTF with mutations in
the critical arginine in the insertion loop are not protected
against trypsin cleavage by the initiator Met-tRNAN (Fig. 3).
Other studies have shown that the insertion loop is important
for formylation and that amino acids in the insertion loop come
close to and interact with the determinants for formylation in
the acceptor stem of the initiator tRNA. For example, (i) a
single trypsin cleavage within the insertion loop results in an
enzyme that is inactive in formylation (ref. 13, D. Mangroo,
S.G., and U.L.R., unpublished results). (ii) Suppressor mutants
of MTF that formylate partially the G72 mutant initiator
tRNA carry a mutation at amino acid 41 within the insertion
sequence (18, 19). (iii) Mutagenesis experiments show that the
basic amino acids Arg-38 and Arg-42 play important roles in
MTF function and that Arg-42 functionally interacts with base
pairs 3:70 and possibly 2:71 in the acceptor stem of the tRNA
(ref. 19; E. Schmitt, M. Panvert, S. Blanquet, and Y. Mechulam, personal communication). Also, comparison of the kinetic parameters of the double mutant G41R兾R42L with those
of the single mutant R42L show that the requirement for an
arginine at position 42 cannot be fulfilled by having an arginine
at position 41 (19). Therefore, although the protection of MTF
by the initiator Met-tRNAN against trypsin cleavage, could, on
its own, be ascribed to the tRNA shielding an otherwise
unstructured and exposed region of MTF in the MTF䡠MettRNAN complex, the combined evidence suggests very strongly
that amino acids 40–45 of MTF undergo a local conformational change in the MTF䡠Met-tRNAN complex in an inducedfit mechanism (41–44). Such an induced conformational
change would be akin to what happens in the HIV-1 Rev
peptide–RRE (Rev response element) interactions (45) or
bovine immunodeficiency virus Tat peptide–TAR RNA interactions (46, 47). In the latter case, a small arginine-rich
peptide segment, normally unstructured and flexible, undergoes a conformational change to a ␤-hairpin on binding to the
corresponding RNA (48, 49).
As mentioned above, protection of MTF against trypsin
cleavage requires the formation of a functional MTF䡠MettRNAN complex. The G72 mutant Met-tRNAN also forms a
complex with MTF (Table 1), however, there is no protection
of MTF against trypsin cleavage, presumably because the
complex is ‘‘inactive.’’ It is also known that MTF can bind
nonspecifically to other tRNAs with comparable affinities (ref.
32; L. Hancox, D. Mangroo, and U.L.R., unpublished results),
although only the initiator Met-tRNA (or Met-tRNAN) is a
substrate for formylation. The notion of a conformational
change within the insertion loop of MTF, subsequent to
binding, only in a functional MTF䡠Met-tRNAN complex is
reminiscent of the situation with aminoacyl-tRNA
synthetase䡠tRNA complexes, in which a conformational
change is triggered by cognate tRNAs but not by noncognate
tRNAs (ref. 50, D. Moras, personal communication).
880
Biochemistry: Ramesh et al.
The results of RNase footprinting show that MTF protects
the acceptor stem and the 3⬘-terminal nucleotides, but not
other regions of the tRNA. We cannot comment on contacts
between MTF and tRNA in the D stem, because the enzyme
probes we have used do not cleave within the D stem. Thus, the
interaction of MTF to the initiator Met-tRNA is localized to
the region that contains the most important determinants for
formylation. Here, too, the formation of a functional complex
is necessary for protection of the tRNA against nucleases. The
G72 mutant initiator Met-tRNAN has approximately the same
affinity for MTF as wild-type initiator Met-tRNAN (Table 1)
but is not protected against RNase V1 cleavage by MTF. The
finding that MTF mutants with a single amino acid mutation
(R42 to K or L) in the insertion loop do not protect the
Met-tRNAN against RNase cleavage highlights the critical role
of the insertion loop in this process. A likely explanation is that
during the formation of a functional MTF䡠Met-tRNA complex, the tRNA is initially bound to MTF mostly through
nonspecific electrostatic interactions involving the basic amino
acids in the C-terminal region (13) that form a positively
charged channel on the surface of the enzyme. This is followed
by interactions between the amino acids in the insertion loop
and the acceptor stem of the initiator tRNA, accompanied by
changes in the local structure of the insertion loop and possibly
of the tRNA in an induced-fit mechanism.
Note Added in Proof. After the submission of this manuscript, a paper
describing the three-dimensional structure of E. coli MTF complexed
with fMet-tRNA, the product of MTF reaction, was published (51).
Our conclusion that amino acids 40–45 undergo an induced fit in the
presence of the initiator tRNA substrate, based on biochemical
analyses described in here and in previous studies (19), is now also
supported by the crystal structure data.
Proc. Natl. Acad. Sci. USA 96 (1999)
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
We thank Dr. Paul Schimmel for comments and suggestions on the
manuscript and Annmarie McInnis for her enthusiasm and care in the
preparation of this manuscript. This work was supported by Grant
R37GM17151 from the National Institutes of Health.
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