Nucleic Acids Research, Vol. 19, No. 4 801
Neighbourhood of the central fold of the tRNA molecule
bound to the E.coli ribosome—affinity labeling studies with
modified tRNAs carrying photoreactive probes attached to
the dihydrouridine loop
Jan Podkowinski and Piotr Gornicki*
Institute of Bioorganic Chemistry, Polish Academy of Sciences, Noskowskiego 12/14, 61704 Poznan,
Poland
Received November 15, 1990; Revised and Accepted January 24, 1991
ABSTRACT
The neighbourhood of the dihydrouridine loop of tRNA
molecule bound to E.coli ribosome has been studied
by affinity labeling, using modified tRNAs carrying
photoreactive azidonitrophenyl probes attached to the
3-{3-amino-3-carboxypropyl)-uridlne located at position
20:1 of Lupin methlonine elongator tRNA. The
maximum distance between the pyrimldine ring and the
azldo group estimated for the two probes employed in
this study is 1 0 - 1 1 A and 1 8 - 1 9 A, respectively.
Cross-linking of the uncharged, modified tRNAs has
been studied with poly(A,U,G) as a message, under
conditions directing uncharged tRNAs preferentially to
the ribosomal P-site. Modified tRNAs bind covalently
to both ribosomal subunits with high yields upon
irradiation of the respective non-covalent complexes.
Proteins S7, L33 and L1 have been consistently found
cross-linked to tRNAs modified with both probes, and
S5 and l_5 to tRNA modified with the longer probe.
Surprisingly, an S5-tRNA cross-linking product is
reproducibly found in a protein fraction prepared from
the purified 50S subunit. Cross-linking to rRNAs is
significant only for the longer probe and is stimulated
2 - 4 fold in the presence of poly(A,U,G). The crosslinking sites are located between nucleotides 1302 and
1398 in 16S rRNA and between nucleotides 2281 and
2358 in 23S rRNA.
INTRODUCTION
The decoding site and peptidyl transferase center are two of the
best characterized sites on the E.coli ribosome. Their locations
define the orientation of the anticodon loop and the aminoacid
end of the ribosome-bound tRNA molecules [1—3]. Important
information has been inferred from affinity labeling experiments
using a tRNA able to form a cyclobutane dimer between its
wobble base and 0,400 of 16S rRNA ([2] and references therein)
or using modified tRNAs carrying reactive probes attached to
the aminoacid residue of aminoacyl-tRNA (see [1] for a review
of earlier results, [4,5]), the 3'-terminus of the tRNA [6, 7] and
the anticodon loop [8, 10]. On the contrary very little information
is yet available concerning the contact sites of other parts of the
tRNA molecule. Protein S19 has been cross-linked to P and Asite bound tRNA via photoreactive probes attached to acp3U47
and sHjg, respectively [2, 11]. Proteins S5, S7 and S9 form
covalent bonds to A 2 |, U45 and U^, respectively, upon UV
irradiation of a AcPhe-tRNAPhe complex with the 30S ribosomal
subunit [12]. Several other proteins have been found cross-linked
to tRNA but the cross-linking sites in tRNA have not been defined
unequivocally (see [1] for review). In another approach, contact
sites between ribosomal RNAs and tRNA bound to the three
ribosomal sites have been mapped by chemical probing [13, 14].
Significant progress in 3-D modeling of ribosomal subunits
including the arrangement of both ribosomal proteins and rRNAs
[15-18] makes analysis of the results concerning the orientation
of tRNA on the ribosome feasible. New details of the functional
three site ribosome model have also emerged [19-20].
As a continuation of our efforts to determine the precise
orientation of tRNA molecules in different sites on the E.coli
ribosome by affinity labeling experiments we report here the
identification of ribosomal components located in the
neighbourhood of the central fold of the tRNA molecule. In this
paper, experiments with uncharged, modified tRNAs carrying
photoreactive probes attached to the dihydrouridine loop are
described. The orientation of this part of the tRNA molecule
would be expected to change substantially during the movement
of tRNA from one ribosomal site to another (more than is the
case with the anticodon loop or the aminoacid end), and therefore
its localization should be extremely helpful in an attempt to
understand the structural organization of the ribosomal tRNA
binding sites (A, P and E).
MATERIALS AND METHODS
tRNAMam from Lupin seeds was isolated as described before
[21 ] by chromatography on BD-DEAE-cellulose and Sepharose
* To whom correspondence should be addressed at Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637, USA
802 Nucleic Acids Research, Vol. 19, No. 4
4B, and was finally purified by HPLC on two TSK-DEAE-2SW
6.4 x 250 mm columns (LKB) using a 4-hour gradient consisting
of 250-280 mM NaCl in a buffer containing 10 mM MgCl2,
10% methanol and 20 mM Tris-HCl (pH 7.4), at room
temperature and with a 0.4 ml/min flow rate. tRNA had a UVabsorbtion corresponding to about 1500 pmoles/A260 unit.
E.coli ribosomes (tight couples, a kind gift from Dr. K.Nierhaus)
were prepared as described previously [22]. Mercaptans were
removed from ribosome preparations by gel filtration on Ultrogel
AcA44 using ribosome binding buffer (below) for elution. 1
unit of ribosomes was taken as 24 pmoles.
Partial removal of the 3 '-terminal adenosine: 1 A26o un it of
tRNA was incubated in 125 n\ of 50 mM Tris-HCl (pH 8.0),
10 mM MgCl2 containing 0.6 ng of snake venom phosphodiesterase (Boehringer, Mannheim) at room temperature for 15
min. After phenol extraction the tRNA was recovered by ethanol
precipitation.
tRNA 3'-end labeling: 1 A260 unit of tRNA with partially
removed 3'-terminal adenosine was incubated at 37°C for 1 h
in 200 /il of 70 mM Tris-HCl (pH 8.0), 20 mM MgCl2, 7 mM
DTT, 70 fiM CTP containing 200-300 /iCi of [a-32P]-ATP
(>400 Ci/mmole, Amersham) and saturating amounts of partially
purified tRNA nucleotidyltransferase ([EC 2.7.7.25.], a kind gift
from Dr. J.Ofengand). The mixture was then incubated for 1 h
at 37 °C after adding ATP to a final concentration of 25 /iM.
Labeled tRNA was purified on a DEAE-cellulose column (ca.
0.5 ml) using 0.25 M NaCl, 50 mM NaOAc (pH 4.5), 10 mM
MgCl2 to wash the column and 1.1 M NaCl in the same buffer
to elute the tRNA.
Modification of the amino group in tRNA: 1 A ^ unit of Lupin
tRNA Ma m was dissolved in 60 /tl of 0.5 M sodium borate (pH
8.2) and 240 /*1 of DMSO. Two 2 - 3 mg portions of N-5-azido2-nitro-benzoyloxysuccinimide or N-succinimidyl-6(4'-azido2'nitrophenyl-amino)-hexanoate (Pierce) dissolved in lO/il of
DMSO were added to the mixture at 45 min intervals and the
reaction was allowed to proceed out at room temperature for 90
min. tRNA was recovered by ethanol precipitation, and the excess
of the reagent was removed by 3 successive ethanol precipitations,
two from 50% DMSO and one from water.
Ribosome binding and cross-linking: 32P labeled tRNA (250
nM), ribosomes (125 nM) and poly(A,U,G) (80 jig/ml) were
incubated at 37°C for 30 min in a buffer containing 20 mM
HEPES-K (pH 7.8, at 0°C), 150 mM NH4C1, 6 mM
Mg(OAc)2, 50 yM spermine and 2 mM spermidine, and then
chilled on ice. tRNA binding was measured by a nitrocellulose
filter assay using 15 mM Mg(OAc)2, 50 mM Tris-HCl (pH 7.5,
at 4°C), 160 mM NH4CI as the wash buffer. Measured values
were corrected for non-specific binding of tRNA to nitrocellulose,
determined from a control sample without ribosomes. The noncovalent complexes were irradiated at 0°C with UV light (using
a photoreactor equiped with four 15 watt 350 run lamps, with
the sample in a pyrex tube), or with visible light (using a slide
projector equiped with a 150W halogen lamp) for 20 and 45 min,
respectively. Covalent binding was calculated from the
cpm/A260 ratio measured after subunit separation (below).
Separation ofribosomal subunits: after irradiation ribosomes were
recovered by ethanol precipitation (0.7 vol), dissolved in
200-400 nl of 20 mM Tris-HCl (pH 7.5), 0.5 mM Mg(OAc)2,
lOOmM NH4CI and centrifuged through a 10-30% linear
sucrose gradient in the same buffer (SW40 rotor, 24,000 rpm,
16 h, 4°C).
Separation ofrRNAs and ribosomal proteins: ribosomal subunits
were recovered from the sucrose gradient fractions by ethanol
precipitation (2.5 vol), and were incubated in 200 /il of 0.4%
SDS, 10 mM EDTA, 20 mM Tris-HCl (pH 7.5), 0.5 mM
Mg(OAc)2, 100 mM NHjCl for 5 min at 37 °C. The samples
were then diluted with 1 volume of 20 mM Tris-HCl (pH 7.5),
0.5 mM MgtOAc^, 100 mM NH4CI and were centrifuged
through a 10-30% linear sucrose gradient in the same buffer
containing 0.1 % SDS and 10 mM EDTA (SW40 rotor, 32,000
rpm, 16 h, 15°C).
Radioactivity measurements: dry filters were counted with 5 ml
of PPO/POPOP/toluene scintillation cocktail or with 10 ml of
'Ready Value' (Beckman). The Cerenkov radiation of the sucrose
gradient fractions was measured, or alternatively aliquots of the
fractions were diluted with water to 0.5 ml and counted with 5
ml of Unisolvel (Koch-Light).
Identification of ribosomal proteins cross-linked to tRNA was
performed with the agarose method of Ref. 23, using the proteintRNA cross-linked complexes from the SDS-containing sucrose
gradients (above), after recovery by ethanol precipitation.
Partial localization of cross-linking sites in ribosomal RNAs was
performed with the RNase H method of Ref. 24, using rRNAtRNA cross-linked complexes from SDS-containing sucrose
gradients (above), after recovery by ethanol precipitation.
RESULTS AND DISCUSSION
The methionine specific elongator tRNA from Lupin contains
the hypermodified nucleoside 3-(3-carboxy-3-aminopropyl)uridine (acr^U) located in the dihydrouridine loop at position
20:1. The free amino group of this nucleoside can be selectively
modified with N-hydroxysuccinimide esters, providing a very
convenient method for introducing different probes into the
central fold of the tRNA molecule. The specificity of the
modification procedure for primary aliphatic amino group has
been tested previously [26, 27]. The structures of the two
photoreactive probes employed in this study are shown in Fig. 1.
The distance between the N-3 atom of acp3U and the azido
group was taken as the maximum extended length of the probe,
and was estimated to be 10— 11 and 18 —19 A for the short and
long probe, respectively. The 2-Nitro-5-azido group of the shorter
probe can be activated by UV light (320-350nm), and the
2-nitro-4-azido group of the longer probe is activated by visible
light. In the following discussion the two modified tRNAs are
referred to as 'tRNA-11 A/20:1' and 'tRNA-19A/20:l' for the
short and long probe, respectively.
An approximate location of the attachment site of the
photoreactive probes can be deduced from the crystal structure
of Yeast tRNAPhc and is shown in Fig.l. We assume that
acr^U^., in tRNAMetm is positioned similarly to G^ in
tRNAptie. A more precise localization is not possible, because
no crystal structure of a tRNA molecule with a dihydrouridine
loop structure similar to that of Lupin tRNAMel is as yet
available.
Nucleic Acids Research, Vol. 19, No. 4 803
3
Dihydrouridine
loop
tRNA-ll8/20:l
COOH
N n
u
"
>U(N-3)-CH_CH_CHNH-C0
acp 3 U
20:1
io D i hydrour i d i ne
loop
tRNA-198/20:l
COOH
XJ (N-3)20:1
18 - 198
Fig.l. Structure (a) and approximate location of the photoreactive probes in tRNA (b). The natural side chain of the hypermodified nucleoside is marked with a
dotted line. In the Yeast phenylalanine tRNA structure [25] (atomic coordinates from Brookhaven Protein Data Bank), the N-7 atom of guanosine 20 is marked
by a full circle (10 A diameter) as the best approximation of the probe attachment site. The amino acid arm of the tRNA molecule shown on the left points away
from the viewer.
The E.coli ribosomes (tight couples) used in this study were
prepared by a procedure without high salt washing [22] to avoid
structural and functional heterogeneity caused by partial removal
of some ribosomal proteins [20]. It has been shown recently that
deacylated tRNAs bind to the ribosomal P site in the presence
of a message containing an internal cognate codon [20]. We
assume that, under the conditions tested in the study quoted above
for a series of different tRNAs, the deacylated tRNAMttm
employed in this study will bind exclusively or at least
predominantly to the P site in the presence of poly(A,U,G) as
a message. The probability of simultaneous binding of two tRNA
molecules to the same ribosome is low for a random A, U, G
copolymer. The ribosomal P site probed in this study by affinity
labeling in a complex containing one single tRNA molecule bound
per ribosome bears a resemblance to the tRNA binding site in
the initiation complex but should also share structural features
of the P site functioning during elongation, especially that in the
post-translocation state [S.Schilling-Bartetzko and K.H.Nierhaus,
personal communication].
About 0.7 molecule of deacylated, unmodified tRNA1** binds
per ribosome in the presence of poly(A,U,G) at a two-fold molar
excess of tRNA. In the absence of poly(A,U,G) binding is about
three-fold lower. The modification has only limited effect on
tRNA binding and at least 85% of the binding ability is retained.
Non-covalent complexes of modified tRNAs formed in the
presence of poly(A,U,G) were irradiated with light of an
appropriate wavelength and then were applied to sucrose gradients
under subunit dissociating conditions. Fig 2. shows the
distribution of cross-linked tRNA between ribosomal subunits,
with non-cross-linked tRNA staying at the top of the gradient.
Binding and cross-linking values measured in the same
experiment are shown in Table 1. The covalent bond formation
is reproducibly very efficient (10—25%) and is higher for
modified tRNA carrying the longer probe. It is light- and probedependent ([27] and data not shown). Cross-linking in the absence
of poly(A,U,G) is equally efficient despite the low tRNA binding
as measured by nitrocellulose filter assay—Table 1. This effect
can be attributed to a rather weak tRNA binding in the absence
804 Nucleic Acids Research, Vol. 19, No. 4
35
CPU (Thousands)
CPU (Thousands)
50
a.
45
30
25
30S
-
A
•
40
30
i
50S
•
10
S
rP
35
A,
20
18
tRNA
a.
/
0
25
20
15
10
23S rRNA
5
B B B it:
10 11 12 13 14 15 18 17 18 19 20 21
0
1 2 3 4 5 6 7 8 9 10 11 12 13 U 15 16 17 18 19 20
Fraction No
Fraction No
- B - +poly(A,U,G)
- a - +poly(A,U,G)
- A - -poly(A,U,G)
CPU (Thousands)
CPU (Thousands)
2 3 4 5 8 7 8 0 10 11 12 13 14 15 18 17 18 19 20
Fraction No
-B-
+poly(A,U,G)
2 3 4 5 6 7 8 9 10 11 12 13 14 15 18 17 18 19 20 21
Fraction No
- A - -po!y(A,U,G)
-B-
+poly(A,U,G)
Fig.2. Sucrose gradient analysis of the distribution of cross-inked tRNA-1 lA/20:l
(a) and tRNA-19A/20i 1 (b) between nbosomal subunits, in the presence (squares)
and in the absence (triangles) of pory(A,U,G). Values in (a) and (b) are corrected
for ribosome recovery and can be compared directly. Fractions containing
ribosomal subunits were identified by A2JO measurements (not shown).
Fig J . Sucrose gradient analysis of the distribution of cross-linked tRNA-19A/20:1
between ribosomaJ RNAs and proteins of the large (a) and small (b) ribosomal
subunit, in the presence of poly(A,U,G). Fractions containing rRNAs were
identified by A26O measurements (not shown).
TaWe 1. Binding and cross-linking of tRNA-11 A/20:1 and tRNA-19A/20:l.
poly(A,U,G) the small ribosomal subunit becomes cross-linked
predominantly. In the latter case the 30S subunit cross-linking
yield is higher than in the presence of the message.
Analyses by SDS-containing sucrose gradient centrifugation
of the distribution of the cross-linked tRNA-19A/20:l between
ribosomal RNAs and proteins are shown in Fig.3.
tRNA-11 A/20:1 is cross-linked only to ribosomal proteins
(>90%, data not shown). In all cases ribosomal proteins are
the major cross-linking target. However, 5S rRNA involvement,
as well as any instability of the covalent product(s), cannot be
excluded on the basis of the sucrose gradient analysis. Substantial
cross-linking to the large rRNAs was observed only for the longer
probe and it was strongly message-dependent (2—4 fold
stimulation in the presence of poly(A,U,G), data not shown).
The tRNA-rRNA covalent products accounted for about 20 and
30% of the total cross-linking to the large and small subunit,
respectively. tRNA-19A/20:1 has been found cross-linked to 23S
rRNA and to one of its large fragments (Fig.3) present in our
tRNA-11 A/20:1
tRNA-19A/2O:l
poly(A,U,G)
+
_
+
—
Binding Cross-linking
(mole tRNA/mole ribosome)
0.65
0.10
0.21
0.13
0.14
0.61
0.25
0.13
%
15
(62)
23
(52)
Binding and cross-linking was measured as described in Materials and Methods.
The cross-linking yields were measured from the sucrose gradients shown in Fig.2.
The last column shows the yield of cross-linking to 70S ribosomes, expressed
as a percentage of the bound tRNA. The values obtained in the absence of
poly(A,U,G) are shown in brackets (see discussion in text).
of poly(A,U,G) which escapes detection by the filter assay but
enables efficient covalent bond formation and product
accumulation. Cross-linking with an > 50% yield seems less
likely. In the presence of poly(A,U,G) both ribosomal subunits
are cross-linked with a similar yield, whereas in the absence of
Nucleic Acids Research, Vol. 19, No. 4 805
Bound tRNA (X)
Bound tRNA (X)
1 8 3 4 6 8 7 / 9 101113 14 15181718193081 B823a4B88a3788Be303a3334
1 2 3 4 S 8 7/ 8 1011131416181718198081388384888827388830333334
Ribosomal protein (L)
Ribosornal protein (L)
Bound tRNA (X)
Bound tRNA (X)
2.5
0.0
1 2 3 4 5 6 7 8 S 10 11 12 13 14 15 18 17 18 19 20 21
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 18 17 18 19 20 21
Ribosomal protein (S)
Ribosomal protein (S)
Fig.4. Identification of large (a) and small subunit (b) proteins cross-linked in
the presence of poly(A,U,G) to tRNA-11 A/20:1. The bars indicate the percentage
of radioactivity (label in tRNA) bound to the antibody in each test performed
for the ribosomal protein indicated. Values from at least two independent
measurements were averaged. L7/ denotes proteins L7 and L12.
ribosome preparation (the fragmentation is unrelated to the
photochemical process taking place during irradiation).
Ribosomal proteins cross-linked to tRNA were identified by
binding them to goat antibodies specific for individual ribosomal
proteins that were immobilized on Agarose-anti-goat IgG
conjugate [23]. Results of some representative antibody assays
are presented in Fig. 4 and 5.
The antibody assay itself is not quantitative and its efficiency
varies substantially. Furthermore, the binding efficiency can be
lowered due to decreased protein-antibody affinity caused by
cross-link formation, and detection of some products can be
affected by their instability, leading to even greater variability,
especially when results of independent cross-linking experiments
are compared. This complicates analysis of the low yield crosslinking products. In fact, some products may escape detection
entirely. The following features of the experimental design were
introduced to overcome these difficulties: i. tRNAs modified with
two different probes were used in all experiments (different
lengths of the probe and different structures of the photoreactive
residue); ii. only those proteins which gave significant signal to
noise ratios in at least four independent antibody assays were
Fig.5. Identification of large subunit (a) and small subunit (b) proteins crosslinked in the presence of poly(A,U,G) to tRNA-19A/20:l (Cf. Fig.4).
considered relevant; iii. results of independent cross-linking
experiments were compared.
Proteins S7, LI and L33 were consistently found cross-linked
to tRNA modified with both probes, and S5 and L5 were
consistently found cross-linked to tRNA modified with the longer
probe (Fig. 4 and 5, and data not shown). Surprisingly, the
S5-tRNA covalent complex was reproducibly present only in the
protein fraction prepared from the 50S subunit purified by sucrose
gradient centrifiigation. It accounted for about 15% of the 50S
protein cross-linking product (no background subtracted),
compared to about 19% found for L33 (Fig. 5a). We are not
aware of any cross-contamination of antibodies used in the assay
that could explain this result.
Proteins S9, SI 1, S13 and L27 were found cross-linked to
tRNA modified with both probes, and L5 to tRNA modified with
the shorter probe but with lower and highly variable efficiency
(Fig. 4 and 5, and data not shown). In some cross-linking
experiments positive identification of these proteins was not
possible (e.g. L27 in experiments presented in Fig. 4a and 5a).
As we pointed out in the discussion above interpretation of such
negative results is especially difficult.
The protein-tRNA cross-linking patterns obtained in the
absence of poly(A,U,G) were very similar, with proteins S7, LI
and L33 being the major products (data not shown).
806 Nucleic Acids Research, Vol. 19, No. 4
a.
o>
5 R 1 I $ I
23S rRNA
i
FIg.6. Polyacrylamide gel analysis of the tRNA-23S rRNA cross-linking product digested with RNase H in the presence of specific oligodeoxynucleotides. Numbers
indicate 23S rRNA nucleotides complementary to the 6th nucleotide of each decadeoxynucleotide in a pair. In (a) pairs are defined by numbers shown on both sides
of each lane. C indicates a control RNase H digestion without any oligodeoxynucleotidc. Arrows and asterisks mark bands discussed in the text.
Cross-linking sites in ribosomal RNAs were localized using
RNase H cleavage directed by pairs of oligodeoxynucleotides
complementary to specific sites in rRNA, followed by analysis
of the size of the RNA fragment cross-linked to 32P-labeled
tRNA by polyacrylamide gel electrophoresis [24]. Results of the
RNase H analysis are shown in Fig.6 and 7. RNase H cleavage
sites, determined by sequences of the oligodeoxynucleotides, were
distributed along the entire length of the rRNA molecule at less
than 300-nucleotide intervals (diagrams in Fig. 6 and 7). Each
oligodeoxynucleotide pair used in the analysis induced cleavages
at adjacent sites. Localization of the tRNA cross-linking site was
based on identification of the pair generating the smallest rRNA
fragment, less than 300 nucleotide long, covalently bound to
tRNA (the fastest-migrating band, marked with an arrow). The
tRNA cross-linking site lies between cleavage sites induced by
this pair of oligodeoxynucleotides. Both cleavages directed with
all other pairs are located on either the 5' or the 3' side of the
cross-linking site and yield larger products containing 3' or 5'
fragments of rRNA bound to tRNA, respectively (a small-size
product can be formed only when the cross-linking site is located
near one of the rRNA termini). Some of these products were
small enough to be separated on the gel and they appeared on
autoradiograms as identifiable bands (marked with asterisks).
Efficiency of RNase H cleavage varied substantially for different
oligonucleotide pairs. In some cases weak non-specific cleavage
was also observed.
The tRNA cross-linking sites were localized between
nucleotides 2235 and 2442 of 23S rRNA, and 1207 and 1398
of 16S rRNA, using 11 pairs of decadeoxynucleotides
complementary to 23S rRNA and 8 pairs of heptadecadeoxynucleotides complementary to 16S rRNA, respectively (Fig. 6a
and 7a, regions marked on the upper part of the diagrams with
a heavy line) .
The same approach with two additional oligodeoxynucleotides
complementary to sequences located within the above boundaries
was then used to localize the cross-linking sites more precisely—
Fig 6b and 7b. Bands marked with arrows represent RNase H
products (identified by their size) generated with pairs of
oligodeoxynucleotides inducing cleavages on both sides of the
tRNA cross-linking site. The site in 23S rRNA lies between
nucleotides 2281 and 2358 (between 2235 and 2358 but not
between 2235 and 2281, Fig. 6b). Some other combinations of
Nucleic Acids Research, Vol. 19, No. 4 807
CO
CM
M
n
o
O
S.
O
O
M
CO
0>
O
co
a
o
CN
o
S2
o
w
a.
169 rRNA
5"'
1'
n a n
* i - uS p 09
co «
2 coa> o CM n
•
'
• •-•—H—^M^
'31
B8 8
Fig.7. Polyacrylamide gel analysis of the tRNA-16S rRNA cross-linking product.digested with RNase H in the presence of specific oligodeoxynucleotides. Numbers
indicate 16S rRNA nucleotides complementary to the 10th nucleotkJe of each heptadecadeoxynucleotide in a pair. Two decadeoxynudeotides were used in this experiment.
Their 6th nucleotides were complementary to nucleotides 1238 and 1302, respectively (b). In (a) pairs are defined by numbers shown on both sides of each lane.
C indiacates control RNaseH digestion without any oligodeoxynucleotide. Arrows and asterisks mark bands discussed in the text.
the oligodeoxynucleotides were also used to analyze the
2235-2442 region of 23S rRNA (data not shown). Results of
the RNase H analysis with pairs including oligonucleotide
complementary to the 2308 site were inconclusive. The cleavage
directed by this oligonucleotide was inhibited, perhaps because
the tRNA molecule was bound covalently within the
complementary sequence, or there were two cross-linking sites
on both sides of the RNase H cleavage directed by this
oligonucleotide. The problem has not been investigated any
further. Otherwise all the data consistently localized the crosslinking site between nucleotides 2281 and 2358, as indicated by
a heavy line in the lower part of the diagram in Fig. 6. Results
obtained for the cross-linking product containing the 23S rRNA
fragment (above) were identical. The tRNA cross-linking site
in 16S rRNA lies between nucleotides 1302 and 1398 . Sizes
of the products generated with all four pairs used in this
experiment (Fig. 7b) agree with the above localization of the
cross-linking site near the 3' end of 16S rRNA (about 200
nucleotides away). The localization was confirmed by an
independent RNase H experiment in which the cleavage was
directed with single oligodeoxynucleotides (data not shown).
Protein S7 as well as S9 and S13 are located on the head of
the 30S subunit (some discrepancies still exist concerning precise
localization of SI3), and protein Sll appears on the platform
in close vicinity to the other three [3, 15, 17]. S19, which can
be also cross-linked to the P or A-site bound tRNA by a probe
attached to the central fold but emerging from the variable loop
or Ug side of the molecule [2, 11] is located on the head of the
30S subunit as well [3, 15, 17]. Cross-linking with a short
bifunctional reagent suggests that it must be also a near neighbour
of S13 [28]. Nucleotides 1302-1398 border on the C l400 region
of 16S rRNA located within the decoding site ([2] and references
therein) and several of the nucleotides are protected from chemical
probes by tRNA binding, suggesting their involvement in building
tRNA binding site(s) [14]. S7, S9 S13 and S19 can be crosslinked to and/or protect nucleotides distributed throughout the
same region of 16S rRNA from chemical or enzymatic probes
(reviewed in [15, 17]). S7 can be also cross-linked to the 3'-end
808 Nucleic Acids Research, Vol. 19, No. 4
of 16S rRNA, which is located on the platform [15]. The
S5-tRNA cross-link is difficult to accommodate within the existing
30S subunit models. In the small subunit map protein S5 is located
about 100A away from the other proteins involved [15].
However, some other data also suggest that this protein can
approach the tRNA binding domain . It can be cross-linked to
SI3 and S9 [29] and to A2] of tRNA bound to the small subunit
[12].
Protein L33 has been placed on the large subunit between
proteins LI and L27, and near G242g to which it can be crosslinked [18]. The tRNA cross-linking site identified in this study
lies between nucleotides 2281 -2358. L5, as well as the other
5S rRNA binding protein L18, and protein L27, cross-link to
the same region of 23S rRNA [18]. A possible 5S rRNA
interaction site within the region has been also suggested [30].
Proteins L5 and LI8, 5S rRNA and nucleotides 2281 -2358 are
located on the central protuberance of the large subunit [3,18].
All the cross-linking sites mentioned above lie within domain V
of 23S rRNA. The domain encompasses nucleotides involved
in building the peptidyl transferase center as well as the LI
binding site ([13, 17] and references therein). Most nucleotides
protected from chemical probes by tRNA binding are located in
this domain [13]. Finally, proteins L5 and L27 were reported
to cross-link to a photoreactive probe attached to the central
domain of the tRNA molecule [2].
Protein-protein cross-linking data further confirm that the
proteins discussed form a distinct and rather compact domain
on the ribosome. LI cross-links to S9, SI 1 and L33; L5 to S9,
S13 and S19; S9 to S7; S13 to S7, Sll and S19 [29]. It is also
worth mentioning that proteins S7, S l l , S13, S19, LI and L5,
and the 16S rRNA fragment 1506-1529 are among ribosomal
components cross-linked to IF3 [31 - 3 3 ] . The IF3 binding site
is believed to bridge the gap between the head and the platform
of the small subunit, and at least partially overlaps with the tRNA
binding site identified in this study.
The results presented in this paper demonstrate that the tRNA
binding domain is located at the ribosome interface and includes
components of both ribosomal subunits. The 30S subunit
components cross-linked to the central fold of the tRNA molecule
(S7, S9, S l l , S13 and 16S rRNA fragment 1302-1398) are
clustered together in a region on the side of the head facing the
cleft and extending towards the cleft (decoding site) and the
platform (3'-end of 16S rRNA). The 50S subunit components
(LI, L33, L5 and 23S rRNA fragment 2281 -2358) are clustered
in a region extending from the LI protuberance towards the
central protuberance. We have already shown previously that
protein S7 and LI are located near the decoding site [10].The
two regions position the 'back' of the tRNA molecule (the region
of the tRNA opposite to the aminoacid arm and extending from
the central fold to the anticodon loop) on the ribosome. The
position leads from the decoding site in the cleft of the small
subunit [26] and near the base of the LI protuberance [10]
towards the head of the small subunit and towards the large
protuberance on the large subunit. In this arrangement the amino
acid arm points away and at an angle from the head of the small
subunit towards the large subunit. This orientation is fully
compatible with the localization of the peptidyl transferase center
near the base of the central protuberance as defined, among other
data, by the localization of protein L27 [ 5 - 7 , 18]. It also accounts
for results from other affinity labeling [2,11] and chemical
probing experiments [13,14], and agrees well with the recent
three-dimentional model of the tRNA binding domain on E. coli
ribosome [18].
ACKNOWLEDGEMENTS
The authors thank R.Brimacombe, J.Ofengand and
W.J.Krzyzosiak for generous support and stimulating discussions,
R.Brimacombe and B.Greuer for assistance with the protein
identification and RNase H experiments, and T.Dymarek-Babs
for technical assistance. This work was supported by the Polish
Academy of Sciences project 04.12.1.3.
REFERENCES
1. Ctfenand,J.,Gornicki,P.,Nurse,K.andBoublik,M. (1984) in Alfred Benzon
Symposium No 19 : Gene expresion. The translational step and its control
(Clark,B.F.C. and Petersen.H.U., eds.), pp. 293-315, Munksgaard,
Copenhagen.
2. OfengamU., CiesiolkaJ., Denman.R. and Nurse,K. (1986) in Structure,
Function and Genetics of Ribosomes (Hardesty, B and Kramer.G., eds),
pp. 473-494, Springer-Verlag, New York and Heidelberg.
3. Stoffler, G. and Stoffler-Meilicke.M (1986) in Structure, Function and
Genetics of Ribosomes (Hardesty, B. and Kramer.G., eds), pp. 28-46,
Springer-Verlag, New York and Heidelberg.
4 Barta.A., Steiner.G., BrosiusJ., Noller.H.F. and Kuechler.E (1984)
Proc.Natl.Acad.Sci.USA 81, 3607-3611.
5. Steiner.G., Kuechler, E. and Barta.A. (1988) EMBO J. 7, 3949-3955.
6. Wower.J., Hixon.S.S. and Zimmermann.R.A. (1989) Proc.Natl.
Acad.Sci.USA 86, 5232-5236.
7. Wower.J., Hixon.S.S. and Zimmermann.R.A. (1988) Biochemistry 27,
8114-8121.
8. Gomicki.P., CiesiolkaJ. and Ofengand.J. (1985) Biochemistry 24,
4924-4930.
9. CiesiolkaJ., Gornicki.P. and Ofengand.J. (1985) Biochemistry 24,
4931-4938.
10. Podkowinski J. and Gornicki.P. (1989) Nucleic Acids Res. 17, 8767-8782.
11. Lin,K.-L., Kahan.L. and OfengandJ. (1984) J.Mol.Biol. 172, 77-86.
12. Abdurashidova.G.G., Tsvetkova.E.A. and Budovsky.E.I. (1989) FEBS Lett.
243-302.
13. Moazed.D. and Noller.H.F. (1989) Cell 57, 585-597.
14. Moazed.D. and Noller.H.F. (1990) J.Mol.Biol. 211, 135-145.
15. Bnmacombe.R., Atmadja,J., Stiege.W. and Schuler.D. (1988) J.Mol.Biol.
199, 115-136.
16. Schuler,D. and Brimacombe.R. (1988) EMBO J. 7, 1509-1513.
17. Stem.S., Weiser.D. and Noller.H.F. (1988) J.Mol.Biol. 204, 447-481.
18. Mitchell,P., Osswald.M., Schueler.D. and Brimacombe.R. (1990) Nucleic
Acids.Res. 18, 4325-4333.
19. Nicrhaus.K.H., Rheinberger.H.-J., Geigenmuller,U., Gnirke.A.,
Saruyama.H. Schilling.S. and Wuimbach,P.(1986) in Structure, Function
and Genetics of Ribosomes (Hardesty, B. and Kramer.G., eds), pp. 454-472,
Springer-Verlag, New York and Heidelberg.
20. Gnirke,A., Geigenmuller,U., Rheinberger,H.-J. and Nierhaus.K.H. (1989)
J.Biol.Chem. 246, 299-302.
21. GomickJ.P., Wolanski.A, Judek,M., Podkowinski J., Gulewkz.K. and
Krzyzosiak.W.K. (1986) Bull.Pol.Ac.:Chem. 34, 15-25.
22. Rheinberger,H.-J., Geigenmuller.U., Wedde.W. and Nierhaus.K.H. (1988)
Methods Enzymol. 164, 658-670.
23. Gulle,H., Hoppe,E., Ostwald.M., Brimacombe.R. and Stoffler.G. (1988)
Nucleic Acids Res. 16, 815-832.
24. Brimacombe.R., Greuer.B., Gulle.H., Kosack.M., Mhchell,P., Osswald.M.,
Stade.K. and Stiege.W. (1990) in Ribosomes and Protein Synthesis; a Practical
Approach (Speeding,G., ed.), IRL Press, Oxford, in the press.
25. Hingerty.B., Brown.R.S. and Jack.A. (1978) J.Mol.Biol. 123, 523-534.
26. Gornicki.P., Nurse.K., Hellman.W., Boublik.M. and OfengandJ. (1984)
J.Biol.Chem. 259, 10493-10498.
27. PodkowinskiJ., Dymarek-Babs, T. and Gornicki.P, (1990) Ada Biochim.
Polon., in the press.
28. Pohl.T. and Wittmann-Liebold.B. (1988) J.Biol.Chem., 263, 4293-4301.
29. Wittmann.H.G. (1983) Ann.Rev.Bicchem. 52, 3 5 - 6 5 .
30. Brimacombe.R. and Stiege,K. (1985) Biochem.J. 229, 1-17.40. L33 xl
31. Pon,C.L., Pawlik.R.T. and Gualrezi.C. (1982) Febs Lett. 137,163-167.
32. Boileau.G., Butkr.P. HersheyJ.W.B. and Traut.R.R. (1983) Biochemistry
22, 3162-3170.
33. Ehresmann.C, Moine.H., Mougel.M., DondonJ., Grunberg- Manago.M.,
EbelJ.P. and Ehresmann.B. (1986) Nucleic Acids Res. 14, 4803-4821.