From the
The interaction between mRNA and Escherichia coli ribosomes has been studied by photochemical cross-linking using
mRNA analogues that contain 4-thiouridine (s
Photoaffinity experiments with suitably derivatized mRNA and
tRNA substrates offer an attractive strategy to determine the location
and properties of the functional sites on the Escherichia coli ribosome. Photo and photochemical cross-links have been made to
the ribosome from the acceptor end
(1, 2) , from
positions in the hinge region
(3, 4) , and from the
anticodon loop of the tRNA
(5, 6) . The general location
of the mRNA track has also been elucidated by photoaffinity
experiments
(7, 8) . These experiments are consistent
with an mRNA track extending around the middle part of the 30 S
subunit, with the sites for the codon-anticodon interactions determined
by the 30 S subunit and with the aminoacyl ends of the tRNAs extending
toward the peptidyltransferase center of the 50 S subunit (see Refs. 2
and 9-11).
Experiments from the groups of Brimacombe and
Bogdanov
(12, 13, 14, 15, 16) have been done with synthetic RNA molecules containing
photoreactive nucleoside s
We reported experiments that used RNA analogues 51 nt in length with
clusters of s
In the present experiments,
the pattern of cross-linking made by mRNA analogues with single
s
RNAs containing
s
Complexes of ribosomes and
s
RNA sequencing was done to
determine the cross-linking site. After cross-linking to nonradioactive
s
Several conclusions made by Stade et al.(12) and Rinke-Appel et al.(13) for
cross-linking by these two mRNA analogues to E. coli rRNA have
been confirmed, including the strong tRNA dependence for cross-linking,
cross-linking to C
Three cross-linking sites in the 16 S rRNA at
U
An important consideration involves the
strategy of performing these experiments. The Brimacombe and
Brimacombe/Bogdanov groups used sedimentation or gel filtration to
first purify mRNA
The location of the mRNA cross-linking sites
in the 30 S subunit can be estimated from available structural
information. The site A
The tRNA
independent mRNA cross-linking sites can also be placed in approximate
locations in the 30 S subunit. The cross-linking site U
The properties of the tRNA-independent and tRNA-dependent
cross-linking indicate a change in the 30 S mRNA analogue interaction
upon tRNA binding. First, under the experimental conditions, mRNA binds
with reasonable stoichiometry to 70 S ribosomes whether tRNA is present
or not. However, there is a large increase in the amount of covalent
cross-linking to the 16 S rRNA upon inclusion of tRNA, much larger than
the binding stoichiometry differences. As an internal control, there is
no significant difference in the amount of cross-linking to 23 S rRNA.
Second, the mRNA bound to the 70 S ribosomes is bound in approximately
the correct location without tRNA, and cross-links occur to all of the
rRNA sites (see ). The Shine-Dalgarno sequence homologies
in the mRNA analogues probably are responsible for the correct
alignment of the mRNA analogues in the ribosome.
There are several
ways to account for the large increase in cross-linking efficiency upon
tRNA binding. One possibility would be an increase in mRNA flexibility
upon tRNA binding since s
Molecules
s
Stoichiometry of binding (mol mRNA
analogue/mol 70 S ribosome) were determined at 5
Extent of cross-linking to 16 S
rRNA and 23 S rRNA is indicated as percent mol mRNA analogue/mol rRNA
species.
Entries indicate the
normalized relative intensity of bands for cross-linked versus control samples. The two values for each are cross-linking without
and with tRNA
We thank Melanie Blackard for providing the mixed
oligomer 16 S-OneB and conditions for its use.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
U) or
s
U modified with azidophenylacyl bromide (APAB), either two
nucleotides upstream or eight nucleotides downstream from the
nucleotide sequence ACC, the codon for tRNA
. The
sequences of the mRNA analogues were described earlier (Stade, K.,
Rinke-Appel, J., and Brimacombe, R.(1989) Nucleic Acids Res. 17, 9889-9908; Rinke-Appel, J., Stade, K., and Brimacombe,
R.(1991) EMBO J. 10, 2195-2202). Under equilibrium
conditions, both of these mRNA analogues bind and cross-link to 70 S
ribosomes without the presence of tRNA
; however, there
are significant increases both in binding and particularly in
cross-linking in the presence of the tRNA
. Four regions
contain cross-linking sites that increase in the presence of tRNA,
C
, A
, A
(and minor sites
around these three positions), and C
/U
.
Three other cross-linking sites, U
, A
, and
U
, show very little change in extent of cross-linking
when tRNA is present. A conformational change in the 30 S subunit
allowing additional accessibility to the 16 S rRNA by the mRNA
analogues upon tRNA binding best explains the behavior of the
tRNA-dependent and tRNA-independent mRNA-16 S rRNA cross-linking sites.
U
(
)
at
different locations with respect to the P site to probe the mRNA
binding track. Cross-links to four sites in the 16 S rRNA have been
reported: A
, C
(and residues G
and A
in its close vicinity), U
,
and the 3`-terminal region. Several specific correspondences between
the location of the photoreactive moiety in the mRNA analogue and the
position of cross-linking in the 16 S rRNA have been drawn. If the
numbering of positions in the mRNA analogue are made from
``+1'' being the first nucleotide of the triplet used in
the P-site, these are +6 in the mRNA to U
in the 16
S rRNA, +7 in the mRNA to C
in the 16 S rRNA, and
+11 in the mRNA to A
in the 16 S rRNA
(15) .
In subsequent reports using additional mRNA analogues, cross-links were
detected from +4 in the mRNA to nucleotides in the interval
1402-1415 in the 16 S rRNA, from +6 to +10 in the mRNA
to C
in the 16 S rRNA, and from +10 to +13 in
the mRNA to A
in the 16 S rRNA
(14) . These
cross-links were strongly tRNA-dependent; in all experiments, complexes
of 70 S ribosomes, mRNA, and tRNA were isolated by sedimentation or gel
filtration to ensure that the most stable complexes were examined.
U in different areas from -20 to
+26 from the first nucleotide of the codon
(17) . These
experiments were done by forming complexes between 70 S ribosomes and
mRNA analogues without or with cognate tRNA and irradiating these
complexes without further purification. Ten cross-links between the
mRNA analogues and 16 S rRNA were detected by primer extension assay.
With the mRNA analogues that were used, only two of the sites
(C
and A
) showed strong tRNA dependence
and the site A
showed a weaker tRNA dependence on
cross-linking for some of the mRNA analogues. The behavior of this set
of mRNA analogues led us to question why there was not tRNA dependence
for a larger number of the rRNA sites.
U residues and the response of the sites with respect to
tRNA binding has been investigated. mRNA analogues with s
U
either at position -2 or +11
(12, 13) were
used because these were shown to exhibit strong tRNA dependence in
binding and cross-linking. Under equilibrium binding conditions, the
pattern of cross-linking can be separated into tRNA-dependent and
tRNA-independent classes. A proposal for a conformation change in the
30 S subunit upon tRNA binding is made to explain the behavior of the
mRNA analogues with the tRNA-dependent 16 S rRNA cross-linking sites.
Preparation of DNA Template and RNA
Synthesis
dsDNA for in vitro transcription was prepared
with Taq DNA polymerase. Single-stranded DNA template
containing the T7 promoter sequence and the sequence for the mRNA
analogue were synthesized, and only left (5`) primers were used since
both molecules contain redundant sequences at their 3` ends. dsDNA was
purified on nondenaturing 8% polyacrylamide gels. In vitro transcription of RNA was done with RiboMax protocols described by
Promega and included 1 mM GMP for partial substitution of GMP
for GTP at the 5` end. In vitro transcription of RNA
containing sU was done with 0.25 mM
s
UTP
(18) and 3 mM concentration each of
GTP, ATP, and CTP.
P-Labeled RNA was made by including 20
µCi of [
-
P]GTP (3000 Ci/mmol, Amersham)
in a reaction that had 0.5 mM cold GTP. Usual yields were
40-60 µg of RNA/100 µl transcription. Transcription
mixtures were purified on Qiagen tips, and RNA was checked on 8%
polyacrylamide, 8 M urea gels. When unlabeled RNA was
transcribed, 50 pmol was used in exchange reaction with
[
-
P]ATP
(19) .
U were used for APAB reaction as described
(12) .
APAB-substituted mRNAs were checked on 8% polyacrylamide, 8 M
urea gels containing 0.003% APM
(20) in which
s
U-containing RNA is retarded, but
s
U-containing RNA substituted with APAB migrates at the
same position as RNA with uridine.
Cross-linking and Analysis
Complexes between
activated 70 S ribosomes and mRNA analogues were made in 10 mM
Tris HCl, pH 7.4, 50 mM KCl, and 10 mM
MgCl(21) . 10 pmol of 70 S ribosomes in 100 µl
were incubated for 30 min at 37 °C with a 5-fold molar excess of
mRNA analogue without or with a 5-fold molar excess of
tRNA
. mRNA binding was determined by nitrocellulose
filter binding as described
(17) . The extent of tRNA binding was
determined by footprinting reactions
(22, 23) comparing
the degree of protection conferred by tRNA
to that
conferred by a known amount of binding of tRNA
(directed
by poly(U)). Cross-linking was carried out for 10 min at 4 °C in an
irradiator producing 320-365 nm light
(24) . Irradiated
samples were digested with proteinase K, phenol-extracted,
ethanol-precipitated, and redissolved in water. Samples were denatured
with formamide before electrophoresis on agarose gels. Primer extension
analysis of the mRNA-rRNA cross-links using reverse transcriptase and
10 DNA oligonucleotide primers was done as described
previously
(17, 25) . Reverse transcripts were analyzed
on 8% polyacrylamide, 8 M urea gels.
Determination of Cross-linking in the 16 S rRNA
3`-Terminal Region
To determine the extent of cross-linking to
P-labeled mRNA at the 3` end of 16 S RNA, 16 S-sized RNA
was purified on 1% agarose gels before use for RNase H reactions. The
oligomer UACTCCUUUGG, complementary to 1426-1437, consisting of
2`-methyl ribonucleosides and deoxynucleotides (underlined) was used to
cleave after position 1435
(26) . Reactions contained 40
mM Tris HCl, pH 7.4, 4 mM MgCl
, 1
mM dithiothreitol, 0.003% bovine serum albumin, 4% glycerol, 1
µg of 16 S rRNA, and 50 pmol of oligomer in 6 µl. Reactions
were done by incubating the mixture for 2 min at 55 °C, slowly
cooling to 37 °C, and then adding 2.5 units of RNase H (Fermentas)
and incubating for 10 min at 37 °C. After phenol extraction and
precipitation, RNA was electrophoresed on composite 4%-12%
polyacrylamide, 8 M urea gels. For sequencing, the 3` ends of
cross-linked and control 16 S rRNA were labeled with
[5`-
P]pCp and T
RNA ligase
(27) according to Krzyzosiak et al.(28) . RNase
H was used to remove the label from the mRNA with additional DNA
oligonucleotides complementary to the mRNA. Partial alkaline hydrolysis
was done in 50 mM NaCO
, pH 9.0, 1 mM EDTA
in the presence of 0.25 µg/µl tRNA and 3`-labeled 16 S RNA
fragment (0.1 µg) in 6 µl for 10 min at 90 °C, then 4.3 mg
of urea were added, and samples were electrophoresed on 8%
polyacrylamide, 8 M urea gels. For sequencing, samples were
digested with RNase T
and RNase U
(29) .
mRNA Binding and Cross-linking
The sequences of
the two mRNA analogues used in this study are shown in .
The stoichiometries of binding to ribosomes under the conditions used
for cross-linking were determined by nitrocellulose filter binding and
are summarized in . In all cases, more than one mRNA binds
per ribosome at saturation. Binding isotherms were examined to estimate
dissociation constants assuming independent sites. For the mRNA
analogue with sU at position -2 with respect to the
sequence ACC (designated s
U(-2)),
K
values of 86 nM (for both
sites) without tRNA
and 19 nM and 77 nM
with tRNA
were determined. For the mRNA analogue with
s
U at position +11 (designated
s
U(+11)), K
values of
104 nM and 178 nM without and 28 nM and 93
nM with tRNA
were determined. To test if the
presence of the two types of mRNA binding sites reflected ribosome
heterogeneity, tRNA binding was measured at an mRNA analogue
concentration that resulted in an average of 1 mRNA analogue/ribosome.
tRNA binding was found to be at least 0.85 tRNA/ribosome, all confined
to the P-site (results not shown). Thus, all ribosomes contain a strong
binding site for these mRNA analogues.
U-containing mRNA or s
U-containing mRNA
derivatized with APAB were irradiated with 320-365 nm wavelength
light. Complexes with mRNA analogues containing uridine were irradiated
as control samples. Electrophoresis of purified RNA from these samples
revealed that mRNA becomes extensively cross-linked to 16 S rRNA with
smaller cross-linking to 23 S rRNA ( Fig. 1and I).
The presence of tRNA increases cross-linking from 3- to nearly 5-fold
in 16 S rRNA for both the(-2) and (+11) mRNA analogues.
Cross-linking to 23 S rRNA is independent of the presence of tRNA.
Binding and cross-linking of s
U-containing mRNA analogues
can be competed by uridine-containing mRNA analogues (results not
shown).
Figure 1:
Agarose gel electrophoresis of RNA
after cross-linking reactions between P-labeled mRNA and
ribosomes. Experiments are shown with mRNA analogues with uridine or
s
U at positions -2 or +11 counting from the
first position of the sequence ACC (the codon for tRNA
).
The mRNA analogues contain uridine (U) for control, s
U, or
s
U modified with azidophenylacyl bromide (APAB). Complexes
contained tRNA
as indicated. The positions of 23 S rRNA,
16 S rRNA, and mRNA are indicated.
Reverse Transcription Analysis
Reverse
transcription experiments were done to determine the location of the
mRNA cross-links in the 16 S rRNA. The mRNA analogue
sU(+11) cross-links in the interval 1389 to 1393 to
C
(the predominant site) and to C
(Fig. 2). The frequencies of all of these increase
significantly when tRNA is present as measured as the ratio of the band
intensity in the cross-linked sample over that in the control sample
(). When s
U(+11)-APAB is used in the
cross-linking reaction, C
and C
are still
present as prominent cross-linking sites and there is a new site at
A
. The presence of tRNA slightly decreases the extent of
cross-linking by s
U(+11)-APAB, except at A
where there is a large increase. For mRNA analogue
s
U(-2), one cross-linking site at U
is
seen in this region, and this does not change significantly in extent
when tRNA is present ( Fig. 2and ). Reactions with
the mRNA analogue s
U(-2)-APAB show lower amounts of
cross-linking at U
, presumably because the
azidophenylacyl moiety interferes with the mRNA-rRNA interaction at
this site, and the appearance of a new cross-link at U
.
Because the stoichiometry of the mRNA binding exceeded 1 mol/mol 70 S,
the experiments were also done with lower mRNA input levels; this
reduces the amount of covalent cross-linking but does not change the
pattern (results not shown).
Figure 2:
Reverse transcription analysis of the
pattern of mRNA cross-linking in the 16 S rRNA region 1417-1365.
Samples were prepared from cross-linking reactions containing mRNA
analogues containing sU, containing s
U
substituted with APAB (written as APA), or containing uridine
and with or without tRNA
as indicated. Reverse
transcription samples in the left part of the gel (lanes
O, A, G, C, and U) were
prepared with total un-irradiated RNA from 70 S ribosomes without
dideoxynucleotide or with ddTTP, ddCTP, ddGTP, or ddATP. Nucleotide
numbering on the right of the gel indicates stopping positions
attributed to the presence of mRNA cross-links; the identity of the
nucleotide 5` to the stopping site is
written.
mRNA analogue sU(+11)
cross-links to sites in the 16 S rRNA at A
and U
with lower intensity sites at A
, U
,
and A
(Fig. 3). All of these show increases when
tRNA is present. Cross-linking in this region is somewhat greater with
mRNA analogue s
U(+11)-APAB but, in this case,
increases in cross-linking upon tRNA addition are smaller.
Figure 3:
Reverse transcription analysis of the
pattern of mRNA cross-linking in the 16 S rRNA region 539-515.
Sample lanes from sU-containing mRNA analogues,
s
U-containing mRNA analogue derivatized with APAB
(APA), or with uridine-containing mRNA analogue are indicated.
The sample O` is a control made with total RNA from irradiated
70 S ribosomes that did not contain mRNA or tRNA. The positions that
indicate cross-links at U
and A
are
indicated. For the sample s
U(+11) with
tRNA
, there are also cross-links at A
and
U
.
Three
additional regions around A, U
, and
A
contain cross-linking sites (Fig. 4). The sites
A
and U
occur with mRNA analogue
s
U(-2) and s
U(-2)-APAB. All of
these show little change in extent when tRNA is present. Finally, a
cross-linking site at A
occurs in mRNA analogue
s
U(+11) (Fig. 4C). This site shows the
same behavior with respect to tRNA dependence as the sites at
C
-A
and around A
. With mRNA
analogue s
U(+11)-APAB, three additional sites
C
, U
, and G
are also seen
and these also increase when tRNA is present.
Figure 4:
Reverse transcription analysis of the
pattern of mRNA cross-linking in three 16 S rRNA regions. A shows the region 860 to 826 containing the cross-linking site at
A. B shows the region 748 to 695 which contains
the cross-linking site at U
. C shows the region
1205 to 1185 which contains the cross-linking site at A
.
In C, positions C
, U
, and
G
are also cross-linking sites for
s
U(+11) derivatized with APAB. The lanes are
indicated as in Fig. 3.
The experiments
involving mRNA sU(-2) have also been performed by
isolating stable complexes by sedimentation before cross-linking. This
decreases the amount of mRNA bound to the 70 S ribosomes particularly
in the absence of tRNA as described before
(13) . However, it was
still possible to detect all cross-links including the tRNA independent
sites (U
, A
, and U
) without
tRNA although there are much lower levels of cross-linking in those
complexes, reflecting the lower levels of bound mRNA in the absence of
tRNA (data not shown).
Determination of Cross-linking Sites in the 3`-Terminal
Region
The terminal 30 nucleotides of the 16 S rRNA cannot be
examined by reverse transcription because of mA
at positions 1518 and A
. Since Rinke-Appel et
al.(13) reported cross-linking to this region with mRNA
analogue s
U(-2), levels of cross-linking were
determined using RNase H cleavage with an oligonucleotide complementary
to 1426-1437. If the 16 S rRNA were cross-linked to
P-labeled mRNA, a fragment longer than expected would be
released and it would be radioactive. This is the case and levels of
cross-linking were determined from an autoradiogram of a polyacrylamide
gel (results not shown). Cross-linking in the terminal 107 nt occurred
only with mRNA analogue s
U(-2); in cross-linking
reactions without tRNA, the extent was 4%, and, in reactions with tRNA,
the extent was 39% ().
U(-2) and purification of 16 S-sized RNA, RNA was
3`-end-labeled. RNase H cleavage at position 1435 of 16 S-sized RNA
from cross-linking reactions releases two prominent products, one of
which has the same gel mobility as the 16 S rRNA terminal 107 nt, and
the other has a significantly slower mobility (Fig. 5, lane
5). When an oligonucleotide complementary to the mRNA was included
in the RNase H digestion to reduce the size of the mRNA and remove its
3` end label, the slower product increases in mobility but still is
different from the terminal 107 nt of the 16 S rRNA (Fig. 5,
lane 6). The control fragment containing the terminal 107 nt
and the fragment containing the cross-linked RNA were isolated and
subjected to partial hydrolysis and RNase T
and RNase
U
digestion to find the site of the cross-link. In this
comparison, the cross-linked sample and control sample should show the
same pattern up to the last nucleotide before the cross-linking site.
The patterns are very similar to position C
and then the
pattern in the cross-linked sample contains a gap (Fig. 6). The
intensity of the partial hydrolysis product at C
is
significantly less than expected indicating a cross-link at C
and the band expected at U
is completely absent.
Given the sequence of the residual mRNA fragment still attached to the
terminal 107 nt of the 16 S rRNA, the patterns of partial RNase T
and RNase U
digestion are consistent with the
assignment. Densitometry was done to estimate the amount of
cross-linking at each of these sites (). The location of
these and the other cross-linking sites are indicated in the 16 S rRNA
secondary structure in Fig. 7.
Figure 5:
Analysis of cross-linking to the
3`-terminal region of 16 S rRNA using RNase H digestion. Control 16 S
rRNA or 16 S rRNA from ribosomes cross-linked with mRNA analogue
sU(-2) were purified, 3`-end-labeled, and then
subjected to RNase H digestion using oligonucleotides complementary to
16 S rRNA (positions 1426-1437) and mRNA (positions) as
indicated. The gel used was a denaturing gel of two parts: 4%
polyacrylamide on top and 12% polyacrylamide on bottom; sections
showing the 16 S rRNA and relevant fragments are shown. RNA from the
bands containing the 107-nt fragment from control 16 S rRNA and
containing the terminal 107-nt fragment cross-linked to the residual
mRNA was isolated for sequencing analysis.
Figure 6:
Partial hydrolysis reaction on the
3`-terminal region of 16 S rRNA to determine the point of mRNA
cross-linking. The control RNA fragment and RNA fragment containing the
mRNA cross-link were subjected to partial hydrolysis
(H), partial digestion with RNase T
(T
), or RNase U
(U
) as indicated. The band that corresponds
to the fragment whose 5`-nucleotide is C
is
indicated.
Figure 7:
Location of mRNA cross-linking sites in
the 16 S rRNA secondary structure (49). The mRNA cross-linking sites
that change in frequency by more than 50% in the presence of tRNA are
shown by solid dots, and those that are independent of tRNA
are indicated by open dots. All of the cross-links are made by
sU at (-2) or (+11) in the mRNA analogues except
those designated with an asterisk which only appeared if the
s
U was additionally derivatized with
APAB.
and A
by the mRNA
analogue s
U(+11), and cross-linking to the 3`-terminal
region by mRNA analogue s
U(-2). There are additional
cross-linking sites in the neighborhoods of these prominent cross-links
detected by reverse transcription (, Fig. 7).
Subsequently, a cross-link at U
was reported from
position +6 in another mRNA analogue
(14) . We do not detect
U
but instead detect A
(this report and
Ref. 17) which is very close to U
in the secondary
structure; these probably represent the same mRNA-16 S rRNA interaction
region.
, U
, and A
are also
detected. All of these sites are made by mRNA analogue
s
U(-2) and its APAB derivative and are nearly tRNA
independent. These sites as well as the tRNA-dependent cross-linking
sites have also been detected using other mRNA analogues
(17) which were both longer and lower in purine base composition
than the molecules s
U(-2) and
s
U(+11). An apparent positional ambiguity of the mRNA
analogues in the mRNA track was determined in our previous experiments
due to the possibility of conformational flexibility in the 30 S
subunit
(17) . The same phenomenon may occur with these two mRNA
analogues; thus, although the mRNA analogue s
U(+11)
makes cross-links with A
, A
, and
C
, this does not necessarily mean these sites are direct
neighbors of one another.
ribosome complexes before cross-linking, and
this decreases the amount of bound mRNA particularly in those complexes
formed without tRNA. In our case, ribosomes in equilibrium with unbound
mRNA and tRNA typically were irradiated, allowing a larger extent of
mRNA binding and cross-linking both with and without tRNA. This has
allowed the recognition of both tRNA-dependent and tRNA-independent
cross-linking sites. The dependence of mRNA binding on tRNA is
consistent with the behavior of poly(U) and tRNA
under
subsaturation conditions observed by Katunin et
al.(21) . Weller and Hill
(30) have shown recently
that competition between oligo(U) and a DNA oligonucleotide
complementary to 16 S rRNA interval 1396-1403 occurs only if
tRNA
is present, again indicating a cooperativity between
mRNA and tRNA binding.
has been associated with protein
S4 by a number of experiments
(31, 32, 33) , and
S4, in turn, has been located in the indentation between the head and
body of the 30 S subunit on the side away from the platform
region
(34) . Sites close to A
have been
associated with protein S5 (see Ref. 35). S5 has been located in the
middle of the subunit on the cytoplasmic side, close to S4
(34) .
The sites at U
to C
are in the vicinity
of the codon-anticodon interaction in the cleft region evidenced by the
anticodon-rRNA cross-link at C
in the 16 S rRNA
(5) and the determination of this site by electron
microscopy
(36) . Likewise, the site at
C
/U
is in the vicinity of the
codon/anticodon interaction because of cross-links between this region
and proteins S7, S18, and S21
(32) and the locations of these
proteins
(34) . Thus, the four tRNA-dependent sites break down
into two groups: the 532 and 1196 regions in the S4 subdomain and the
1395 and 1533 regions close to the P-site codon/anticodon interaction.
There has been some differing information about the closeness of these
two regions. On the one hand, a large distance between protein S4 and
S7 has been determined by neutron scattering (34) and electron
microscopy
(37) . On the other hand, there is evidence intimately
linking the 530 region with the decoding process including evidence
that tRNA in both the A-site and P-site produces footprints in the
interval 529-532
(23) , that nucleotide changes in the 523
to 530 region strongly affect translational proofreading and ribosome
function (38-42), and that the 530 region is involved in EF-Tu
function
(43, 44) . In addition, Alexander et
al.(45) have recently shown that protein S7 and the
3`-terminal region of the 16 S rRNA can be labeled by a photoaffinity
probe annealed to the 16 S rRNA in the interval 518-526,
indicating a distance of no greater than 24 Å between C
and the 16 S terminal region and protein S7.
is in the region associated with protein S7
(32, 46) the location of which has been determined on the side of
the head of the subunit facing the cleft region. The cross-linking
sites U
and A
are in the rRNA regions
associated with S6, S18, S15, and S8
(32) , and these have been
located in the side of the body next to the decoding
region
(34) . There are large distances between S7 and the other
group of proteins suggesting that this part of the mRNA track is quite
wide.
U cross-linking efficiency is
greater at interactions that have structural mobility
(47) , but
this would be contrary to the increase in mRNA binding strength and the
fact that the mRNA derivatized with APA behaves in a similar manner.
Another possibility is that tRNA binding subtly alters the orientation
of the mRNA analogue on the subunit. A specific change in the
arrangement of s
U at -2 with respect to the
U
/C
site may occur because tRNA binding,
together with the Shine-Dalgarno interaction, would constrain the mRNA.
However, a constraint imposed by tRNA binding is unlikely to account
for the behavior of s
U(+11) because of the distance
between s
U at position +11 and the ACC codon. The last
possibility is that there is a conformational change in the 30 S
subunit upon tRNA binding that results in greater exposure of the
reactive 16 S rRNA sites. This would account for the increase in
cross-linking of s
U at position +11 as well as at
position -2 and would also be consistent with the increase in
mRNA binding strength, if there is an associated increase in favorable
mRNA-30 S subunit interactions. Since the tRNA-dependent mRNA
cross-links are located at the interface between the body and head of
the 30 S subunit, some movement of the head of the 30 S subunit may be
involved in this change to alter the accessibility of these rRNA sites.
Huttenhofer and Noller
(48) recently determined the protection
of mRNA analogues on the 30 S subunit from hydroxyl radical cleavage
and found that the protection increased from about 40 nt to 54 nt when
tRNA was present. Their interpretation was that the codon-anticodon
interaction influenced the mRNA pathway, particularly past position
+6 from the codon. Their observation is also consistent with a
tRNA-dependent strengthening and alteration in the mRNA-30 S subunit
interaction indicated by the present cross-linking results.
Table:
Sequences of the mRNA analogues
sU(-2) and s
U(+11)
U(-2) and s
U(+11) are the same
sequences as the molecules ``mRNA No. 1'' from Stade et
al. (12) and ``I.'' from Rinke-Appel et al. (13). Numbering of the positions in the mRNA analogues is from the
first nucleotide of codon for tRNA
.
Table:
Stoichiometry of binding mRNA
sU(-2) and s
U(+11) to 70 S ribosomes
without and with cognate tRNA
molar excess of
mRNA analogue over 70 S ribosome. When present, tRNA
was
at 5
molar excess over 70 S ribosomes.
Table:
Extent of cross-linking mRNA analogues
sU(-2) and s
U(+11) to 70 S ribosomes
without and with tRNA
Table:
Summary of the 16 S rRNA cross-linking
sites and relative extent of cross-linking
. For all cross-linking sites, it was
assumed that reverse transcriptase stops completely at the
3`-nucleotide to the cross-link. Positions that had an increase in
relative band intensity values of at least 1.5 either without or with
tRNA are listed. Empty areas indicate no cross-links under any
condition. ND, is not determined. The most frequent cross-linking site
in each group is underlined.
U, 4-thiouridine; APAB (or APA),
azidophenylacyl bromide; APM,
[(N-acryloylamino)phenyl]mercuric chloride; dsDNA,
double-stranded DNA; nt, nucleotide(s); TBE, 89 mM Tris, 89
mM boric acid, 10 mM EDTA.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.