(Received for publication, October 3, 1994)
From the
Ribosomal protein S4 protects a characteristic set of bases in
16 S rRNA from attack by chemical probes. Use of hydroxyl radical as a
probe of the RNA backbone shows that ribose residues in these same
regions are also protected by S4, confirming the localization of its
interactions with 16 S rRNA to the junction of five helical elements in
the proximal region of the 5` domain. At 0 °C, the nucleotides
protected by S4 from base-specific probes are confined almost
exclusively to the two compound helices formed by residues
404-499. After subsequent heating of the complex briefly at 30 or
42 °C, nucleotides in the three adjacent helices are additionally
protected, resulting in a pattern of protection that is identical to
that which is observed when S4 is incubated with 16 S rRNA under in
vitro reconstitution conditions. Preincubation of the protein or
the RNA (or both) separately at elevated temperature does not
substitute for heating the S4RNA complex. The regions in the RNA
affected by the heat step are known to interact with proteins S12 and
S16, both of which depend upon S4 for their binding to the RNA. Thus,
the finding that S4 recruits additional sites of interaction in the RNA
following its initial binding suggests a possible mechanism to insure
the sequential addition of proteins during ribosomal assembly.
Ribosomal protein S4 is one of the most extensively studied of
the small subunit proteins that bind directly and independently to 16 S
rRNA. In addition to its central role in ribosome assembly, it also
functions as a translational repressor by binding to its own mRNA
(Nomura et al., 1984) and has long served as a model for the
study of protein-RNA interaction (for review, see Zimmermann(1980) and
Garrett et al.(1984)). The first experiments attempting to
localize the RNA binding site for S4 involved partial ribonuclease
digestion of S416 S rRNA complexes and sequence analysis of the
resulting protein-protected RNA fragments (Schaup et al.,
1971; Zimmermann et al., 1972; Ungewickell et al.,
1975). The protected RNA encompassed several hundred nucleotides of the
5` region of 16 S rRNA, unexpectedly large to be the binding site of a
protein of mass 23,000. However, almost exactly the same subfragments
of 16 S rRNA were protected in the absence of protein, under somewhat
milder digestion conditions, using carrier-bound nuclease (Ehresmann et al., 1977). This result suggested that much of what had
been attributed to protection of 16 S rRNA by protein S4 was in fact
due to self-structuring of the RNA; its 5` region was inherently
nuclease-resistant (Garrett et al., 1977). There remained the
question of which specific structural features of the 5` region
actually interact with S4.
Two approaches were devised to address
this question. Development of rapid chemical and enzymatic probing
methods (Moazed et al., 1986; Stern et al., 1988b)
made it possible to examine protein-RNA interactions at
single-nucleotide resolution using microgram amounts of material.
Examination of the S416 S rRNA complex using this approach showed
that only a modest number of bases in the 5` domain were protected by
bound S4 (Stern et al., 1986, 1988a). The protected bases were
confined to a limited region of the domain comprising the junction of
five helical elements, which was termed the ``S4 junction.''
This region of the RNA lies within the RNase-resistant fragments
characterized in the earlier nuclease protection studies, but it
defines a compact RNA substructure that could more plausibly constitute
the actual binding site for a protein of the size of S4. Direct
cross-linking of S4 to one of the helices that forms the S4 junction
supports this assignment (Greuer et al., 1987).
Using another approach, Vartikar and Draper(1988) studied the RNA binding site requirements for protein S4 by constructing synthetic subfragments of 16 S rRNA using in vitro transcription and testing the ability of the synthetic rRNA analogues to bind S4. They found that deletion of regions of the RNA containing two of the five implicated helical elements had little or no effect on the specific binding of S4 to the RNA, in apparent contradiction to our findings.
In the
present study, interaction of S4 with 16 S rRNA is reexamined. The
results of hydroxyl radical protection, which probes interactions
involving the RNA backbone, strongly support the conclusions from our
earlier studies; all of the S4-dependent protections of the RNA
backbone are localized to the S4 junction region. In addition, we find
that S4RNA interaction involves two separable steps; using
base-specific probes, one set of protections is observed at 0 °C,
while an additional set is seen only after incubation of the complex at
elevated temperature. The 0 °C effects involve only nucleotides
that are located in the subfragments identified by Draper and
co-workers (Sapag etal., 1990), while the
heat-dependent protections include all of the protected nucleotide
positions that lie outside of their RNA binding fragments. Since the
latter authors carried out their binding reactions at 0 °C, our
data appear to resolve the apparent contradiction. The ability of
protein S4 to participate in two distinct and separable interactions
with different structural features of 16 S rRNA raises new questions
concerning mechanisms of RNA-protein interactions in the ribosome.
Ribosomes, 30 S ribosomal subunits, 30 S ribosomal proteins,
and 16 S rRNA were isolated as described previously (Stern et
al., 1988b). Complexes between 16 S rRNA and S4 were formed at 0
°C by combining 5 µg of 16 S rRNA with 4 mol eq of purified S4
protein in 25 µl of reconstitution buffer containing 80 mM potassium Hepes (pH 7.6), 20 mM MgCl, 300
mM KCl, 6 mM 2-mercaptoethanol, 0.01% (w/v) Nikkol
detergent (octaethylene glycol mono-n-dodecyl ether; Nikko
Chemical Co. Ltd.), followed by incubation at either 0 °C for 60
min, 30 °C for 10 min, or 42 °C for 30 min. The resulting
complexes were incubated for an additional 30 min at 0 °C prior to
the addition of chemical modification reagents. For comparison, 5
µg of naked 16 S rRNA was also probed in parallel, following
incubation first at 42 °C for 30 min and then at 0 °C for 30
min.
Dimethyl sulfate and kethoxal modifications were performed at 0
°C as described previously (Stern et al., 1988b).
Fe(II)-EDTA-generated hydroxyl radical cleavage reactions were
performed essentially as described by Cech and co-workers (Latham and
Cech, 1989; Celander and Cech, 1990) and by Tullius and co-workers
(Tullius and Dombroski, 1986; Burkhoff and Tullius, 1987). 1 µl of
each of the following four solutions (prepared freshly prior to each
experiment) was added to the top of a 1.5-ml Eppendorf tube, containing
25 µl of sample, and mixed by centrifugation for 2-5 s in an
Eppendorf microcentrifuge (all procedures were carried out at 4
°C): 1) 50 mM Fe(NH)
(SO
)
6H
O
(final concentration, 2 mM); 2) 100 mM EDTA (final
concentration, 4 mM); 3) 250 mM ascorbic acid (final
concentration, 10 mM); 4) 2.5% H
O
(final concentration, 0.1%). Samples were then incubated on ice
for 10 min, and the cleavage reaction was stopped by addition of 10
µl of 0.1 M thiourea. Samples were precipitated by the
addition of 25 µl of 0.3 M sodium acetate (pH 6.5) and 200
µl of 95% ethanol. After modification, 16 S rRNA was repurified by
phenol extraction, and the sites of modification and cleavage were
determined by primer extension and gel electrophoresis as described
previously (Stern et al., 1988b).
Figure 1: a-e, results of DMS and kethoxal (KE) probing of 16 S rRNA following incubation with protein S4 at different temperatures. Lane1, naked 16 S rRNA; lane2, 16 S rRNA + S4, incubated at 0 °C; lane3, 16 S rRNA + S4, incubated at 30 °C; lane4, 16 S rRNA + S4, incubated at 42 °C. f, control experiment demonstrating that the effects observed with 16 S rRNA + S4 at 30 °C and 42 °C require heating both the RNA and protein together. Lane1, unmodified 16 S rRNA; lane2, naked 16 S rRNA; lane3, 16 S rRNA + S4, incubated together at 0 °C; lane4, 16 S rRNA incubated alone at 42 °C and then incubated with S4 at 0 °C; lane5, S4 incubated alone at 42 °C and then incubated with 16 S rRNA at 0 °C; lane6, 16 S rRNA and S4 each heated separately at 42 °C and then incubated together at 0 °C; lane7, 16 S rRNA + S4, incubated together at 42 °C. Samples in lanes2-7 were modified with DMS at 0 °C. Dots on the left are positioned every 10th nucleotide. Primers used are as follows: a and f, 683; b, 480; c, 162; d, 1046; e, 1257.
Maximum
binding of S4 to 16 S rRNA has been shown to require heating of the
complex to temperatures above 30 °C (Schulte et al.,
1974). The temperature-dependent differences that we observe do not
simply reflect an overall increase in the number of stable complexes
formed, however, as complete protection of many residues in 16 S rRNA
is observed in the complex formed at 0 °C, including
A, G
, and A
(Fig. 1a; Table 1). Interestingly, the
reactivities of A
and G
are enhanced in the
presence of S4 at 0 °C but are then protected when the complex is
heated (Fig. 1, a and c, respectively),
suggesting that transient protein-induced conformational alterations in
RNA structure accompany assembly of S4.
A trivial explanation for these effects is that the heating step renatures either S4 or 16 S rRNA or both. To test this possibility, complexes were formed after heating one or both components individually at 42 °C, followed by incubating them together at 0 °C. Only when the RNA and protein are heated together are these additional protections and enhancements observed (Fig. 1f; compare lanes3-6 with lane7; for simplicity, only DMS probing data for the 500 region are shown). These results demonstrate that the heat step is not required simply for renaturation of one of the components but rather for a structural rearrangement in the assembling ribonucleoprotein.
Figure 2:
Results of Fe(II)-EDTA generated hydroxyl
radical probing of the S416 S rRNA complex. Lanes1 and 4, unmodified 16 S rRNA; lanes2 and 5, naked 16 S rRNA; lanes3 and 6, 16 S rRNA + S4. Samples where incubated either at 0
°C (lanes1-3) or 42 °C (lanes4-6) followed by modification with hydroxyl
radicals at 0 °C, as described under ``Experimental
Procedures.'' Primers used are as follows: a, 480; b, and c, 565; d, 161. Other symbols are as
described in the legend to Fig. 1.
When naked 16 S rRNA is treated with hydroxyl radicals, strand
scission occurs at essentially every position, generating a nearly
uniform cleavage pattern (Fig. 2; lanes2 and 5). Following incubation with S4 at 42 °C, strong
protection against cleavage is observed around position A (Fig. 2a). Several other regions in the 5` domain
also show significant protection by S4 (Fig. 2). Significantly,
all residues protected by S4 from hydroxyl radical attack are located
exclusively within the S4 junction (see Fig. 4). Unlike the
behavior observed for the base-specific probes, protection of the RNA
by S4 from hydroxyl radical attack was independent of temperature;
identical results were obtained whether the probing was done at 0 or 42
°C (Fig. 2).
Figure 4:
Schematic secondary structure of the S4
junction in the 5` domain of 16 S rRNA (Stern et al., 1986),
summarizing results of probing the S416 S rRNA complex with
hydroxyl radicals. Circles indicate reduced reactivities in
the S4-16 S rRNA complex versus naked 16 S rRNA. Smallsymbols indicate weak effects. Similar results
were obtained when the complex was formed at 0 or 42 °C. Data are
from Fig. 2.
Weak protection of bases in the 500 region
by S4 against hydroxyl radical attack was surprising in light of the
strong protections this protein affords against base-specific probes
(compare Fig. 1a and Fig. 2c). To test
the possibility that hydroxyl radicals disrupt the 16 S rRNAS4
complex, we first modified the complex with Fe(II)-EDTA, followed by
treatment of this same sample with DMS. No decrease in the level of
protection of residues in 16 S rRNA from DMS attack by S4 is observed
after prior modification of the 16 S rRNA
S4 complex with
Fe(II)-EDTA, indicating that hydroxyl radicals do not, in fact, perturb
the interaction between 16 S rRNA and S4 (data not shown).
These findings confirm our earlier conclusions that the
principle interactions between protein S4 and 16 S rRNA are localized
to a relatively compact region of the 5` domain, which we call the S4
junction (Stern et al., 1986, 1988a) ( Fig. 3and 4). In
our earlier work, a significant number of S4-dependent changes in the
chemical probing pattern involved bases outside of the S4 junction. We
reasoned that most of the latter were unlikely to interact with S4,
based on the findings that they 1) were not contained within the large
nuclease-resistant S4 binding-site fragments or 2) showed increased
reactivity in the presence of bound S4, indicating an S4-dependent
conformational change. Conversely, we argued that clusters of strongly
protected bases would be likely indicators of S4RNA interactions.
Use of hydroxyl radical as a probe of the RNA backbone is a method that
is complementary to base-specific probing and carries with it the
advantage that it is not susceptible to certain ambiguities of
interpretation of the latter approach such as, for example,
protein-dependent base pair formation. However, the fact that tertiary
folding of tRNA results in some protection of its backbone from
hydroxyl radical attack (Latham and Cech, 1989) shows that some degree
of caution must be maintained. In this work, we observed no
S4-dependent protection of 16 S rRNA from hydroxyl radical outside of
the S4 junction region (Fig. 4). This is consistent with our
earlier interpretation that this region of the RNA contains most of the
important contacts with S4. It is still possible, however, that the
protein could interact with certain RNA bases without affecting the
reactivities of their associated or neighboring ribose moieties.
Figure 3:
Schematic secondary structure of the S4
junction in the 5` domain of 16 S rRNA (Stern et al., 1986),
summarizing results of probing the S4-16 S rRNA complex with
base-specific probes DMS and kethoxal. 0 °C, reactivity
differences in the S416 S rRNA complex formed at 0 °C versus naked 16 S rRNA; 42 °C, reactivity
differences in the S4
16 S rRNA complex formed at 42 °C (or 30
°C) versus the complex formed at 0 °C. Circles and triangles indicate reduced and enhanced reactivities,
respectively. Smallsymbols indicate weak effects.
Data are from Fig. 1and Table 1.
Another approach to analysis of the S4 binding site was taken by Vartikar and Draper(1989). These authors constructed a series of 12 fragments of 16 S rRNA by in vitro transcription of deleted versions of the 16 S rRNA gene and tested the ability of the fragments to bind S4. Their findings were generally consistent with our interpretation, with three main exceptions. 1) Full binding activity was observed with a fragment spanning positions 1-559 in which residues 501-544 had been deleted; this deletion removes several S4-protected nucleotides in the 501-510 region, involving one of the five junction helices (Fig. 3). 2) Full binding activity was also observed for a fragment comprising residues 39-559; this fragment excludes another of the junction helices, which contains protections involving positions 30-33 (Fig. 3). 3) A fragment spanning residues 27-559, but missing residues 53-352, showed sharply decreased binding, although the deleted regions lie outside the junction proper (Fig. 3).
One
significant difference between the experiments of Vartikar and
Draper(1989) and our own studies concerns the temperature of the
binding reaction. Binding was carried out in one case at 0 °C
(Vartikar and Draper, 1989) and in the other at 30 or 42 °C,
similar to temperatures used for in vitro reconstitution of 30
S subunits (Stern et al., 1986, 1988a). When we reexamined the
protection of 16 S rRNA from base-specific probes by S4 as a function
of the temperature of the binding reaction, the results were striking (Fig. 3). At 0 °C, almost no effects are observed in the two
helices (30/550 and 510/540 regions, respectively) that were disrupted
in two of the experiments of Vartikar and Draper(1989); after
incubation at 30 or 42 °C, an extensive set of additional
nucleotides is protected, many of which are found in the two helices in
question (Fig. 3). Thus, the results of Vartikar and
Draper(1989) are entirely consistent with the results of chemical
probing for the 0 °C complex. However, it is clear that there is
also a second, heat-dependent event, which must involve conformational
rearrangement of the protein-RNA complex, since preincubation of the
isolated components at 42 °C prior to binding is insufficient to
effect the second step of S4RNA interaction. Whether this second
step affects the association constant for the interaction is not
addressed by our chemical probing studies. However, early experiments
by Schulte et al.(1974) show that heating of the S4
RNA
complex to temperatures of 30 °C or higher was required for maximal
binding, suggesting that the second step may contribute to the
stability of the interaction.
In contrast to results using base-specific probes, temperature has no detectable effect on the protection by S4 from hydroxyl radicals (Fig. 2). Since hydroxyl radicals are believed to attack the ribose moiety of RNA, we conclude that the heat-dependent conformational change in the S4 ribonucleoprotein results in no changes in the accessibilities of riboses. This suggests that the temperature-dependent differences seen for the base-specific probing are caused by changes in base-base or base-protein interactions.
Ofengand and co-workers (Weitzmann etal., 1993) have recently investigated the ability of a synthetic RNA fragment, corresponding to residues 1-526 of 16 S rRNA, to self-assemble into a ribosomal subdomain, following reconstitution with total 30 S proteins at 42 °C. Four proteins were found to bind specifically to this RNA fragment, S4, S16, S17, and S20; however, the stoichiometry of S4 was found to be significantly lower compared with the other three proteins. Since the 30/550 and 510/540 helices are disrupted in this RNA, these findings are consistent with their possible importance for the stable binding of S4 to 16 S rRNA.
It is less obvious why deletion of residues 53-352 from the 27-559 fragment should cause such a strong decrease in S4 binding if in fact its binding site lies completely within the boundaries of the junction region. One possible explanation is that some features of the lower half of the 5` domain make RNA-RNA interactions with the S4 junction region that help to form or stabilize its S4-compatible conformation. Indeed, partial nuclease digestion experiments suggested that the upper and lower halves of the 5` domain could be held together by noncovalent interactions (Ehresmann et al., 1977). Another possibility is that the deleted fragment folds anomalously, thereby perturbing the S4 binding site indirectly. Such an explanation might also account for the deleterious effects of other deletions in 16 S rRNA outside of the S4 junction (Sapag et al., 1990).
The temperature-dependent conformational change
reported here provides support for an earlier suggestion for the
mechanism of sequential assembly of ribosomal proteins (Stern et
al., 1989). Proteins S12 and S16 depend on prior binding of S4 to
16 S rRNA for their own assembly (Held et al., 1974). Among
the characteristic effects of these two proteins is protection of
groups of bases in the 520 and 360 regions, respectively (Stern et
al., 1989). However, these bases are unreactive in naked 16 S
rRNA, and only become reactive during the heat-dependent step of S4
assembly (Table 1). Thus, it may be that the conformational
rearrangement of the S416 S rRNA complex observed here is an
obligatory step leading to the assembly of the latter two proteins.
Finally, it seems likely that similar effects will be found upon
detailed examination of other ribosomal protein-rRNA interactions, and
may even turn out to be a common device in orchestrating an orderly,
sequential pathway for ribosome assembly.
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