(Received for publication, August 8, 1995; and in revised form, September 13, 1995)
From the
Xenopus ribosomal protein L5 was expressed in Escherichia coli and exhibits high affinity (K = 2 nM) and
specificity for oocyte 5 S rRNA. The pH dependence of the association
constant for the complex reveals an ionization with a
pK
value of 10.1, indicating that
tyrosine and/or lysine residues are important for specific binding of
L5 to the RNA. Formation of the L5
5 S rRNA complex is remarkably
insensitive to ionic strength, providing evidence that nonelectrostatic
interactions make significant contributions to binding. Together, these
results suggest that one or more tyrosine residues may form critical
contacts through stacking interactions with bases in the RNA. In order
to locate recognition elements within 5 S rRNA, we measured binding of
L5 to a collection of site-specific mutants. Mutations in the RNA that
affected the interaction are confined to the hairpin structure
comprised of helix III and loop C. Earlier experiments with a rhodium
structural probe had shown that the two-nucleotide bulge in helix III
and the intrinsic structure of loop C create sites in the major groove
that are opened and accessible to stacking interactions with the metal
complex. In the present studies, we detect a correlation between the
intercalative binding of the rhodium complex to mutants in the hairpin
and binding of L5, supporting the proposal that binding of the protein
is mediated, in some part, by stacking interactions. Furthermore, the
results from mutagenesis establish that, despite overlapping binding
sites on 5 S rRNA, L5 and transcription factor IIIA utilize distinct
structural elements for recognition.
The metabolism of Xenopus 5 S ribosomal RNA during
oogenesis provides the opportunity to study the interaction of several
different proteins with the same nucleic acid. Moreover, these
individual ribonucleoprotein complexes appear to determine the
intracellular translocation of 5 S rRNA. Initially, the primary
transcripts are transiently associated with the La antigen in the
nucleus(1, 2) . However, the synthesis of 5 S rRNA and
ribosome assembly are discontinuous in the early stages of oogenesis,
so that much of the RNA is stored in the cytoplasm complexed either
with transcription factor IIIA (TFIIIA) ()or with the
protein p43 as part of a large multicomponent 42 S RNP
complex(3, 4) . Coincident with the expression of the
ribosomal proteins during vitellogenesis, an increasing amount of
cytoplasmic 5 S rRNA becomes associated with ribosomal protein
L5(5) . This latter complex then moves to the nucleolus, where
it becomes integrated into nascent ribosomes(1, 6) .
Mutant forms of 5 S rRNA that are unable to bind to TFIIIA or L5 are
retained in the nucleus of the oocyte, indicating that the RNA can only
be exported to the cytoplasm in the form of an RNP complex(2) .
Likewise, the ultimate return of cytoplasmic 5 S rRNA to the nucleolus
depends on the formation of the complex with
L5(6, 7) . It is not known how the formation of a
particular RNP particle creates a signal for nucleocytoplasmic
transport.
The interaction of TFIIIA with oocyte 5 S rRNA is the most thoroughly characterized of the four complexes. The factor makes multiple contacts over much of the nucleic acid through the nine zinc finger domains of the protein(8, 9) . Recognition is mediated by the secondary structure of the RNA with the major determinants located in a central domain composed of helix II-loop A-helix V-loop E(10) . The other protein involved in the storage of cytoplasmic 5 S rRNA, p43, also contains nine zinc finger domains; however, it shares only 33% amino acid identity with TFIIIA(11) . Similar to the case with TFIIIA, the identity elements for p43 appear to be dispersed over a considerable amount of the 5 S rRNA molecule including helices II, IV, and V as well as loop D(12) . Contacts made by p43 to helical regions of the RNA appear to be more sequence-dependent compared with those made by TFIIIA(12) . Notwithstanding the structural similarities between TFIIIA and p43 and their overlapping binding sites on 5 S rRNA, the determinants of high affinity binding are not identical for the two proteins.
Protection (13, 14, 15, 16) and interference (17) experiments have been used to determine the binding site
for rat ribosomal protein L5 or its yeast equivalent, YL3, on 5 S rRNA.
The cumulative results are in good agreement and indicate that helices
I, II, IV, and V, as well as loops D and E provide the major contact
surface for the ribosomal protein. Despite having nearly congruent
binding sites on 5 S rRNA, L5 and TFIIIA share no similarity in the
sequences of their amino acids(5, 18) . Thus, two
proteins have arisen independently to bind to the same RNA molecule and
present a means for the comparative analysis of RNA recognition by two
disparate proteins. In order to identify the important recognition
elements for Xenopus L5, we have measured binding of the
protein to a collection of site-specific mutants made in oocyte 5 S
rRNA. Additionally, we have taken the occasion to compare these results
with those from analogous experiments involving TFIIIA. ()
In standard binding assays, approximately 10 µg of fL5 in affinity column buffer was digested with 0.25-1.0 µg of Factor Xa at 0 °C for 1 h. Benzamidine was then added to a final concentration of 5 mM to inhibit further proteolysis. This protein solution was then diluted with binding buffer to the desired protein concentration.
Figure 1:
Expression and purification of fL5.
Protein samples were analyzed on a 10% SDS-polyacrylamide gel stained
with Coomassie Blue dye. Lane 1, molecular mass standards of
69, 45, and 29 kDa; lanes 2 and 3, extract prepared
from E. coli cells before and after induction of expression of
fL5 with isopropyl-1-thio--D-galactopyranoside,
respectively; lane 4, fL5 eluted from amylose affinity column; lane 5, digestion products of fL5 after treatment overnight at
0 °C with factor Xa (weight ratio of fL5 to Xa was 100). The
positions of the MBP polypeptide (a), L5 (b), and Xa (c) are indicated.
The fusion protein is prone to proteolytic degradation; therefore, cells collected by centrifugation were suspended in buffer containing a mixture of protease inhibitors. In addition, samples were continuously kept on ice during all steps of the purification. L5 appears to be easily oxidized, resulting in denatured, insoluble protein. For this reason, all buffers were degassed and contained 1 mM DTT. Cells were lysed using a French press rather than by sonication, since the latter method also appeared to promote oxidation of the protein. Despite these precautions, preparations of the protein, on average, were between 50 and 60% active as determined by Scatchard analysis of 5 S rRNA binding activity (results not shown). Protein that was below this level of activity or that yielded high molecular weight complexes in mobility shift gel assays was discarded.
After lysis and centrifugation to remove cellular debris, the crude cell extract was passed through an amylose affinity column, which was subsequently washed with buffer overnight. The retained fusion protein was eluted with buffer containing 10 mM maltose (Fig. 1, lane 4). Digestion of the purified fL5 with Factor Xa produces two polypeptide products having molecular masses of 42 and 34 kDa, which correspond to the MBP domain and L5, respectively (Fig. 1, lane 5). Generally, a small portion of fL5 was resistant to Xa cleavage, presumably due to denaturation and/or aggregation of the protein, which prevented access to the cleavage site. Intact L5 was purified after digestion of the fusion protein by chromatography over DEAE Sephacel as described in ``Experimental Procedures.'' No contaminating E. coli 5 S rRNA could be detected in samples of affinity-purified fL5 upon staining SDS-polyacrylamide gels with silver.
Figure 2: Binding of fL5 and L5 to 5 S rRNA. A, internally radiolabeled 5 S rRNA (10 nM) was incubated with 5, 20, 50, or 100 nM L5 (lanes 2-5) purified from the cleaved fusion protein by chromatography on DEAE-Sephacel or with the same concentrations of fL5 (lanes 6-9). Samples were analyzed by electrophoresis on 8% nondenaturing polyacrylamide gels followed by autoradiography. B, 5 S rRNA (10 nM) was incubated with 50 nM (lanes 1 and 2) or 20 nM (lanes 3 and 4) MBP in the absence (lanes 1 and 3) or presence (lanes 2 and 4) of an equal concentration of L5; lane 5, binding assay containing 50 nM fL5.
Figure 3:
Dependence of the K for the L5
5 S rRNA complex on pH and magnesium
concentration. A, apparent K
values were determined from binding isotherms measured at
appropriately buffered values of pH. The curve through the data points
represents the best fit for a process containing two ionizations with
pK
values of 5.4 and 10.1. The data point
at pH 9.5, represented by an open circle, was not included in
the curve fitting. B, apparent K
values were determined in standard binding buffer containing
increasing concentrations of MgCl
. Note that the abscissa of the plot is logarithmic
scale.
Magnesium moderately enhances binding of L5
to 5 S rRNA at low concentrations, but a decrease in affinity is
detectable at concentrations greater than 2 mM (Fig. 3B). At 8 mM magnesium, the
apparent K decreases by more than an factor of 10.
Yeh et al.(27) have shown that high concentrations of
magnesium promote dissociation of the yeast YL3
5 S rRNA complex.
Our standard binding assays contained 0.5 mM MgCl
.
Since the zinc finger domains of TFIIIA mediate binding of the factor
to nucleic acids, micromolar concentrations of zinc are generally
included in buffers for this protein. We found that binding of L5 to 5
S rRNA is severely inhibited by micromolar concentrations of zinc with
no detectable binding above 30 µM ZnCl
.
Consequently, this divalent cation was not included in the standard
binding buffer.
The interaction of L5 with 5 S rRNA is insensitive
to ionic strength. The apparent K of the complex
remains constant over a range of 0-0.4 M KCl (data not
shown). Only a 2-fold decrease in binding affinity was observed at a
monovalent salt concentration of 0.6 M. These results indicate
that an appreciable contribution to binding comes from nonelectrostatic
interactions, which is surprising given that L5 is a highly basic
protein with a net charge of +22(5) . However, this
behavior has been observed for other RNA-binding proteins(28) ,
including ribosomal proteins(29) . The absence of an
appreciable dependence on salt concentration is particularly meaningful
in light of the pH profile that suggests the involvement of tyrosine
and/or lysine residues in binding. These results indicate that it is
the former amino acid that accounts for the pK
of
10.1. Thus, contacts in which this aromatic residue is stacked upon the
base moiety of a nucleotide may contribute significantly to the overall
free energy of binding of the ribosomal protein to 5 S rRNA.
A
concentration of 0.1 M KCl was chosen for standard assays of
L5, since this is comparable with the conditions that were used to
measure binding of TFIIIA to these same mutants in 5 S rRNA. Moreover, higher salt concentrations appear to stabilize the
nucleic acid binding activity of the protein.
Figure 4:
Specificity of fL5 binding to 5 S rRNA.
fL5 (40 nM) and radiolabeled 5 S rRNA (5 nM) were
incubated with either 5, 25, 67, 125 nM unlabeled, competitor
5 S rRNA (), or 10, 50, 250, 500 nM unlabeled tRNA
(
). The autoradiograph was scanned with a densitometer to
quantitate the amount of bound radiolabeled 5 S
rRNA.
The binding affinities of fL5
for the mutant 5 S rRNAs were measured by titrating 1 nM internally radiolabeled RNA with increasing concentrations of
protein. Free and bound 5 S rRNA were separated by electrophoresis on
nondenaturing polyacrylamide gels (Fig. 5). Autoradiographs of
these gels were scanned with a laser densitometer, and the integrated
volumes of the individual bands were entered into the program
EZ-Fit(24) , which generates binding isotherms (Fig. 6)
and a value for the K of the complex. We used at
least two different preparations of each RNA and performed each assay
in duplicate, so there is a minimum of four assays for each mutant. In
each series of experiments, the dissociation constants of the mutants
were determined relative to that for wild-type 5 S rRNA measured in the
same experiment in order to control for any differences in the activity
of fL5 (or L5) from one series of assays to another. For those mutants
that have a greatly reduced affinity for L5 (e.g.
A
), it is not possible to saturate
binding; aggregation of L5 in binding buffer at concentrations above
100 nM begins to interfere with the electrophoretic assay.
Therefore, titrations did not exceed this concentration, and values for
the dissociation constants of these mutants were calculated by the
EZ-Fit program without complete binding of the RNA. The dissociation
constant measured here for the L5
5 S rRNA complex in optimized
conditions is 2 nM. The dissociation constants for each mutant
relative to wild-type 5 S rRNA are presented in Table 1. The
binding of TFIIIA (isolated from Xenopus oocytes) to these
mutant RNAs has also been measured using the same electrophoretic
assay
; these data are included in Table 1in order to
compare the recognition elements in the RNA for the two proteins.
Figure 5:
Mobility shift assays for binding of fL5
to mutant 5 S rRNAs. Autoradiographs for a selection of mutants are
presented. In each assay 1.0 nM 5 S rRNA internally labeled
with [P]GTP was incubated with increasing
concentrations of fL5, which ranged from 0 (first lane of each
gel) to 160 nM (for WILD TYPE, A42C,
A83) or 300 nM (for T43A/C44G,
A49/50, T76G). The two dots on the
autoradiograph for T76G mark the two equilibrium conformations
of this RNA; the upper form corresponds to the native
conformation.
Figure 6:
Binding isotherms derived from RNA
mobility shift assays. Autoradiographs of the nondenaturing
polyacrylamide gels were scanned with a laser densitometer to
quantitate the intensity of the individual bands. Exposures were within
the linear response range of the film. Curves were fit to the data by
nonlinear regression analysis(24) . A, wild-type
(); A
C (
); T
A/C
G (
). B, wild-type (
);
A
(
);
A
,
(
);
A
(
). C, wild-type (
); G
C/G
C/G
C/G
C (
); T
G (
).
The mutations in 5 S rRNA that affect binding of L5 are located in
the hairpin structure encompassing helix III-loop C. Deletion of the
two bulged adenosines at positions 49 and 50 of the helix caused a very
marked decrease in affinity as did a mutation in loop C at the
stem-loop junction (T
A/C
G).
Another mutation in loop C (A
C) has no impact on
binding. The only mutation outside of this region that affects binding
of L5 is the transversion T
G located in loop E. We
have shown earlier that this substitution engenders an alternative
conformation in the RNA that is in equilibrium with the wild-type
structure.
These two forms of 5 S rRNA are resolved on
mobility shift gels (Fig. 5) with the alternative structure
migrating faster than the wild-type conformation. Since no other
mutations in this domain of the RNA, including the quadruple
substitution G
C/G
C/G
C/G
C, has an effect on L5 binding, we
believe that the observed conformational perturbation induced by
T
G accounts for the large decrease in binding
rather than disruption of an essential contact to this site. This
result suggests that the structural changes induced by T
G extend well beyond the site of the mutation and result
in a global rearrangement of the RNA.
We have expressed the eukaryotic ribosomal protein L5 in E. coli and have shown that it has high affinity and
specificity for 5 S rRNA. In the past, this protein has been difficult
to characterize due to its notorious insolubility, especially when
freed of its cognate RNA (see (16) and references therein).
Consequently, most experiments to date have used the L55 S rRNA
complex that is released from ribosomes upon treatment with high
concentrations (25 mM) of EDTA. The expression of sufficient
amounts of active protein enable us to avoid this procedure and to
reconstitute the complex directly. Scatchard analysis has shown that
preparations of fL5 never exceeded 60% binding activity, indicating
that some solubility problems may still exist for the protein expressed
in bacteria. Since the presence of the MBP domain in the chimeric
protein appears to stabilize the 5 S rRNA binding activity, protein is
stored in this form and only cut with Xa protease immediately before
use in binding assays. Most important, we have never detected any
effect of the MBP domain on the RNA binding activity or specificity of
L5.
Characterization of the binding of L5 to the collective mutants
of 5 S rRNA identifies the hairpin structure composed of helix III and
loop C as the major determinant of recognition. The large changes in K values for these mutants indicate that much of
the free energy of binding is derived from contacts made to this
region. This is in contrast to TFIIIA, where point mutations, or even
block mutations, seldom produce more than a 3-fold decrease in binding,
suggesting that this latter interaction is directed by several weak
contacts dispersed over a large amount of the RNA secondary structure (8, 10, 30) .
The results of
these binding experiments are unexpected, since much of the earlier
work to define the site on 5 S rRNA for the cognate ribosomal protein
all indicated that the arm of the RNA molecule composed of helix
IV-loop E-helix V as well as helices I and II comprise the contact
site(13, 14, 15, 16, 17, 33, 34) .
However, there is some evidence for contact between L5 and the helix
III-loop C region(2, 14, 15) , although in at
least one case this interaction appeared to be weak and secondary to
those in other regions of the RNA(14) . -Sarcin, a
purine-specific ribonuclease, was used in footprinting experiments with
the 7 S RNP complex released from rat ribosomes(16) . In accord
with earlier work, L5 protected a substantial region of the RNA that
includes helices I, II, IV, and V and loop E.
-Sarcin only cuts at
positions 37, 41, and 42 in the segment of 5 S rRNA extending from
nucleotide 28 to 46, leaving a significant amount of this region of the
RNA untested by the nuclease. There is no detectable protection of
these three nucleotides nor of the run of 5 purines from positions
47-51. Contacts between L5 and the stem-loop junction will not be
detectable using
-sarcin, since this is a blind spot with respect
to the nuclease. It is difficult, however, to understand why protection
from
-sarcin is not seen in helix III, unless all contacts with L5
are to the 5`-strand of the helix (i.e. nucleotides
27-32) leaving the 3`-strand exposed and susceptible to the
nuclease. On the other hand, no mutation, excepting T
G, within the footprint for L5 has an effect on binding.
Of particular interest is the quadruple mutant (G
C/G
C/G
C/G
C) that should destabilize both helices IV and V, and probably that
entire arm of 5 S rRNA. This mutation causes a 20-fold increase in the
dissociation constant for TFIIIA. Remarkably, this mutant binds L5 with
wild-type affinity.
In other studies, the 3` terminus of 5 S rRNA
was chemically cross-linked to rat L5, suggesting that helix I is in
close proximity to the protein(33) . Fragments of 5 S rRNA that
remained associated with rat L5 after digestion of the 7 S RNP complex
with ribonucleases included oligoribonucleotides encompassing positions
1-21, 77-102, and 106-120, which is in concordance
with the experiments using -sarcin(14) . Interestingly,
small amounts of fragments extending from nucleotide 41 to 52 or 56
were recovered in some experiments. Experiments with RNase T
showed protection by L5 at nucleotides 37, 47, 48, and
89(15) , providing the only supporting evidence for an
interaction between the helix III-loop C structure and the ribosomal
protein. Comparable experiments with the yeast homologue, YL3, have
yielded similar results. Fragments encompassing residues 1-12 and
79-121 remain bound to the protein after digestion with
ribonuclease(13) . Modification interference experiments
identified important contact sites in helices I, II, and
IV(17) . We note that we have not assessed helix I in our
mutagenesis experiments.
Experiments that specifically address the Xenopus L55 S rRNA complex, however, are in good
agreement with the results presented here(2) . RNP complexes
were immunoprecipitated from oocytes injected with mutant 5 S rRNA
genes that were predicted to delete or disrupt particular elements of
secondary structure. The only variant RNA that could not be recovered
by immunoprecipitation with anti-L5 antibodies was a mutant missing
nucleotides 11-41; this deletion removes the domain of the RNA
comprised of helix II-loop B-helix III-loop C. Mutants that disrupted
the structure of the other arm of 5 S rRNA had no detectable effect on
binding. Microinjection of somatic and oocyte 5 S rRNA into oocytes
revealed preferential association of the somatic-type RNA with L5; five
of the six nucleotides that differ between these two RNAs are located
in helix III and loop B(7) .
It is difficult to resolve the
apparent discrepancies mentioned here. L5, like TFIIIA, may protect a
substantial amount of 5 S rRNA from digestion by ribonucleases; yet,
unlike the transcription factor, it may contact only a small number of
nucleotides within this protected region. This would account for the
large impact particular mutants in the RNA have on the affinity of L5
compared with the much smaller effects seen with TFIIIA (Table 1). Missing nucleoside experiments in combination with
chemical footprinting should help to resolve this issue(9) . It
is worth noting that the earlier studies used samples of the L55
S rRNA complex that is released from ribosomes treated with EDTA. It
will be important to determine whether this procedure somehow alters
the integrity of the native structure. It is unlikely that there are
significant differences between the interactions of the rat and Xenopus proteins with 5 S rRNA, since the two have 92%
identity with respect to the sequences of their amino acids.
The
higher order structure of Xenopus oocyte 5 S rRNA has been
examined using the metal complex
Rh(phen)(phi)
(35) . This probe,
which binds to nucleic acids on the basis of shape selection, promotes
strand cleavage upon photoactivation. The rhodium complex is sterically
excluded from double-stranded regions of RNA because the major groove
of an A conformation helix is too narrow, nor does the probe bind to
unstructured single-stranded regions that are devoid of base stacking
interactions. Rh(phen)
(phi)
does target
positions within RNA molecules where the major groove is opened and
accessible to stacking interactions with the metal complex, including
stem-loop junctions, noncanonical or mismatched base pairs, base
triples, and bulges of more than one
nucleotide(35, 36) . Positions cleaved by
Rh(phen)
(phi)
are indicated on the
secondary structure of 5 S rRNA in Fig. 7. Major sites of
cleavage are clustered in loop E and along the helix III-loop C
hairpin. The conformation of loop E has been determined by NMR
spectroscopy and it approximates an A-form helix(37) . However,
a reverse Hoogsteen A:U pair, possibly involved in a base triple
interaction with an adjacent bulged guanosine residue, and three
consecutive mismatched appositions open the major groove, while base
stacking maintains helical structure. These structural features of loop
E account for the strong sites of cleavage by the transition metal
complex. Loop E is an essential determinant for the binding of TFIIIA
to 5 S rRNA, presumably because of its unique geometry and because
contacts with the protein are possible through the major
groove(9, 32) .
Figure 7:
The secondary structure of 5 S rRNA with
designations of the sites of cleavage by
Rh(phen)(phi)
. The arrows indicate positions of strand scission with the length
corresponding to the relative intensity of cleavage by the metal
complex(35) . Mutations made at nucleotide positions shown in boldface lettering decrease binding of L5 to 5 S rRNA. Dashed lines indicate proposed base pairings in loop
C(39) . The inset displays the cleavage pattern of
Rh(phen)
(phi)
on mutants in loop C. Open arrowheads denote sites of reduced cleavage, and filled arrowheads denote sites of increased cleavage; asterisks mark sites where cleavage intensity remains the same
as wild-type RNA.
The major groove of the
helix III-loop C hairpin is accessible to
Rh(phen)(phi)
at the site of the two
bulged adenosines and at the stem-loop junction. There is evidence that
the structure of loop C is highly
ordered(35, 38, 39) . On the basis of data
from NMR spectroscopy, Li et al.(38) found evidence
for two additional base pairs in wheat germ 5 S rRNA that they proposed
formed between U
:A
and
C
:G
. Chemical and enzymatic probes were used
to characterize block mutations in this domain of Xenopus oocyte 5 S rRNA; Brunel et al.(39) proposed a
Hoogsteen base pair between U
:A
and a
Watson-Crick base pair between C
:G
. The
extended sites of cleavage by Rh(phen)
(phi)
into loop C provide evidence for base stacking interactions
within the loop that may be due either to these putative base pairs or
to the apposition of noncanonical pairs stacked into the loop.
Regardless of whether the bases are hydrogen-bonded, it seems likely
that loop C is folded in such a way as to form a tetraloop
(GUCU
), which would contribute to the stability of the
structure(40, 41) . Stacking of C
onto
G
, in an arrangement comparable with that seen in the
structure of the UUCG tetraloop (40) would account for cleavage
by Rh(phen)
(phi)
at C
.
A
correlation between cleavage of the mutants by
Rh(phen)(phi)
and binding affinity for L5
is evident. Deletion of A
and A
converts the
stem of the hairpin to a canonical A-form helix, significantly reducing
accessibility to the major groove. This mutation causes the greatest
decrease in binding of L5. In model studies, it has been shown that
complex formation between double-stranded DNA and aromatic amino acid
side chains occurs by partial insertion that bends the
helix(42) . Single- or multiple-nucleotide bulges will induce a
bend or kink into duplex RNA(43) ; however, only bulges of two
or more nucleotides increase the accessibility of the major
groove(35, 44) . Thus, this two-nucleotide bulge
provides a structure that is compatible with and that could facilitate
a stacking interaction with an aromatic amino acid.
The mutation
T
A,C
G results in diminished
cleavage at A
, C
, C
, and
G
, indicating decreased accessibility of the major groove
and/or loss of critical stacking interactions. Furthermore, the
complete absence of cleavage at C
in this mutant provides
evidence for a change in the structure of the putative tetraloop. This
mutant also has a substantially reduced affinity for L5. Alternatively,
the transversion A
C binds L5 with wild-type
affinity. Although this latter mutation alters the cleavage pattern of
Rh(phen)
(phi)
in loop C and does diminish
cleavage at C
, C
, C
, and
C
, enhanced cleavage is measured at A
and a
new cleavage site is now observed at C
. Thus, the loop
region of this mutant remains opened to stacking interactions with the
metal complex, which is reflected in the high affinity binding of L5.
It is important to note that these mutations change only the local
structure of the RNA; no long-range effects were detected(35) .
There appears to be three sites in the hairpin that can accommodate
binding of Rh(phen)
(phi)
; these are found
at the bulged adenosines, at the stem-loop junction, and within the
loop possibly stacked on the putative Hoogsteen A:U pair. This number
can be compared with the two residues indicated by the slope of the pH
curve at pK
10.1. The close parallel between
intercalative binding of Rh(phen)
(phi)
and binding of L5 to mutants of 5 S rRNA adds strong support to
the suggestion that binding of the ribosomal protein through the major
groove of the helix III-loop C hairpin structure is mediated, at least
in part, by stacking interactions involving tyrosine residues.
Experiments with the fluorescent probe bisanilinonaphthalenesulfonic
acid detected the presence of hydrophobic sites on the surface of yeast
YL3 that become exposed upon disruption of the RNP
complex(27) . Increased turbidity upon dissociation of the
complex also indicated exposure of hydrophobic regions on the ribosomal
protein. Like Xenopus L5, the yeast protein is rich in
tyrosine residues, despite there being only 45% homology between the
two amino acid sequences.
L5 and TFIIIA both utilize sites on 5 S
rRNA where major groove structures are opened and
accessible(9, 35) . However, in all other respects the
interactions of these two proteins with the nucleic acid are different.
The binding of TFIIIA depends on the overall secondary structure of 5 S
rRNA with weak contacts dispersed over a large surface of the
RNA(8, 45) . For this reason, individual mutations in
5 S rRNA generally have small effects on the binding affinity of the
transcription factor(8) . Disruption of the helix
IV-loop E-helix V domain of 5 S rRNA by the quadruple mutant G
C/G
C/G
C/G
C has a substantial impact on TFIIIA because of the
cumulative effect of disrupting several contacts that occur through
this region of the RNA; yet, this same mutant has no effect on L5.
Those mutations that do influence binding of L5 cause a considerable
decrease in affinity and are confined to a relatively small region of 5
S rRNA. Of most significance, perhaps, are the differences in the
physicochemical properties of the two complexes. Like many
protein-nucleic acid interactions, the pH profile of TFIIIA binding to
5 S rRNA is rather featureless, exhibiting a modest decrease in
affinity above pH 8.0 (19) . An analysis of apparent binding
constants as a function of ionic strength indicates that there are
approximately five ion pairs formed between the transcription factor
and 5 S rRNA, although there is an additional contribution from
nonelectrostatic interactions to the free energy of
binding(19) . In marked contrast is the distinct pH profile for
L5 and the exceptional insensitivity of binding to ionic strength that
point to the importance of one or more tyrosine residues. These results
indicate that stacking or intercalative, rather than electrostatic,
interactions are the major determinants of the L5
5 S rRNA
complex. Our earlier experiments with
Rh(phen)
(phi)
demonstrate that sites for
such contacts are present in the helix III-loop C hairpin
structure(35) .