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(Received for publication, June 21, 1995; and in revised form, September
6, 1995) From the
Binding of transcription factor IIIA (TFIIIA) to site-specific
mutants of Xenopus oocyte 5 S rRNA has been used to identify
important recognition elements in the molecule. The putative base
triple G
Transcription factor IIIA (TFIIIA) ( In addition to
being a potentially important aspect of the developmental regulation of
5 S rRNA synthesis, the binding of TFIIIA to both RNA and DNA is of
considerable interest with respect to protein-nucleic acid recognition.
TFIIIA possesses nine zinc finger domains (6) that mediate
mutually exclusive interactions with the two nucleic acids(4) .
The binding sites for the factor on the gene and on 5 S rRNA are
similar(7, 8, 9) , which led to speculation
that the protein associates with the two nucleic acids through common
determinants. However, studies using truncated or mutated forms of
TFIIIA have demonstrated that the nine zinc finger domains, while all
contributing to some degree to DNA and RNA binding, are not
functionally equivalent. Peptides containing the three amino-terminal
finger domains bind to the gene with an affinity nearly equal to that
of the intact factor(10, 11) . Moreover, variants of
TFIIIA in which fingers 2 or 3 have been deleted or disrupted exhibit
considerably reduced DNA binding affinity without a corresponding
decrease in RNA binding(12, 13, 14) .
Nuclease protection (11, 15, 16, 17) and missing
nucleotide experiments (18) indicate that these three fingers
bind in the major groove of the internal control region (ICR) from
approximately nucleotide position 79 to 92. Both hydroxyl radical (16) and DNase I (15, 17) footprinting reveal
a change in the orientation of the factor relative to the DNA helix in
the vicinity of base pair 78 which likely signifies the exit of the
protein from the major groove with finger 4 crossing the minor groove.
The triplet of fingers 4-6 runs parallel to one side of the helix
for nearly 20 base pairs. The factor then re-enters the major groove
near base pair 60 and the three carboxyl-terminal fingers appear to be
contiguously aligned in a manner similar to the amino-terminal fingers.
The distinct orientation of the central three fingers on the gene may
reflect their role as the primary mediators of RNA binding (12, 19) . A polypeptide comprised of zinc fingers
4-7 binds with high affinity to 5 S rRNA and deletion of two or
more of these fingers from the factor significantly reduces binding to
RNA. Thus, a model has emerged in which two subsets of fingers within
TFIIIA confer differential specificity for binding of the factor to DNA
(fingers 1-3) or RNA (fingers 4-6). Viewed in this way, the
interactions of TFIIIA with the 5 S gene and 5 S rRNA are quite
different. However, this oversimplification minimizes the contributions
that the remaining fingers clearly make to the free energy of binding
in both complexes (12, 13, 14) . A variety
of studies have used mutagenesis to define the interaction of TFIIIA
with 5 S rRNA genes and 5 S
rRNA(20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32) .
The cumulative results of these studies establish that the binding site
for TFIIIA on the 5 S rRNA gene is composed of three sequence elements
which seemingly reflect the disposition of the protein along the DNA
described above. As a consequence of disrupting sequence-specific
interactions, mutations made in box C (bp 80-90) and the
intermediate element (bp 67-72) have the most substantial effect
on the binding of TFIIIA. On the other hand, binding of the factor to 5
S rRNA is most notably altered by changes in the higher order structure
of the nucleic acid(12, 27, 33) . In this
study we have examined the effects of point mutations made at critical
locations within the structure of 5 S rRNA. Binding of TFIIIA to the
corresponding mutations in the gene was also measured. The influence of
most changes introduced into 5 S rRNA can be explained on the basis of
the molecule's secondary structure. In particular, the integrity
of loop E, which appears to be determined by a base triple
interaction(34) , is especially important for binding of the
factor. While the results presented here support earlier work
establishing that the primary determinants of binding are located in
nonhomologous regions of the two nucleic acids, helix V and its
corresponding sequence in the gene, the intermediate element, appear to
be common sites of recognition for the factor.
Figure S1:
Scheme 1.
Quantitative DNase I footprinting experiments were carried out with
plasmids linearized with EcoRI and end-labeled on the coding
strand using Klenow fragment and [ Binding of TFIIIA to mutant 5 S RNAs was
measured by a shift in the mobility of the RNA in nondenaturing 8%
polyacrylamide gels run at room temperature at 100 V(38) .
Binding assays were carried out in the same buffer used to measure
binding of TFIIIA to the 5 S gene except for the addition of
ribonuclease inhibitor (RNasin, Promega; 0.4 unit/µl) and
acetylated bovine serum albumin (0.1 µg/µl). The binding
reactions contained 1.5 nM 5 S rRNA (internally labeled with
[
Figure 1:
The
nucleic acid binding sites for TFIIIA. A, the secondary
structure of 5 S rRNA and the sites of mutagenesis. The positions
marked on the secondary structure of the RNA designate the locations of
point mutations; boxes enclose multiple substitutions or
deletions. Additional mutants that contain multiple changes are listed
below the secondary structure. For a given mutant the first letter
indicates the wild type nucleotide and the letter following the
position number indicates its replacement.
The binding affinity of TFIIIA for the mutant 5 S
rRNAs was measured by titrating a constant amount of internally labeled
RNA with increasing amounts of the protein. Free and bound 5 S rRNA
were separated by electrophoresis on nondenaturing polyacrylamide gels (Fig. 2). Autoradiographs of the gels were scanned with a laser
densitometer and the integrated volumes of the individual bands entered
into the program EZ-Fit (46) which generates binding isotherms (Fig. 3) and a value for the K
Figure 2:
Mobility shift gel assays for binding of
TFIIIA to mutant 5 S rRNAs. Autoradiographs for a selection of mutants
are presented. In each assay 1.5 nM 5 S rRNA internally
labeled with [
Figure 3:
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(46) . A:
The
binding of TFIIIA to the 5 S rRNA variants was also measured in
competition assays. The exchange of radiolabeled wild type 5 S rRNA
into the native 7 S RNP particle was measured in the presence of
increasing concentrations of each mutant. Samples were analyzed by
electrophoresis followed by autoradiography. The competition binding
data, likewise obtained by densitometry, were processed using the
program LIGAND(48) . The relative dissociation constants
measured in these assays were very similar to those determined in the
direct binding assays, providing an independent determination of
binding strength. Quantitative DNase I footprinting was used to
measure the binding of the factor to the mutant 5 S rRNA genes in the
same conditions used above for assays with RNA. Autoradiographs of
sequencing gels were scanned with a densitometer and binding isotherms
were again constructed using the EZ-Fit program (Fig. 4). The
value of the Hill number was not constrained, which resulted in
nonhyberbolic binding curves giving the best fit to the data. Only a
single molecule of TFIIIA binds to the 5 S rRNA
gene(36, 51) ; however, sigmoidal binding isotherms
for TFIIIA have been observed in other
studies(35, 52) . In our experiments this behavior may
reflect the limitations of the footprinting method to detect small
amounts of complex at low concentrations of protein. The dissociation
constant measured for TFIIIA and the oocyte gene in these experiments
is 3 nM. The dissociation constants for each mutant relative
to wild type are presented in Table 1.
Figure 4:
Quantitative DNase I footprint assays for
binding of TFIIIA to mutant 5 S rRNA genes. A, autoradiographs
for a selection of assays is presented that exhibit quantitative and/or
qualitative effects on the binding of TFIIIA. In each assay 1.8 nM 5 S rRNA gene was incubated with increasing amounts of TFIIIA. The number above each lane indicates the nanomolar concentration
of protein. In each panel the coding strand is shown with numbers on the left indicating nucleotide positions
determined by the Maxam-Gilbert reaction for guanosine. Lanes marked C represent untreated DNA. B and C, binding isotherms derived from footprint titrations are presented
for selected mutants. Autoradiographs of the sequencing gels were
scanned with a densitometer to quantitate the intensity of individual
bands at each concentration of TFIIIA. Curves were fit to the data by
nonlinear regression analysis(46) . B:
The N3 positions of both U Loop D of Xenopus 5 S rRNA belongs to the family
of tetranucleotide loops having the consensus sequence
GNRA(57) . Structures of these loops determined by NMR
spectroscopy reveal that their exceptional stability is a consequence
of base pairing between the first and fourth bases in the loop,
stacking of the bases in the loop, and putative base-phosphate hydrogen
bonds(57, 58) . There are examples, particularly in
ribosomal RNAs, where the GNRA loop is an essential component of a
protein binding site(57) . However, substitutions made at
position 86, which will enlarge the size of the loop D, or at position
87, which will eliminate the stabilizing hydrogen bonds between the
first and fourth positions of the tetraloop, do not alter binding of
TFIIIA. Numerous chemical and enzymatic protection experiments
indicate that TFIIIA is in close proximity to nucleotides within the
helix IV-loop D region of 5 S
rRNA(7, 8, 9, 49, 50, 59) .
Nonetheless, we do not detect any significant thermodynamic
contributions to binding in this region of the RNA upon making
substitutions that will, in most cases, change local secondary
structure in addition to sequence. The fortuitous increase in affinity
seen with A83U does indicate that the protein is contiguous with this
segment of the nucleic acid. However, our results are in accord with
those from experiments in which helix IV was disrupted by block
mutations or was truncated without appreciable decreases in binding
TFIIIA(30, 60) . The mutation C79G, however, does
decrease TFIIIA binding. This substitution creates two adjacent
mismatched pairs proximal to loop E and may express its influence on
binding through the latter structure (see later). The binding of the
factor to the corresponding mutations in the 5 S rRNA gene clearly
establishes not only the differences between the two TFIIIA-nucleic
acid complexes, but also the importance of bp 81-96 (box C) for
binding to the DNA. Whereas the deletion of A
In particular, the three nucleotides (G The
A The
mutation C79G, which results in a 3-fold increase in K None of the mutations
corresponding to the loop E region has an effect on the binding of
TFIIIA to the 5 S rRNA gene, indicating again that the primary
determinants for binding to the two nucleic acids are different.
Methylation interference experiments
indicate that G
Similarly, the binding of
TFIIIA to box A is not significantly influenced by point mutations made
in this element. Both DNase I and hydroxyl radical footprinting
experiments (74) as well as missing nucleoside experiments (18) demonstrate that TFIIIA is closely associated with this
region of the ICR. In contrast, no essential contacts within box A were
identified in chemical interference experiments(61) . Studies
using polypeptides representing subsets of zinc fingers derived from
TFIIIA have demonstrated that binding affinity is not strictly
correlated with the loss of contacts made to this region of the
ICR(72) . The interaction of fingers 7-9 with box A
appears to be complex and it has been proposed that the free energy of
binding may be counterbalanced by other processes such as bending of
the DNA (72) or unfavorable interactions between certain finger
domains (13) . The apparently small energetic contribution made
by fingers 7-9 is well illustrated by mutations made at position
56. A transversion at this site from A (major oocyte) to C (trace
oocyte) abolishes over one-third of the TFIIIA footprint, yielding a
protection pattern similar to that found on the trace-oocyte gene (bp
62-94). We have measured a 2-fold decrease in binding for the A
to C transversion made at this site. The alternative transversion,
A56T, has even less effect on the affinity for TFIIIA; yet, this mutant
also exhibits a footprint diminished by approximately 20 base pairs (Fig. 4). It is difficult to understand how the association
between TFIIIA and box A can be maintained based upon such a small
change in free energy. One possible explanation is that disruption of
the contacts between the factor and the 5`-end of the ICR relieves some
high energy structure induced in the protein and/or nucleic acid upon
complex formation. Indeed, there is evidence that the 5 S rRNA gene is
markedly bent in the presence, but not in the absence, of
TFIIIA(75, 76) . Based upon this observation and the
unusually short linker sequences that lead to and from finger 6, Berg (77) has offered a model of the complex in which fingers
1-5 and 7-9 form contacts on the 3` and 5` sides,
respectively, of the bend that occurs at the binding site for finger 6.
It is possible that mutations at position 56 disrupt the interaction of
the three carboxyl-terminal fingers that bind the 5` end of the ICR,
obviating the protein-dependent bending of the DNA which, in turn,
would compensate energetically for the reduced binding surface in the
mutant. Deletion mutants of TFIIIA missing fingers 7-9 generate
DNase I footprints very similar to those of the two mutants at position
56 (72, 74) . The 5 S rRNA gene possesses an
unusual amount of polymorphism in its helical
structure(38, 70, 78) . CD spectroscopy and
topoisomer band shift experiments showed that, on average, the helix is
moderately underwound(38) . Cleavage of duplex DNA by DNase I
is sensitive to the dimensions of the minor groove, being dampened
where the width becomes either larger or smaller than the mean
12-Å cross-strand spacing(79) . Digestion profiles of DNA
generated with this nuclease, therefore, provide a measure of the local
structural heterogeneity of the helix (80, 81) .
Changes in the DNase I cleavage patterns of several mutants relative to
the wild type gene exemplify the potential importance of
sequence-generated conformation. For example, inverting the G:C pair at
position 81 mitigates the strong cutting that normally occurs at this
site, while changing T
Zinc fingers 4-6 make the greatest contribution
to RNA binding(12, 19) . The mutations made here in
loop E have some of the largest effects measured on RNA binding and
implicate the putative G Helix V
of 5 S rRNA corresponds to the intermediate element of the ICR. These
are the respective binding sites for finger 5. The various mutations we
have made at nucleotides 70 and 105 affect binding to RNA and DNA
similarly and provide evidence that this finger makes comparable
interactions through the major grooves of both nucleic acids . The
three carboxyl-terminal fingers of TFIIIA associate with the helix
II-loop B-helix III region of 5 S rRNA or the corresponding box A
element of the ICR. We find that only substitutions made at stem-loop
junctions in this arm of 5 S rRNA have any detectable effect on binding
and these are rather small. Likewise, these mutations have little
effect on the affinity of TFIIIA for the gene; yet, as demonstrated by
substitutions made at A
Volume 271,
Number 2,
Issue of January 12, 1996 pp. 869-877
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
:U
:A
appears to
determine the conformation of the loop E region whose integrity is
especially important for binding of the factor. Proximal substitutions
in helices IV and V indicate that the proper folding of loop E is also
dependent on these structures. Mutations in helix V affect binding of
TFIIIA to 5 S rRNA and to the gene similarly and provide evidence that
zinc finger 5 makes sequence-specific contact through the major groove
of both nucleic acids. Although fingers 1-3 are positioned along
helix IV and loop D, mutations in this region, including those that
disrupt the tetraloop or close the opening in the major groove of the
helix created by the U
:U
mismatch, have no
impact on binding. Substitutions made at stem-loop junctions in the arm
of the RNA comprised of helix II-loop B-helix III display minor
decreases in affinity for TFIIIA. Despite the alignment of the factor
along nearly the entire length of 5 S rRNA, the essential elements for
high affinity binding are limited to the central region of the
molecule. Analysis of the corresponding mutations in the gene confirm
that box C and the intermediate element provide the high affinity sites
for binding of the factor to the DNA. Despite the small thermodynamic
contribution made by contacts to box A, mutations made in this element
can cause substantial changes in the orientation of the
carboxyl-terminal fingers along the 5`-end of the internal control
region.
)is a positive
regulator of 5 S rRNA gene transcription(1) . Together with
factors TFIIIB and TFIIIC, it forms an initiation complex on the
intragenic promoter of these genes which directs transcription by RNA
polymerase III(2) . In previtellogenic Xenopus oocytes
the factor serves a second function, forming a complex with 5 S rRNA
until this nucleic acid is used for the assembly of ribosomes in the
later stages of oogenesis(3, 4) . Thus, TFIIIA has the
distinctive ability to bind specifically to both the gene and its
transcript. It is not clear whether the dual nucleic acid binding
activity of TFIIIA is involved in the control of transcription of 5 S
rRNA genes(4, 5) . Sequestering the factor into RNP
particles may restrict the number of active transcription complexes
and, thereby, abate synthesis of 5 S rRNA. In this case TFIIIA would be
the mediator of an autoregulatory loop limiting the amount of 5 S rRNA
that is ultimately synthesized during oogenesis.
Preparation of TFIIIA and 5 S rRNA
The 7 S RNP
particle of TFIIIA bound to 5 S rRNA was prepared from immature ovaries
of Xenopus laevis (Nasco, Fort Atkinson, WI)(35) .
TFIIIA was isolated from the RNP particle by the method of Smith et
al.(36) with the following modifications. The 7 S
particle was digested with RNase A (15 µg/mg particle) and RNase
T (200 units/mg particle) for 1 h at room temperature.
The sample was loaded onto a BioRex-70 column equilibrated with buffer
A (50 mM HEPES, pH 7.5, 5 mM MgCl
, 1
mM DTT, 10 µM ZnCl, 20% glycerol)
containing 0.1 M KCl. TFIIIA was eluted with buffer A
containing 1 M KCl; one drop fractions were collected and
stored at -80 °C. All steps were performed at room
temperature and none of the buffers contained urea. Xenopus 5
S rRNA was prepared from the 7 S RNP particle by extraction with
(NH
)SO(37) followed by
extraction with phenol. Escherichia coli 5 S rRNA was prepared
from a crude fraction of ribosomes by methods described
previously(38) . The tRNA was purchased from
Boehringer Mannheim.
Construction and Cloning of a Xenopus Oocyte 5 S rRNA
Gene Transcription Unit
A 153-bp fragment containing a T7 RNA
polymerase promoter immediately upstream of a Xenopus oocyte 5
S rRNA gene was assembled from five complementary pairs of synthetic
oligonucleotides (Fig. S1). Three of these double-stranded
segments had protruding ends which were complementary to those of
adjacent segments. One nmol of oligonucleotides a, b, B, c, C, d,
D, and E were phosphorylated at the 5` termini using T4
polynucleotide kinase. Equal amounts of oligonucleotides A and e were added to the phosphorylated oligonucleotides and the
mixture was annealed by slow cooling after heating to 85 °C for 2
min. The sample was incubated at 16 °C for 24 h in the presence of
T4 DNA ligase (5 units) to seal nicks. The resulting 153-bp fragment
was purified by electrophoresis in a 4% Nusieve GTG-agarose gel (FMC
Bioproducts) and ligated through the EcoRI and BamHI
restriction sites into pUC119 to generate the plasmid pT75S. The
sequence of the cloned transcription unit was confirmed by sequencing.
In Vitro Synthesis of 5 S rRNA
Transcription
reactions were carried out with minor modification of reported
procedures(39) . Reaction mixtures containing 0.5 µg/µl
plasmid linearized with DraI in 40 mM Tris
HCl,
pH 7.5, 6 mM MgCl, 10 mM DTT, 2 mM spermidine, 2 mM each of ATP, CTP, and UTP, 0.2 mM GTP, 10 µCi of [
-P]GTP, 0.2
unit/µl RNasin (Promega), and 5 units/µl T7 RNA polymerase
(Epicentre Technologies) were incubated at 37 °C for 90 min. At the
conclusion of the synthesis, RNase-free DNase I (1 unit) was added and
the incubation continued for an additional 30 min. Full-length 5 S rRNA
was purified by electrophoresis on 12% polyacrylamide gels containing 8 M urea and subsequently renatured as described
previously(40) . The sequence of the wild-type transcript was
determined using enzymatic sequencing methods to establish that
authentic full-length 5 S rRNA had been made.
Site-directed Mutagenesis
Oligonucleotide-directed
mutagenesis was performed according to the procedure described by
Kunkel(41) . E. coli strain BW313 (dut,ung
) was
transformed with pT75S and superinfected with M13K07 to stimulate
production and preferential packaging of single-stranded pT75S that was
enriched with uridine. In each case a 5`-phosphorylated primer was
annealed to 1 µg of the uridine-enriched template (20:1 molar
ratio, respectively); the solution was then adjusted to 20 mM HEPES, pH 7.8, 2 mM DTT, 10 mM MgCl
,
0.5 mM dATP, dGTP, dCTP, and TTP, and 0.8 mM ATP.
Five units each of T7 DNA polymerase and T4 DNA ligase were added and
the mixture was kept on ice for 15 min, followed by incubation at 25
°C for 5 min, and then at 37 °C for 2 h. One-fifth of the
extension reaction was used to transform E. coli (DH5).
Mutants were identified by sequencing plasmids prepared from randomly
selected colonies.Binding Assays
Protein concentrations were
determined by the method of Bradford (42) using bovine serum
albumin as the standard. Plasmids were prepared by the method of Holmes
and Quigley (43) with some modifications and were purified over
two successive CsCl gradients. The concentration of all DNA samples
used in the TFIIIA binding assays was determined by a fluorometric
method (44) specific for DNA in order to eliminate interference
from any trace contamination of residual RNA. Sonicated calf thymus DNA
used as the standard for the fluorometric assays was prepared by
treating the nucleic acid with ribonucleases T and A,
extracting several times with phenol and phenol/chloroform, and then
dialyzing extensively. The concentrations of RNA samples were
determined spectrophotometrically at 260 nm using an extinction
coefficient of 22.2 (mg/ml) cm
.
-P]dATP
in the presence of 0.5 mM ddTTP. The DNA was then digested
with BamHI to generate the 153-bp fragment containing the
oocyte 5 S rRNA gene which was purified by electrophoresis on 6%
polyacrylamide gels(45) . The fragment was eluted from the
excised gel slices, extracted twice with phenol, once with
phenol:chloroform (1:1), twice with chloroform, and then precipitated
with ethanol. The assay mixture for footprinting contained 20 mM HEPES, pH 7.5, 70 mM NH
Cl, 7 mM
MgCl, 5 mM DTT, 10 µM ZnCl, 0.01% Nonidet P-40, 1.8 nM 5 S rDNA in
the form of linearized plasmid, and the indicated concentration of
TFIIIA. DNase protection experiments were performed as described
earlier(24, 38) . The autoradiographs of the
sequencing gels were scanned with a laser densitometer to quantitate
the intensity of the bands. A minimum of three bands inside the
protected region were used to measure the amount of DNA bound to TFIIIA
and two bands outside the ICR were used to normalize each lane relative
to one another. The binding activity of TFIIIA was determined from
Scatchard plots; however, wild type plasmid was included in every
series of binding assays as a control for the activity of each sample
of protein. Data for each mutant acquired from the densitometer scan
was used to determine a dissociation constant by nonlinear regression
analysis using the program EZ-Fit(46) . Binding assays were
performed in duplicate and repeated with a minimum of two different
preparations of DNA.-P]GTP) and the indicated concentration of
TFIIIA in a total volume of 10 µl. Samples were incubated at room
temperature for 30 min and, after the addition of 0.5 µl of sample
buffer (0.25% bromphenol blue, 0.25% xylene cyanol, and 60% sucrose),
were loaded onto the gels. Autoradiographs were scanned with a laser
densitometer and this binding data was also processed using the EZ-Fit
program(46) . Binding assays were performed in duplicate with a
minimum of two different preparations of RNA. Alternatively, binding of
TFIIIA to mutant 5 S rRNAs was measured using the exchange reaction
described by Andersen and Delihas(47) . In this case the
incorporation of
P-labeled wild type 5 S rRNA into 7 S RNP
complex was measured in the presence of unlabeled mutant 5 S rRNA. The
reactions contained 50 mM Tris
HCl, pH 7.5, 3 mM DTT, 15 µg/ml bovine serum albumin, 6.5 µM 7 S
RNP particle, 1.3 nM internally labeled wild-type 5 S rRNA and
increasing amounts of unlabeled, competitor mutant RNA in a volume of
20 µl. The exchange reaction comes to equilibrium within 30 min at
20 °C(47) ; in these assays the reactions were kept at room
temperature for 2 h. The products of the exchange reaction were
analyzed by electrophoresis on nondenaturing polyacrylamide gels. The
amounts of bound and free wild type 5 S rRNA were determined either by
excising individual bands from the gels and measuring the radioactivity
by Cherenkov counting or by scanning autoradiographs with a
densitometer. The competition binding data was analyzed using the
program LIGAND(48) . This program is specifically designed for
the analysis of data from competition binding assays. It uses an exact
mathematical model of the ligand binding system and a weighted least
squares algorithm for curve fitting.
The Structure of 5 S rRNA and Sites of
Mutagenesis
A total of 42 site-specific mutations were made in
the X. laevis oocyte 5 S rRNA gene. In vitro transcription of these genes using a promoter for T7 RNA
polymerase enabled us to measure binding of the factor to the
corresponding 5 S rRNA molecules. The majority of mutants were
single-nucleotide substitutions that were chosen with regard to the
structure of the RNA molecule. Several mutations changed features of
the secondary structure of the nucleic acid which could be expected to
be utilized by the protein for recognition and binding, e.g. bulged nucleotides and mismatched nucleotide pairs. Other mutants
tested the importance of sequence, e.g. the repeated
pentanucleotide CCUGG that flanks the two sides of loop E. The
substitutions are displayed on the secondary structure of 5S rRNA in Fig. 1.
denotes a deletion.
The structure of loop E is that proposed by Wimberly et al.(34) based upon data from two-dimensional NMR
spectroscopy. B, the ICR of the Xenopus oocyte 5 S
rRNA gene. Nucleotides protected by TFIIIA from digestion with DNase I
are designated by bars above or below the noncoding and coding
strands, respectively, while sites hypersensitive to the nuclease in
the presence of TFIIIA are marked by asterisks. The positions
where the somatic and oocyte sequences differ are indicated by placing
the somatic-specific nucleotides above the noncoding strand. Guanine
residues important for binding TFIIIA, determined in methylation
interference experiments(61) , are represented by boldface
letters. The locations of box A, the intermediate element (I.E.), and box C are defined by brackets(23) .
of the
complex. In those instances where a mutation has an effect, the
magnitude is small, being generally 3-fold or less. In an effort to
minimize the problems of measuring such small differences, we used at
least two different preparations of each RNA and performed each assay
in duplicate. Thus, there is a minimum of four assays for each mutant.
In each series of experiments the dissociation constants of the mutants
are presented 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 TFIIIA from one series of assays to another. The dissociation
constant measured here for the TFIIIA5 S rRNA complex is 2 nM which is in good agreement with values reported
elsewhere(49, 50) . In addition, we found no
difference in the affinity of TFIIIA for 5 S rRNA synthesized in
vitro and native 5 S rRNA purified from 7 S RNP particle.
P]GTP was incubated with
increasing amounts of TFIIIA. The nanomolar concentration of factor is
given below each lane of the gel. The two dots on the
autoradiograph for T76G mark the two conformations of this RNA; the
upper form corresponds to the native
conformation.
, wild
type;
, A56T;
, T76G. B:
, wild type;
, T96A;
, G70C.
, wild
type;
, A;
, G81C,C95G. C:
, wild type;
, A56T; ()
T55C.
Helix IV-Loop D
There is accumulating evidence
that specific features of secondary structure such as bulged
nucleotides, non-Watson-Crick or mismatched base pairs, and base
triples are important for the recognition of RNA by
proteins(53, 54) . These elements can distort the
usual A conformation of RNA helices, making the major groove accessible
to proteins. Helix IV contains a U:U mismatch and a bulged adenosine
flanked by a G:U pair that could be potentially utilized by TFIIIA.
Experiments with chemical and enzymatic probes indicate that the bulged
nucleotide at position 83 is external to helix IV, as are the other
bulged nucleotides at positions 49, 50, and
63(8, 55) . Additionally, the metal complex
Rh(phen)(phi)
, which targets widened
major grooves in RNA helices such as occurs at base triples and
mismatched pairs, does not cleave at A(56) . This
demonstrates that the bulged nucleotide is not involved in a base
triple interaction with the adjacent G:U pair, but rather is in accord
with the nucleoside being external to the helix. Baudin and Romaniuk (28) have shown that deletion of any of the bulged residues in Xenopus oocyte 5 S rRNA has no effect on the binding of
TFIIIA. We also observe no appreciable effect upon deletion of A
or substitution by G or C. However, a transversion to U at
position 83 results in a 2-fold increase in the affinity of TFIIIA for
the RNA.
and U
do not react with CMCT(8, 55) , suggesting a
2-carbonyl-N3, 4-carbonyl-N3 mismatched pair between these two bases.
Cleavage by Rh(phen)
(phi) at positions
U and G
establishes that the major groove is
accessible at the site of this mismatch(56) . Mutations that
convert this site to either a Watson-Crick pair (U96A) or a wobble pair
(U96G) have no effect on the binding of TFIIIA; however, cleavage by
the rhodium probe is eliminated by these substitutions, indicating that
the helix now approximates a canonical A-type conformation with a major
groove that is inaccessible to the metal complex. Inversion of the
G
:C
pair that flanks the U:U mismatch to
C
:G
has no influence on binding of the
factor, despite having some subtle effect on the geometry of the
helix(56) . Substitution of C
by G, which creates
a second mismatch adjacent to the U:U pair and should have an
appreciable effect on the structure of helix IV, also binds TFIIIA with
wild type affinity. The only single nucleotide substitution that we
have made in helix IV that has a notable effect on binding is proximal
to loop E at C
. A transversion to G, which generates a G:G
mismatch, increases the K
3-fold relative to wild
type; however, a transition to U, which generates a G:U wobble pair in
place of G:C
, does not change binding
affinity.
has no
impact on the binding of TFIIIA to 5 S rRNA, there is a significant
3.5-fold decrease in binding to the equivalent mutation in the DNA.
Moreover, substitutions made at this position affect RNA and DNA
binding differently. This position is not important for factor binding
to the RNA; however, all three possible substitutions have a negative
effect on binding of TFIIIA to the gene. Mutations at G
and G
which are silent in 5 S rRNA, likewise, have a
pronounced influence on binding of the factor to the DNA. Notably, at
the former position a transversion to C markedly effects binding while
a transition to A is well tolerated, suggesting contact occurs through
the N7 position of the purine. These latter guanine residues are among
those positions along the ICR identified in methylation interference
experiments(61) . The double mutant G81C,C95G, which simply
inverts a base pair in helix IV, has little impact on binding of the
factor to 5 S rRNA; however, it has a very marked effect on binding to
the gene. This is most likely due to the change at residue 81, since
the single mutation, G81C, has already been shown to significantly
decrease binding of TFIIIA to the gene(23) . Zinc fingers 1
through 3 associate with box C of the ICR (11, 15, 16, 17, 18) and
the helix IV-loop D region of 5 S rRNA(12) . The effects of
mutations characterized here support previous data that this subset of
fingers is of primary importance in mediating binding to DNA and makes
only a small contribution to binding to 5 S
rRNA(10, 12, 19, 62) .
Loop E
In ``missing nucleoside''
experiments we determined that loop E provides a critical structure for
recognition and binding of TFIIIA to 5 S rRNA(9) . Studies with
the structural probe Rh(phen)(phi)
demonstrated that loop E possesses a helical structure, due to base
stacking interactions, with an opened major groove(56) . The
conformation of a 27-nucleotide duplex which represents loop E has been
determined by NMR spectroscopy (34) and reveals that this
domain is comprised of several non-Watson-Crick pairings as well as a
reverse-Hoogsteen pair forming a base triple with G (Fig. 1A). The structure, which closely resembles
an A-form helix, is stabilized by significant base-stacking
interactions and, perhaps, interstrand hydrogen bonding. Of the single
nucleotide mutants tested in the present experiments, those in loop E
have the greatest negative effect on binding of TFIIIA to the RNA.
,
U
, and A
) of the putative base-triple
comprise an important element for recognition by TFIIIA. The conversion
of the reverse-Hoogsteen A:U pair to a G:U pair (mutant A100G) has a
modest influence on binding; however, a change to a C:U mismatch
(mutant A100C) has a much more pronounced effect. Alternatively, when
the Hoogsteen pair is disrupted by a substitution at U
,
the consequences are far more acute; quantitatively, the 9-fold effect
of the mutant U76G is significantly greater than the many block
mutations that have been used to characterize the TFIIIA
5 S rRNA
complex(31) . The greater impact of a substitution at
nucleotide 76 relative to its partner at 100 may reflect the fact that
the former is also paired to G in the base triple and the
latter is not. The U76G mutation engenders an alternative conformation
in 5 S rRNA that is in equilibrium with the wild-type structure and
this accounts for the exceptionally large effect on binding of the
protein. The two forms are resolved on the nondenaturing polyacrylamide
gels used for the binding assay (Fig. 2) with the alternative
structure migrating ahead of the wild-type conformation. Although the
two conformations are in equilibrium, their distinct mobility suggests
that the global higher-order structures of the two forms are
considerably different. The identity elements in 5 S rRNA for ribosomal
protein L5 are confined to the hairpin structure composed of helix
III-loop C. No mutations in the helix IV-loop E-helix V arm, including
the quadruple mutation G70C, G71C,G81C,G82C, alter binding of L5; the
only exception is U76G which has a greatly reduced affinity for L5. (
)These results indicate that structural changes in U76G
occur in regions of the RNA distal to the site of the mutation. In
``missing nucleoside'' experiments removal of U had the most deleterious impact on binding of TFIIIA to 5 S
rRNA(9) . Additionally, U
becomes cross-linked to
G
upon irradiation of 5 S rRNA with ultraviolet light (63) and the strong NOEs between these two bases (34) indicate that the helix is greatly overwound at the step
between the Hoogsteen pair and the flanking G
:A
mismatched pair. This is borne out by the absence of cleavage by
Rh(phen)
(phi) at this end of the
loop(56) . McBryant et al.(64) have shown
that deletion of the bulged G from 5 S rRNA causes a
severe decrease in the binding of a four-fingered peptide derived from
TFIIIA. This result further supports the contention that the base
triple in loop E is either used directly by TFIIIA or creates a higher
order structure essential for recognition.
:A
mismatch pair immediately flanking the
base triple structure is unexpectedly insensitive to mutagenesis;
substitutions at either position do not disturb binding of TFIIIA. NMR
studies (34) indicate that the ribose of A
(and
G
) is predominantly C
-endo and that
this residue is in a reversed conformation, so that the direction of
the two strands is parallel at this position. If substitutions at
A
change this local conformational perturbation, it does
not influence the binding of TFIIIA. The structure generated at the
Hoogsteen base pair, however, may be favored strongly enough to put any
nucleotide at position 74 into a reversed conformation, maintaining
this distortion in the helical structure of this strand.
, creates consecutive mismatched pairs in helix
IV, whereas conversion to a G:U wobble pair (C79T) has no effect. The
consequence of the former substitution may be direct; however, it is
equally possible that this disruption in the structure of the helix
propagates itself into loop E. Indeed, a quadruple mutant
G70C,G71C,G81C,G82C that will alter the structures of both helices IV
and V binds TFIIIA with an affinity comparable to E. coli 5 S
rRNA. This result attests to the importance of this arm of the molecule
for high affinity binding and at the same time provides evidence that
the intricate structure of loop E is critically dependent on the
integrity of the flanking stem structures.Helix V
Disruption of this helix by block
substitutions can increase the dissociation constant of the complex up
to 3-fold; however, in most cases a second mutation that restores a
base paired helical structure, but not the wild type sequence, can
restore the binding affinity(30) . These results indicate that
recognition occurs primarily through the higher order structure of the
helix. Interestingly, we find that a transition at position 70 (G70A),
which results in an A:C pair in helix V, is well tolerated by TFIIIA.
An A:C pair, in which the imino nitrogen of adenine is protonated, has
been shown to fit quite well into an A-form RNA double helix with
virtually no distortion of the backbone(65) . The geometry of
this non-Watson-Crick pair is similar to a G:U base pair (65) and, indeed, we find that conversion to a G:U pair (C105T)
at this position of helix V also has no detrimental effect on binding.
The transversion mutant G70C, however, produces a 3-fold increase in K. The magnitude of the effect of this point
mutation equals or exceeds those of block mutants designed to disrupt
the entire helix, suggesting that G70C, rather than simply disrupting
the secondary structure of the helix, removes an important site for a
sequence-specific contact with the protein. The fact that substitution
by adenosine (G70A) has no effect supports this contention and provides
evidence that contact at position 70 could occur through the N7
position of the purine base. is also a critical contact point in the
gene (61) . The consequences of substitutions made at this
position are similar for both the RNA and DNA, indicating that this
could be a common site of recognition shared between the two nucleic
acids. A transition to A has no effect on the binding of TFIIIA to
either nucleic acid, while transversion to C causes a decrease for
both. The slightly greater effect of G70C on binding of the factor to
RNA relative to DNA can be explained by the pyrimidine:pyrimidine
apposition resulting in the former. Various mapping experiments
indicate that finger 5 is contiguous to base pairs centered around
position 70 both in the gene (11, 13, 15, 16, 18) and 5
S rRNA(19, 64) . Results from interference (61) and missing contact (18) experiments as well as
the differences noted here between transition and transversion
mutations all point to an interaction at this site through the major
groove (possibly at the N7 of the purine) of both nucleic acids. Zinc
fingers 4-6 of TFIIIA run parallel to the helical axis of the
DNA, rather than following the trajectory of the major
groove(15, 18) . Although finger 5 appears to bind
through the major groove of the DNA, missing nucleoside experiments
indicate that its orientation relative to the helix is different from
the other major groove fingers, i.e. fingers 1-3 and
7-9(18) . This distinct alignment could reflect the fact
that this finger may also be required to penetrate the less sterically
accessible major groove of an A-form RNA helix. Thus, contacts to the
nucleic acids may occur through amino acid side chains of the
-sheet or tip region rather than the residues of the -helix
that dominate the interactions seen in the Zif 268(66) ,
GLI(67) , and Tramtrack (68) co-crystal structures. In
this instance it is relevant that the structure of the DNA helix at the
binding site for individual zinc finger domains frequently has a deep,
but wide, major groove that can be characterized as intermediate
between canonical A and B conformations(67, 69) . We
have shown that the helical structure of the Xenopus 5 S rRNA
gene is highly polymorphic, containing elements with A-like
conformation(38, 70) . This feature in the structure
of the DNA may also explain how the protein could make an equivalent
contact to sites on both DNA and RNA.Loop A
A model for oocyte 5 S rRNA based on
chemical reactivity data has helices II and V nearly co-axial; this
orientation is dependent on a triple interaction among nucleotides
A, G
, and U
(71) . This
model has been tested by an exhaustive set of mutations in loop A which
show that substitutions that decrease the flexibility of this
``hinge'' region often have a negative effect on the affinity
of TFIIIA(27) . We have changed U
to a C which
can then form a Watson-Crick pair with G
, extending helix
V toward loop A. This change results in a 2-fold increase in K
which has been reported by Baudin et
al.(27) . It is clear from their work that a specific
three-dimensional structure, dictated by the hydrogen bonding pattern
within loop A, is an essential recognition element for this
interaction. Zinc fingers 4 and 6 are especially important for binding
of TFIIIA to 5 S rRNA (12, 19) and appear to be
positioned at loops E and A, respectively(64) . It has been
proposed that these two fingers, unlike the others, extend across the
minor groove of the ICR when the factor is bound to the
gene(15, 17, 18) , reflecting their
functional difference relative to the other fingers. The only portion
of loop A that has corresponding nucleotides in the ICR are positions
65-67. There is no data from the many mutagenesis or chemical and
enzymatic probing experiments that indicate these nucleotides are
important for the binding of TFIIIA to the gene.Helix II-Loop B-Helix III
This arm of 5 S rRNA
encompasses the binding site for fingers 7-9 which make
corresponding contacts to the box A element (bp 50-64) of the
ICR. These three fingers are not essential for binding of the factor to
either nucleic acid(12) . Although dispensable, these fingers
still make thermodynamic contributions to the free energy of binding to
both 5 S rRNA(19, 64) and the
gene(13, 72) . Contacts made by TFIIIA to this region
of the RNA appear to utilize the secondary structure of the nucleic
acid. Disruption of helix II with block mutations lowers affinity for
TFIIIA as much as 3-fold; restoration of duplex structure, but not
sequence, with complimentary changes in the opposite strand returns
binding to wild type affinity(30) . Likewise, point mutations
at several positions in helix II lower binding of the
factor(12) , but these effects can also be reversed by
secondary mutations that restore Watson-Crick pairing. Linker (25, 29, 30) and point (12) mutations
in the remaining domains of this arm (i.e. loop B, helix III,
and loop C) have only small effects, if any, on the affinity of TFIIIA
for 5 S rRNA. Of the mutants we have made in this arm of the RNA, many,
including the deletion of the two bulged adenosine residues in helix
III, have no impact on binding of TFIIIA. However, substitutions at
positions 43/44, 53, and 56, which occur at stem-loop junctions, do
have modest effects. These results are in accord with chemical and
enzymatic protection data (8) which suggest that TFIIIA
utilizes the accessible, widened major groove that is known to occur at
stem-loop junctions in RNA(73) . to C abolishes the internal
hypersensitive site on the coding strand at position 51 (Fig. 4). Mapping experiments with metal complexes that bind to
DNA on the basis of shape complementarity revealed distinct, localized
openings in the major groove due to base pair tilt(70) .
Modeling experiments, based upon data from the conformational mapping,
indicated that an opened major groove which is also underwound (A-like
in structure) provides the maximal amount of surface overlap between a
zinc finger domain and the DNA. Subsequently, these structural features
of the helix were observed in the co-crystal structures of two
different zinc finger peptides bound to their cognate
sequences(67) . Interactions, such van der Waals forces,
resulting from surface complementarity between the opened major groove
and the zinc finger domain could potentially contribute to the binding
of TFIIIA to the 5 S rRNA gene and may account, in some part, for the
apparent complexity of the interaction.
Conclusions
Mutations in helix IV and loop D have
no appreciable effect on the binding of TFIIIA, despite evidence that
the protein is closely associated with this region of the
RNA(7, 8, 9, 12) . The regular A
type conformation of helix IV should disallow contact through the major
groove. The U:U
mismatch does create an
opening in the major groove(56) ; however, when it is closed by
mutagenesis (T96G,T96A) there is no change in binding of the factor.
These results indicate that fingers 1-3 are weakly associated
with the RNA through contacts to the ribose-phosphate backbone and/or
sites in the minor groove, an arrangement different from that in the
DNA complex.
:U
:A
base triple as a major recognition element for TFIIIA. Our results,
however, cannot be used to conclude whether contacts with finger 4
occur through the major or minor groove of this structure. Finger 6
also contacts a loop structure (loop A) and substitutions at this site,
likewise, have a considerable impact on binding of TFIIIA to the RNA (27) . Fingers 4 and 6 appear to be specifically designed for
recognition of the unique geometries presented by loops E and A,
explaining both their importance for binding of the factor to 5 S rRNA
and their relatively subordinate interaction with the gene.
, they can cause considerable
disruptions between the 5`-end of the ICR and the carboxyl-terminal
fingers of the factor. Thus, although the apparent thermodynamic
contribution of these three fingers to binding is not large, these
interactions are important for properly orienting the factor on the
ICR.
)(phi),
bis(phenanthroline)(phenanthrenequinone diimine)rhodium(III); CMCT,
1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide
metho-p-toluene sulfonate; bp, base pair(s); DTT,
dithiothreitol.)
We are grateful to Dr. Elliott Rosen for
oligonucleotides, Drs. James McLinden, Elliott Rosen, and Joseph
O'Tousa for providing the necessary strains and plasmids for
site-directed mutagenesis, and to members of our laboratory for
critically reading the manuscript.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
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