(Received for publication, July 14, 1994; and in revised form, December 18, 1994)
From the
The bacteriophage T4 regA protein is a translational repressor
that regulates the synthesis of >12 T4 proteins. Earlier studies
demonstrated that photocross-linking of the 122-residue regA protein to
(dT) occurs at two sites, with the major site occurring at
Phe-106. Amino acid substitutions were introduced at Phe-106 to
evaluate its role in nucleic acid binding. Binding affinities of
mutants F106C, F106V, and F106Y for nonspecific and specific RNA
ligands indicated little difference between the K
of the mutants and wild type regA protein, for either poly(U) or
for a specific gene 44 oligoribonucleotide. Thus, Phe-106 does
not contribute measurably to the overall free energy of binding.
Partial proteolysis of regA protein was carried out to further probe
its domain structure. Chymotryptic cleavage produced a fragment of
11,095 Da that has reduced affinity for poly(U) and that contains the
first 93 residues of regA protein. Interestingly, proteolysis of regA
protein is reduced in the presence of the specific target, gene 44 RNA.
Two deletion mutants, 1
94 and 1
109, have also been cloned
and purified. The binding affinities of these deletion mutants
indicated a 100-1000-fold reduction in their affinities for poly(U).
These studies indicate the last 13 amino acids in regA protein make a
significant contribution to RNA binding.
The bacteriophage T4 regA protein regulates the expression of at least 12 T4 genes, while also regulating its own synthesis at the level of translation (for review, see Miller et al.(1994)). It has been shown that regA protein acts by binding to specific mRNAs and competing with the formation of initiation complexes by ribosomes (Winter et al., 1987; Unnithan et al., 1990). We have previously shown that repression of T4 gene 44 by regA protein involves the recognition of a specific RNA element, in which apparently both the sequence and structure are of importance (Webster and Spicer, 1990; Szewczak et al., 1991).
More recent studies have focused on
structure-function relationships in regA protein in an effort to
identify domains and specific amino acid residues involved in RNA
recognition and binding (Webster et al., 1992; Jozwik and
Miller, 1992). Photochemical cross-linking has identified amino acid
residues at the interface of a regA protein: nucleic acid complex
(Webster et al., 1992). Two sites of cross-linking to
p(dT) were identified: the major site of cross-linking was
at Phe-106 and the minor site was the peptide spanning 31
41, with
the site of cross-linking tentatively assigned to Cys-36 (see Fig. 1). A COOH-terminal peptide (containing residues
95
122) produced by CNBr cleavage of regA protein was found to
retain the ability to bind to nonspecific RNA and to be
photo-cross-linked to p(dT)
(Webster et al.,
1992). In addition, Jozwik and Miller(1992) have examined mutations in
T4 and the related phage RB69 regA proteins and have found that two
regions are particularly sensitive to mutations: the regions from
Val-15 to Ala-25 and between Arg-70 and Ser-73. Finally, amino acid
similarities to other RNA-binding proteins have been identified for two
regions of regA protein; one in the NH
-terminal region
between residues Val-15 to His-37 containing a potential
helix-turn-helix structural motif (Jozwik and Miller, 1992) and the
other in the COOH-terminal region between residues Leu-97 and Lys-113
(Webster et al., 1992) (see Fig. 1).
Figure 1: A, Amino acid sequence of T4 regA protein represented by the one-letter code. Asterisks indicate the sites of photo-cross-linking. B, schematic summary of fragments of regA protein used in this study.
In this study,
the role of residues in the COOH-terminal region of regA protein have
been examined further. In order to determine if residue Phe-106, the
major site of photo-cross-linking, plays a significant role in
RNA-protein interactions, site-specific mutagenesis was used to
introduce three substitutions at codon 106. To assess the role of
residues neighboring Phe-106, two truncation mutants were constructed
containing residues 1109 and 1
94 (Fig. 1). In
addition, we have examined the sensitivity of regA protein to partial
proteolysis by chymotrypsin and trypsin.
The apparent association constant (K) was calculated using the equivalence point
of the titration and a binding site size of n = 9
residues (assuming non-overlapping sites) for regA protein binding to
polynucleotides, as determined previously (Webster and Spicer, 1990).
Direct determination of the binding site size for regA protein
fragments 1
94 and 1
109 binding to poly(U) was not possible
because of the low affinities of the proteins for nucleic acids. A
simplifying assumption was made that the site size for both protein
fragments is likely to be smaller than that of WT regA protein since
the fragments correspond to approximately two-thirds the size of regA
protein. Accordingly a proportional site size of 6 nucleotides was
assumed. If the occluded site sizes of the deletion mutants were 9
nucleotides, then the K
would be 2-fold higher
than estimated. For oligonucleotide gene 44-4, a single binding site
was assumed for both regA protein and the protein fragments. The
maximal percent quenching (%Q
) was determined by
two methods (a) the observation of a constant plateau in
protein fluorescence quenching after successive additions of lattice to
regA samples or (b) for oligonucleotide gene 44-4, in cases
were a stable plateau was not reached, %Q
was
derived from double-reciprocal analysis in which 1/
F was
plotted versus 1/(free oligo) (Kelly et al., 1976).
The y intercept of the plot yields 1/
F
.
Laser desorption mass spectrometry (LDMS) analyses were performed on
a VG TOFSPEC (Fisons) mass spectrometer. For determination of tryptic
and chymotryptic peptide masses, the mass spectrometer was scanned from m/z = 500 to m/z = 17000. The samples
were run in an -cyano matrix. The instrument was calibrated using
-lactoglobulin (18,281 Da and its doubly charged ion, 9,141 Da),
insulin (5,734 Da), and gramicidin (1,142 Da). All LDMS analyses were
performed by the W. M. Keck Foundation Biotechnology Resource
Laboratory at Yale University. Samples submitted to LDMS analysis were
prepared by 10% trichloroacetic acid precipitation, as described by
Stone et al.(1989). The precipitate was then resuspended in
deionized H
O.
Comparison of the CD spectra of the mutant
proteins with that of wild type regA protein, shown in Fig. 2A, indicate that substitutions of Cys and Tyr for
Phe-106 do not produce large alterations in the protein's
secondary structure. Based on variations observed in the
200-215-nm region of repeated CD scans of WT regA protein (Fig. 2B), the variations in CD spectra observed for
the F106C and F106Y proteins are relatively small. Although the
difference between the F106V and the WT scan appears larger than that
of the other two mutants, we note that the shape of the F106V and WT
scans are extremely similar and that the difference in absolute molar
ellipticity of these two proteins is less than the ±10% average
error in determining their concentrations (see ``Materials and
Methods''). Prosec analysis of these spectra generally confirms
these qualitative conclusions. As shown in Table 1, the F106Y and
F106V mutants have predicted -helical contents that are within two
standard deviations of the range seen for the WT protein. In the case
of F106C, Prosec analysis predicts this substitution decreases the
-helical content by less than 8%. That substitution of Phe-106 by
these three mutations does not result in any large change in the
protein's secondary structure is further confirmed by limited
proteolysis studies. That is, comparative partial proteolysis using
either chymotrypsin or trypsin (see below for proteolysis of WT regA
protein) did not reveal any observable differences in sensitivity to
proteolysis, consistent with the overall conformations of the three
substitution mutants being similar to that of WT.
Figure 2:
Circular dichroism spectra of wild type
and Phe-106-substituted regA proteins. A, molar (residue)
ellipticities are plotted as a function of wavelength. Protein
concentrations were between 5 and 10 µM in buffer C (pH
7.2), plus 75 mM NaCl. Following data collection (at 25
°C) protein concentrations were determined by duplicate amino acid
analysis. Line representations are WT regA (-), F106C
(--), F106V(- - - - -), and Fl06Y
(-
-
). B, comparison of the four repeat
scans of WT regA protein to the averaged scan (solid
line).
As a first step
toward determining if the mutant proteins bind to nucleic acids in a
manner similar to that of wild type regA protein, the ability of the
proteins to be photo-cross-linked to
[P]p(dT)
was examined. As shown in Fig. 3, all three mutant proteins retain the ability to be
photo-cross-linked to p(dT)
, although with variable
efficiency. As judged by gel electrophoresis and densitometry of
autoradiograms, the efficiency of cross-linking of the proteins with
the three substitutions was Cys > Phe > Val > Tyr with
corresponding efficiencies of 1.2:1.0:0.7:0.3. The variation in
efficiencies presumably is due to differences in reactivity of the four
amino acids and may also be a reflection of changes in the topology of
protein:DNA contacts.
Figure 3:
Photochemical cross-linking of wild type
and Phe-106-substituted regA proteins to p(dT). Proteins
(10 µM) were preincubated with
[
P]p(dT)
(15 µM) in
buffer C plus 75 mM NaCl at 4 °C and exposed to UV light
at a dose of 1
10
ergs/mm
. Samples were
boiled in sample loading buffer and then electrophoresed on an SDS-15%
polyacrylamide gel. The cross-linked products were visualized by
autoradiography and quantitated by densitometry. The lower molecular
weight cross-linked species in the F106C lane may result from
proteolysis or nuclease degradation of the
p(dT)
F106C complex.
To further examine the functional activity of
the mutant proteins, the ability of the proteins to inhibit translation
of three T4 genes was examined. For these assays, plasmid pTL45W, which
contains T4 genes rpbA, 45, and the 5` half of gene 44 (g44) under the transcriptional control of the
P
promoter, was used as a template in in vitro coupled
transcription-translation assays (see Webster et al., 1989).
Based on these studies (data not shown), it appears all three Phe-106
mutant regA proteins retain the ability to inhibit translation of the
three regA-controlled genes on this plasmid.
To quantitatively
assess the effect of the Phe-106 amino acid substitutions on the
functional activity of regA protein, nonspecific and specific RNA
binding affinities were measured. Fluorescence quenching assays were
used to measure the apparent equilibrium binding constants (K) of the three mutants for poly(U) and for the
gene 44-4 16-mer RNA (5`-AAUGAGGAAAUUAUGA-3`), corresponding to the
binding site for regA protein on gene 44 mRNA (Webster and Spicer,
1990). The reverse fluorescence titration curves, shown in Fig. 4A, demonstrate that the affinity of the Phe-106
regA substitution mutants for the nonspecific RNA target poly(U) is
very similar to that of wild type regA protein. The corresponding
salt-back titrations for the Phe-106 substitution mutants, shown in Fig. 4B, indicate that binding of all three mutant
proteins is slightly more sensitive to ionic strength than WT regA
protein binding. Reverse titrations for specific binding to the target
gene 44-4 RNA, shown in Fig. 5, demonstrate binding affinities
that are, again, very similar to that of the wild type regA protein.
The affinity constants derived from these binding curves, given in Table 2, indicate that the substitutions at Phe-106 do not
significantly affect the ability of regA protein to bind to either
nonspecific or specific RNA.
Figure 4:
A, reverse titrations of
Phe-106-substituted mutants and wild type regA protein with poly(U).
The quenching (%Q) of intrinsic protein fluorescence upon
binding to nucleic acid is plotted as a function of the molar ratio of
uridine monophosphate (P) to protein. Titrations were
performed with 0.2 µM protein in buffer C plus 10 mM NaCl, at 25 °C. Fluorescence measurements were acquired using
= 282 nm and
= 347
nm. WT regA protein (
) and mutant proteins are designated F106C
(
), F106Y (
), and F106V (
). B, fluorescence
``salt-back'' titrations of Phe-106-substituted mutants and
wild type regA protein bound to poly(U). Titrations were made using
increasing additions of 5 M NaCl to samples saturated with
poly(U) (A). The percent of initial fluorescence was estimated
from comparison to a photobleach control of each protein, which was
corrected for dilution effects. Symbols are the same as in A.
Figure 5: Reverse titrations of Phe-106 substituted mutants and wild type regA protein with specific target RNA, gene 44-4 (16 mer). Titrations were performed at 0.2 µM protein in buffer C plus 150 mM NaCl (25 °C). Symbols used are the same as in Fig. 4.
To evaluate
whether the fragments were folded in a near native conformation, CD
spectra were measured for the deletion mutants (Fig. 6A). The CD spectra of a synthetic peptide
corresponding to COOH-terminal residues 95122 was also measured (Fig. 6B). Summation of the CD spectra of the 1
94
fragment and the synthetic peptide 95
122 resulted in a composite
CD spectra (Fig. 6B) which was found to overlap closely
with that of the unmodified wild type protein. Prosec analysis of these
spectra suggest that while the 95-122 synthetic peptide contains
no detectable
-helix the 1
94 fragment contains 57%
-helix. The latter corresponds to about 54 residues of
-helix, as opposed to the
66 residues predicted in the native
protein (Table 1). Thus, while cleavage at Met-94 may lead to the
loss of
12 residues of
-helical structure, it does not appear
to substantially alter the overall regA protein secondary structure.
Figure 6:
Circular dichroism spectra of wild type
and truncation mutants of regA protein. Molar (protein or fragment)
ellipticities are plotted as a function of wavelength. In the case of
the regA fragments, the molar ellipticities were adjusted based on the
relative size of the fragments as compared to regA protein (i.e. so the molar ellipticity reflects the contribution of the
fragments to the regA CD spectrum). Protein concentrations and
conditions were identical to those in Fig. 2. A,
individual spectra of WT regA protein (-), 1109(- -
- - -), 1
94 (
-
-) and CNBr6 synthetic
peptide corresponding to residues 95
122
(
). B, comparison of the summed
spectra of fragments 1
94 and 95
122 with the spectrum of WT
regA protein. Line representations are WT regA (-) and
combined 1
94 and 94
122 fragments spectrum(- - - -
-).
To evaluate the RNA binding affinities of regA proteins 194
and 1
109, reverse titration fluorescence quenching assays were
performed. As illustrated in Fig. 7, the observed fluorescence
quenching of 1
109 and 1
94 upon poly(U) binding were both
dramatically reduced relative to that of WT regA protein (Fig. 4A). The estimated %Q
for
poly(U) titrations of 1
94 and 1
109 were 16 and 35%,
respectively. This reduction in %Q
, from the 75%
value observed with WT regA protein, suggests that the individual
contributions of the three tryptophans (Trp-78, Trp-81, and Trp-112) to
regA protein fluorescence quenching may not be equal and that Trp-112
may be a major contributor to the fluorescence quenching observed upon
poly(U) binding.
Figure 7:
Reverse
titrations of deletion mutants 194 and 1
109 with poly(U).
Titration conditions were the same as in Fig. 5. Protein
designations are: (
) 1
109; (
)
1
94.
The reduced level of fluorescence quenching and
weaker binding (see below) observed for the regA protein fragments made
estimation of %Q and subsequent calculation of K
somewhat less accurate than for regA protein.
Nevertheless, approximations of K
could be made
from double reciprocal plots (for gene 44-4 oligonucleotide) or from
curve fitting (poly(U)). The K
derived from
these analysis, given in Table 3, indicated that the binding
affinities of 1
94 and 1
109 for poly(U) and gene 44-4 RNA
are approximately 100-1000-fold lower than that of wild type regA
protein.
Figure 8: Partial chymotryptic proteolysis of WT regA protein in the absence and presence of 44-4 RNA. The reaction was carried out in 20 mM Tris (pH 8.0), at 37 °C, with a ratio of 25:1 (w/w) of regA protein to chymotrypsin. Reactions labeled (+ gene 44) were carried out in the presence of an equimolar amount of gene 44-4 RNA. Note that early time points give an indication of intermediate cleavage sites in the COOH terminus.
To determine the
site(s) of chymotryptic cleavage that produced the 11-kDa fragment,
fragments eluted from a poly(U) agarose column were separated by
SDS-PAGE and then electro-transferred to an Immobilon P membrane.
NH-terminal micro-sequencing was performed on the membrane
section containing the 11-kDa polypeptide. The sequence of the first 4
residues revealed that the fragment has the same
NH
-terminal sequence as the intact protein. Determination
of the masses of the chymotryptic and tryptic core fragments by LDMS
revealed that the chymotryptic fragment (11,095 Da) (Fig. 9) is
approximately 200 daltons larger than the tryptic fragment (10,898 Da).
These mass determinations and NH
-terminal sequencing data
together indicate that the site of cleavage by chymotrypsin producing
the 11-kDa fragment, is at Phe-93, while that of trypsin is at Arg-91.
In addition, LDMS analysis of chymotryptic digestion products indicated
4-5 smaller peptide masses (see Fig. 9), indicating that
multiple proteolytic cleavages occur in the COOH-terminal region. The
possible sites of chymotrypsin cleavage (after aromatic residues) in
this region are at Phe-93, Phe-101, Phe-106, Trp-112, and Tyr-118. As
shown in Table 4, four of the observed chymotryptic fragments
have masses corresponding to peptides predicted from cleavages at three
of these sites (i.e. after Phe-93, Phe-106, and Tyr-118).
While these cleavages may occur after a COOH-terminal fragment has been
released from regA protein, the observation of two intermediate
proteolytic products by SDS-PAGE (Fig. 8) suggests that these
cleavages can occur in the intact protein. Taken together with the CD
studies, these results suggest the NH
-terminal three
quarters of regA protein are folded into a structured, protease
resistant core, while the COOH-terminal one quarter is significantly
more flexible and exposed to solvent.
Figure 9: LDMS analysis of chymotryptic fragments of regA protein. The top panel shows fragments in the mass range between 1,000 and 6,400 m/z; bottom panel presents fragments observed in the 5,000-18,000 m/z range. regA protein was digested for 30 min with chymotrypsin (25:1 (w:w) ratio protein to chymotrypsin) and then trichloroacetic acid-precipitated, prior to LDMS analysis (see ``Materials and Methods'').
The functional activities of three regA protein mutants with
substitutions at Phe-106 were assayed by UV-induced photo-cross-linking
and fluorescence titrations of RNA binding. regA F106C and F106V
proteins were found to retain a near native ability to be
photo-cross-linked to p(dT), while F106Y demonstrated a
reduced cross-linking efficiency. Coupled with the negligible effect of
the F106Y mutation on RNA binding affinity, this result suggests that
the preponderance of proteins that have been found to be cross-linked
at Phe (Williams and Konigsberg, 1991), as opposed to Tyr, results from
the unique ability of Phe to participate in these photochemical
reactions, as opposed to any unique ability of Phe to participate in
interactions with single-stranded nucleic acids.
The nonspecific and specific RNA binding affinities of all three mutants were found to be very similar to that of wild type regA protein. However, RNA binding of all three substitutions was found to be slightly more salt sensitive than wild type regA protein suggesting slight changes in the types of RNA: protein interactions. Previous fluorescence binding studies (Webster and Spicer, 1990) demonstrated that RNA recognition by WT regA is weakly salt-sensitive and that as few as two ionic interactions may be involved in RNA binding by WT regA protein. Taken together, these results indicate that while Phe-106 is at the interface of a regA protein-nucleic acid complex, it does not contribute significantly to the overall free energy of RNA binding.
Partial proteolysis studies
suggest that the NH-terminal three quarters of regA protein
are folded into an ordered, protease-resistant core, while the
COOH-terminal one quarter is more exposed to solvent and/or less
structured. The crystal structure of regA protein solved by C.-H. Kang,
A. Rich and co-workers (
)reveals that residues 90-96,
as well as a number of additional COOH-terminal residues, are on the
surface of the protein. Interestingly, RNA binding to regA protein
results in a reduction in susceptibility to proteolytic cleavage. This
is presumably due either to a direct shielding of the chymotryptic
cleavage sites on regA protein by the bound RNA or to a conformational
change in the COOH-terminal region induced by RNA binding. Such a
reordering of an unstructured region has been proposed for a region of
the RNP domain of U1 sn-RNP A protein (Nagai et al.,
1990) and has been observed in a number of other nucleic acid binding
proteins (Steitz, 1990).
Previous studies (Webster et al. 1992) demonstrated that a COOH-terminal fragment of regA protein
(CNBr 6) containing residues 95-122 retained the ability to be
photo-cross-linked to p(dT) and to bind poly(U) (K
= 3
10
M
in 10 mM NaCl). To further
evaluate the relative contribution of COOH-terminal residues to RNA
binding, a complimentary fragment containing residues 1-94 was
produced and tested for RNA binding. This fragment has an affinity of
approximately 3
10
M
for poly(U) (in 10 mM NaCl) (Table 3). Thus, both
fragments have weak affinities for RNA. Summation of the free energies
of RNA binding for the two fragments is somewhat higher than the free
energy for intact regA protein binding to poly(U). That is, the free
energy of regA protein binding is
10.0 kcal, compared to
12.2
kcal for the sum of the free energies of binding of the two fragments.
This discrepancy may arise from inaccuracies in estimation of the
binding site size and/or %Q
for both fragments.
Also, the CNBr6 peptide may bind somewhat tighter when it is free in
solution than as part of intact regA protein (see Nadler et
al., 1992).
These binding studies indicate that the
NH-terminal 1
94 fragment of regA protein retains the
ability to bind to RNA, although with significantly weaker affinity
than intact regA protein. The 1
109 fragment, containing 15
additional C-terminal residues (including Phe-106) had a somewhat
higher affinity for poly(U) than 1
94. However, the 1
109
fragment binds RNA with a 100-fold lower affinity than WT regA protein,
indicating that a large part of the fragments' observed defects
in RNA binding is due to the absence of the COOH-terminal 13 residues.
Interestingly, this region was identified by Jozwik and Miller(1992) as
being one of three mutationally sensitive regions of regA protein.
Based on the relative affinities of 1
109 and WT regA protein for
poly(U) (2
10
versus 2
10
M
), the C-terminal 10% of regA
protein contributes approximately 28% of the overall free energy of RNA
binding. Thus, although other regions of regA protein also must play
important roles in RNA binding, the proteolysis and truncation studies
reported here demonstrate that COOH-terminal residues make significant
contributions to RNA:regA protein interactions. The crystal structure
of regA protein
has revealed similarities between
NH
-terminal residues of regA protein and the RNP domain
found in a number of RNA-binding proteins (Bandziulis et al.,
1989), including the U1 sn-RNP A protein (Nagai et
al., 1990). We anticipate that future mutagenesis and x-ray
structural studies of regA protein will reveal how both the
NH
- and COOH-terminal regions of regA protein are
associated with RNA in protein-RNA complexes.