(Received for publication, August 17, 1995)
From the
It was shown that Cys-168 is required for RNase T function and
thermostability and that its hydrophobic properties are important for
this role (Li, Z., Zhan, L., and Deutscher, M. P.(1996) J. Biol
Chem. 271, 1127-1132). To understand the molecular basis for
these findings, further studies of Cys-168 and RNase T structure were
carried out. Treatment of RNase T with the sulfhydryl-modifying agent
5,5`-dithiobis-(2-nitrobenzoic acid) leads not only to inactivation,
but also to monomerization of the protein. Similarly, specifically
converting Cys-168 to either serine or asparagine leads to loss of
activity and to monomer formation at 37 °C. However, at 10 °C
the serine mutant remains as a dimer and retains full RNase T activity,
whereas the asparagine derivative shows only a low level of activity
and of dimer formation. These data show a strong correlation between
activity and the dimer form of RNase T. The importance of dimer
formation was also shown in vivo using genetic studies. An
inactive mutant of RNase T, termed HA2, which exists as a dimer at 37
°C in vitro, completely suppresses endogenous RNase T
activity in vivo and in vitro when introduced into a
RNase T cell on a multicopy phagemid, most likely as a
consequence of inactive heterodimer formation. Introduction of the HA2
gene on a single-copy plasmid, as expected, leads to a proportionally
smaller effect on endogenous activity. The dominant negative effect
displayed by the HA2 protein can be relieved by an additional mutation
in HA2 RNase T that abolishes its ability to dimerize. An inactive
mutant asparagine derivative of Cys-168, which also does not dimerize,
also shows little of the dominant negative phenotype. Thus, these data
demonstrate that RNase T dimerizes in vivo, that the dimer
form is required for RNase T activity, and that Cys-168 is needed for
dimerization of the enzyme.
RNase T, one of eight exoribonucleases identified in Escherichia coli, plays an important role in both tRNA and 5 S RNA metabolism(1, 2, 3) . In a companion study(4) , we showed, by chemical modification, site-directed mutagenesis, and activity measurements in vivo and in vitro, that the enzyme contains an essential cysteine residue at position 168, as well as a second cysteine at position 112 that also contributes significantly to RNase T function. Interestingly, Cys-168 does not appear to participate directly in substrate binding or catalysis. Rather, this residue contributes a hydrophobic group that is important for RNase T structure, affecting the protein's thermostability and ultimately its activity.
In this study, we examine the structural role of Cys-168 in more detail. Our data demonstrate that treatments which modify or alter this residue convert RNase T to an inactive monomer, and that restoration of activity is related to dimer formation. Moreover, inactive RNase T can display a dominant negative effect in vivo, but only if the mutant protein is capable of dimerizing. These findings indicate that Cys-168 is involved in the dimerization of the RNase T subunits and that dimer formation is required for RNase T activity.
Figure 1:
Gel filtration of RNase T treated with
DTNB. One mg of wild type or DTNB-treated wild type S100 extract was
applied to an Ultrogel AcA44 column (51 0.6 cm) preequilibrated
with buffer A (without dithiothreitol for DTNB-treated S100) at 37
°C. DTNB treatment was carried out by adding DTNB (5 mM)
to 10 ml of 100 mM NaPO
buffer, pH 7.3 containing
1 mg of the S100 fraction. The sample was incubated for 30 min at 25
°C, concentrated to 0.1 ml, and adjusted to 1 M KCl prior
to loading the column. Fractions of 0.23 ml were tested for RNase T
protein by immunoblotting as described under ``Experimental
Procedures.'' The amount of RNase T protein from wild type S100
(
) or DTNB-treated S100 (
) are presented in arbitrary
densitometric units.
Figure 2:
Immunoblotting of RNase T from cell
extracts and gel filtration. Cell S100 extracts (5 µg, except HA2,
10 µg, and Ha2-1, 20 µg) or gel filtration peaks containing an
equivalent amount of RNase T were separated on a 12% SDS-PAGE gel,
transferred, and detected by RNase T antibody as described under
``Experimental Procedures.'' Purified RNase T was run as the
standard to determine the size of the RNase T
bands.
The second RNase T peak generated by DTNB modification actually is slightly larger than the dimeric form of the enzyme (Fig. 1). SDS-PAGE and immunoblotting of the material in this peak indicated that the RNase T polypeptide present also is of normal size (Fig. 2). However, SDS-PAGE run under nonreducing conditions revealed that RNase T is also present in several bands of various sizes that are larger than the dimeric form of the enzyme (data not shown). We suspect that these represent cross-linked products between RNase T and other proteins that form by disulfide interchange with the DTNB-modified protein.
As noted above (Fig. 1), wild type RNase
T elutes at the dimer position at 37 °C, and as shown in Fig. 3A, it elutes at the identical position at 10
°C. In contrast, the C168S mutant protein elutes at the monomer
position at 37 °C, but it elutes at the dimer position at 10 °C (Fig. 3B). At 20 °C the protein elutes in between
the monomer and dimer position, undoubtedly reflecting an equilibrium
between the two forms of the protein (Fig. 3B).
SDS-PAGE and immunoblotting of the 37 °C peak fraction indicated
that the RNase T polypeptide is of normal size (Fig. 2). The
RNase T activity of the C168S mutant protein correlates very well with
its dimerization state. At 10 °C, when the mutant protein is a
dimer, nuclease activity in a S100 extract is >90% that of the wild
type; this decreases to 75% at 20 °C and, as already noted, to
<10% at 37 °C, when the protein is a monomer.
Figure 3:
Gel
filtration of RNase T wild type and Cys-168 mutants. S100 extracts of
wild type, C168S, and C168N mutant rnt genes on pBS(+) in
a T host were analyzed by gel filtration as described
for Fig. 1except that 5 mM dithiothreitol was present
in each run. The curves show the amount of RNase T protein in fractions
of gel filtrations performed at 10 °C (
), 20 °C (
),
or 37 °C (
). A, wild type; B, C168S; C, C168N.
Study of the
C168N mutant protein leads to the same conclusion, i.e. dimerization is required for activity. Gel filtration at 37
°C, at which the enzyme is inactive, shows only the monomer form (Fig. 3C). Even at 10 °C, the C168N mutant elutes
primarily between monomer and dimer, with at most a small shoulder
extending into the dimer position. For this mutant protein, RNase T
activity at 10 °C is at most 25% of wild type. Thus, the C168N
mutant, which contains a more hydrophilic substitution at position 168,
and is less active at each temperature, also is more
temperature-sensitive for dimerization. The valine mutant protein, on
the other hand, runs on gel filtration as the dimer form at 10, 20, and
37 °C (data not shown). These data support the conclusions that the
Cys residue at position 168 participates in dimer formation, that the
hydrophobicity of the residue at this position is important, and that
RNase T activity depends on the dimer form of the enzyme.
Screening of RNase T clones was carried out in CCA
cells that lack
the enzyme tRNA nucleotidyltransferase, as described in our companion
study(4) . In a CCA
background, lowered RNase
T activity leads to faster growth. Thus, after phagemid infection, the
largest colonies were assayed for RNase T activity in vitro.
By this method, one mutant, termed HA2, with very low RNase T activity,
was selected. Based on DNA sequencing (data not shown), the HA2 rnt gene contains two mutations that would lead to Arg-15
His
and Gly-28
Arg in RNase T. The HA2 protein is normal with regard
to its amount and size (see Fig. 2; as noted in the legend,
twice as much HA2 protein was loaded), and it exists as a dimer at 37
°C (Fig. 4). However, the HA2 protein is essentially devoid
of RNase T activity. In a RNase T
cell, the HA2 gene
in the multicopy vector pBS(+), results in a level of RNase T
activity equivalent to only 13% of that present in a RNase T
cell containing the single chromosomal copy of rnt (Table 1); no RNase T activity is detectable when the HA2
gene is introduced into a RNase T
cell on the single
copy plasmid pOU61 (Table 1). Thus, the HA2 gene results in a
RNase T with considerably < 1% of wild type activity.
Figure 4:
Gel filtration of HA2 and HA2-1 mutant
RNase T. S100 extracts of the HA2 and Ha2-1 genes on pBS(+) in a
T host were analyzed by gel filtration on an AcA44
column preequilibrated and run with buffer A at 37 °C. RNase T
protein was detected by immunoblotting as described under
``Experimental Procedures.'' HA2 (
) and HA2-1
(
).
Of most
interest, however, is that the presence of this HA2 protein exerts a
dominant negative effect on the endogenous RNase T activity present in
the RNase T cell (Table 1). Thus, when HA2 is
present on pBS(+), it completely suppresses the activity arising
from the chromosomal rnt
gene (Table 1), and all that is seen is the 13% activity due to the
HA2 protein. Likewise, when HA2 is present on pOU61, it decreases RNase
T activity over 70%. These findings strongly suggest that the HA2
protein must form a heterodimer with the wild type RNase T monomer
resulting in an inactive protein. Indeed, the level of RNase T activity
remaining (28%) when HA2 is introduced into the RNase T
cell via pOU61, is just what would be expected for the amount of
wild type dimer that should be formed (33%) upon random association of
monomers, considering that pOU61 is present at
2
copies/cell(4) .
The dominant negative effect of the HA2
protein can also be examined in vivo by determining how it
influences the growth rate of a CCA T
cell. It has already been noted that the growth of
CCA
cells is exquisitely sensitive to the level of
RNase T activity present(4) . As can be seen in Table 2,
introduction of the HA2 gene into a CCA
T
cell has little effect on growth, whether present on pBS(+)
or pOU61. However, introduction of HA2 into the CCA
T
cell leads to much faster growth, and the effect is
greater when HA2 is present on the multicopy plasmid. These data show
that in vivo the presence of the HA2 protein greatly
suppresses RNase T activity, in agreement with the in vitro measurements.
As shown in Table 1, the HA2-1 protein shows
much less of the dominant negative effect exhibited by HA2 when present
in pBS(+), decreasing RNase T activity only 30%. Likewise, HA2-1
has much less effect on the growth of CCA T
cells than HA2 (Table 2). These data
show that elimination of the dimerization capabilities of HA2 also
greatly relieves its dominant negative phenotype, supporting the
conclusion that the effects of HA2 are dependent on its ability to
dimerize with wild type RNase T monomers.
The data presented in this study relate the dimerization state of RNase T protein, RNase T activity, RNase T thermostability, and the Cys residue at position 168. RNase T normally is isolated as a dimer, and the information presented here indicates that it also exists as a dimer in vivo. Moreover, there is a strong correlation between the dimer form of RNase T and its catalytic activity. Treatments or alterations which were shown to lead to a loss of RNase T activity(4) , also result in dissociation of RNase T to the monomer form. Thus, DTNB modification or conversion of Cys-168 to hydrophilic residues, each of which causes loss of RNase T activity, also leads to monomerization of the protein. Most convincingly, lowering the temperature to which RNase T is exposed, stabilizes the dimer form to different degrees depending on the mutant, and results in a concomitant retention of catalytic activity. These findings strongly support the conclusion that it is the dimeric RNase T that is the catalytically competent form.
This conclusion is strengthened by the
data indicating that the dimer is present in vivo. Many RNase
T mutants when present on the multicopy phagemid
pBS(+) lead to faster growth when introduced into
CCA
T
cells, implying a reduction in
the endogenous RNase T activity by the excess of inactive enzyme (data
not shown). With one mutant, HA2, endogenous RNase T activity can be
completely suppressed, and the degree of suppression depends on the
amount of mutant RNase T made. These data are most consistent with the
formation of heterodimers between wild type monomers and the excess of
mutant monomers present. Moreover, when the ability of the mutant to
dimerize is lost, such as with the HA2-1 mutant, its dominant negative
effect also is greatly decreased. These data, which agree completely
with the in vitro gel filtration results, indicate that dimers
form in vivo as well.
What is the role of Cys-168 in the
dimerization process? Clearly, this residue is important because its
modification by DTNB or its alteration by mutagenesis can result in
monomerization. On the other hand, other hydrophobic residues, such as
Val or Ala, also retain more or less activity, and presumably can
maintain the dimeric state. Yet, based on the temperature sensitivity
of their RNase T activity(4) , these alternate residues at
position 168 lead to somewhat less stable dimers. Perhaps, a Cys
residue at this position can lead to transient disulfide formation with
the corresponding Cys on the other monomer which can help to stabilize
the dimer. Analysis of the RNase T sequence by the methods of Chou and
Fasman (7) and Garnier et al.(8) suggest that
Cys-168 is located near the end of a region (residues 151-168)
rich in hydrophobic moieties. Helical wheel analysis shows that Cys-168
is part of a hydrophobic helical face that conceivably could function
as a dimerization domain (Fig. 5). Additional mutations in this
region might be useful for addressing this question. Interestingly,
another mutation, Gly 206 Ser, which is present in HA2-1, also
affects dimer formation in the background of the other HA2 changes.
Whether residue 206 is close in the three-dimensional structure to the
hydrophobic region encompassing Cys-168 remains to be determined.
Figure 5: Helical wheel analysis of the region near Cys-168. Residues 151-168 of RNase T are shown in the helical wheel format in which each residue is offset from the preceding one by 100 °. Hydrophobic and nonpolar residues are boxed. The helical wheel file in the GCG software package was used for the analysis.
The studies presented here raise some interesting points regarding
the active site of RNase T. The dominant negative phenotype exhibited
by certain of the mutants suggest that both subunits must be active in
order to generate a functional RNase T and that RNase T monomers are
not active. One possibility to explain these observations is that both
subunits contribute to a single binding site for tRNA. In support of
this suggestion is the finding presented in our companion study (4) that tRNA stabilizes the C168S mutant against temperature
inactivation. Since the serine mutant monomerizes at 37 °C (Fig. 3), and the dimer is needed for RNase T activity, this
observation suggests that tRNA maintains the enzyme in the dimer form.
This would also explain the binding of tRNA to the C168S mutant at 37
°C determined by fluorescence quenching, shown in our companion
study(4) . Thus, in a situation in which the monomer and dimer
forms of RNase T are in equilibrium, binding of tRNA only to the dimer
form would drive the protein in that direction. This conclusion also
predicts that the monomer would bind tRNA weakly, or not at all. In
fact, the C168N mutant, which is largely monomeric even at low
temperatures (Fig. 3), binds relatively poorly to Affi-Gel blue. ()Affi-Gel blue is known to strongly bind tRNA binding
proteins(9) . The fact that the C168N mutant, which is a
monomer, binds to this matrix considerably more weakly than either the
wild type or C168S mutant protein, is consistent with poor nucleic acid
binding by the monomer.
On the other hand, because the HA2 mutant is inactive, but still dimerizes, a functional active site is not simply generated by dimerization. It is possible, but less likely, that each subunit contains its own tRNA binding site, and that correct interaction between the two subunits is necessary to render these sites active. Clearly, further structural work on this important RNA processing enzyme will be necessary to resolve these points.