(Received for publication, August 17, 1995)
From the
Escherichia coli RNase T, which is responsible for the
3` processing and end-turnover of tRNA and the maturation of 5 S RNA,
is extremely sensitive to sulfhydryl reagents and to oxidation,
suggesting a role for cysteine residues in its activity. Titration of
homogeneous RNase T with 5,5`-dithiobis-(2-nitrobenzoic acid) revealed
that the 4 cysteine residues present in each of the two protein
subunits are in a reduced form and that 1 or 2 of them are important
for activity. To identify these residue(s), each of the cysteines in
RNase T was changed individually to either serine or alanine. The
serine mutant at position 168 is greatly reduced in RNase T activity
both in vivo and in vitro; likewise, the serine
mutant at position 112 and the alanine mutants at positions 112 and 168
also display decreased RNase T activity. Mutations at the other
cysteine positions show little or no change. Kinetic analyses of the
mutant enzymes showed that the K values
of C168S and C168A are increased considerably, whereas their V
values are reduced only slightly compared to
the wild type enzyme. The other mutant enzymes are little changed.
Additional amino acid replacements at position 168 showed that the in vivo and in vitro activities of RNase T are in the
order Cys
Val > Ala
Ser
Asn
Asp, which closely
follows the relative hydrophobicity of these amino acid residues.
However, the affinity for tRNA, determined by fluorescence quenching,
is not altered in C168S, suggesting that Cys-168 is not directly
involved in substrate binding. Interestingly, proteins altered at
position 168 showed increased temperature sensitivity as the residue at
that position became less hydrophobic. These data indicate that Cys-168
contributes a hydrophobic group that influences the structure and
ultimately the catalytic activity of RNase T.
RNase T is one of the eight distinct exoribonucleases known to
be present in Escherichia coli(1) . The enzyme was
originally identified based on its ability to remove the 3` terminal A
and penultimate C residues from tRNA(2) . The subsequent
isolation of a mutant strain deficient in RNase T (3) demonstrated that this enzyme, together with tRNA
nucleotidyltransferase which repairs the -CCA sequence, is responsible
for the process of end-turnover of tRNA that is known to occur in
vivo in both prokaryotic and eukaryotic cells. Subsequent studies
showed that RNase T also participates in the 3` processing of tRNA
precursors, being most effective in removal of the residue immediately
downstream of the -CCA sequence(4, 5) . ()Interestingly, although the role of RNase T in maturation
of tRNA can be circumvented by the presence of other
exoribonucleases(4, 5) , cells devoid of RNase T still
grow 5-10 min slower than wild type(6) , suggesting an
additional role for this RNase in RNA metabolism. Very recently, we
have shown that RNase T also is required for the maturation of 5 S RNA;
no mature 5 S RNA is made in the absence of this enzyme. Rather, a
molecule with 2 extra residues at the 3` terminus accumulates, and this
incompletely processed 5 S RNA is incorporated into
ribosomes(7) . Based on these observations, it is clear that
RNase T plays an important role in RNA metabolic processes.
RNase T
has been purified to homogeneity(8) . The enzyme is an
dimer of approximately 47 kDa. The sequence of the rnt gene encoding RNase T has also been elucidated (9) . RNase T is a 3`
5` exoribonuclease that initiates
attack at a free 3` terminus of tRNA; aminoacyl-tRNA is not a
substrate. RNase T is extremely sensitive to sulfhydryl reagents and to
air oxidation(8) , suggesting that one or more of the 4
cysteine residues present in each subunit may be important for the
enzyme's activity. Inasmuch as -SH groups have not
previously been implicated in the action of RNases, these observations
are of considerable interest.
In this study we use chemical modification, site-directed mutagenesis, and activity measurements in vitro and in vivo to investigate the role of the individual cysteine residues in RNase T. Our data indicate that Cys-168 plays an important role in RNase T activity by contributing a hydrophobic residue that is important for the structure of this enzyme.
Figure 1: SDS-PAGE of RNase T. Electrophoresis was performed using a 12% gel and proteins were visualized after silver staining as described under ``Experimental Procedures.'' Lane 1, 1 µg of Ultrogel AcA44 peak fraction of wild type RNase T. Lanes 2-5, samples from different steps of C168S purification in which 0.5 µg of Ultrogel AcA44 column sample (lane 2), 1.1 µg of hydroxylapatite column sample (lane 3), 3.4 µg of Affi-Gel blue column sample (lane 4), or 13.2 µg of S100 (lane 5) were loaded. The positions of the protein size standards are shown on the left.
Fig. 2shows the relationship between the degree of sulfhydryl modification and the activity of RNase T. RNase T activity decreased rapidly in the first few minutes after mixing with DTNB, in concert with the increase in the degree of -SH modification. Modification by DTNB reached a maximum in 5-10 min (varies with DTNB concentration), at which point RNase T was totally inactivated, indicating that the presence of free -SH groups are essential for RNase T activity. The maximum modification corresponds to close to 4 -SH groups per subunit of RNase T. Thus, the 4 cysteine residues in each monomer are in a reduced form, and all of them are accessible to DTNB in the native protein. The completeness of the modification was confirmed by the reaction of DTNB with protein denatured with either 7.8 M urea, 6 M guanidinium chloride or 0.5% SDS. In each case the same maximum degree of modification observed with the native protein was seen within 30 s after mixing the denatured proteins with DTNB (data not shown). The slower modification of the native protein suggests that the -SH groups may be partially buried, but still accessible enough to result in complete reaction in a short time. It was not possible to determine from these studies whether some -SH residues might be more reactive. Rather, the -SH residues appear to be close to randomly modified, as the reaction is pseudo first-order and monophasic. Assuming random modification and using the binomial distribution, it is estimated that one or two cysteine residues are important for RNase T activity.
Figure 2:
Effect of sulfhydryl modification and
regeneration on RNase T activity. Dialyzed RNase T was mixed with DTNB
at zero time as described under ``Experimental Procedures.''
The number of modified -SH groups at the indicated times were
determined by the increase in A using a molar
extinction coefficient of 14,150. The amount of RNase T protein was
determined colorimetrically and by A
and agreed
within 10%. DTT was added at 20 mM at the time indicated by
the arrow. RNase T activity is expressed as a percentage of
the activity at zero time.
The importance of the cysteine residues was further demonstrated by the complete restoration of RNase T activity upon regeneration of the sulfhydryls with excess DTT (Fig. 2). The half-time of reactivation was about 14 min at 26 °C, much longer than that needed for the half-time of inactivation (<1 min), even with a much higher (80-fold) DTT concentration demonstrating the extreme sensitivity of RNase T to oxidation, as noted earlier(8) . The reactivation by DTT indicates that the loss of RNase T activity was caused solely by the formation of disulfide bonds, and not by any irreversible changes in the protein structure. However, by these methods it was not possible to distinguish whether reduction of a particular modified -SH had more of an effect than others. Thus, other studies, such as site-directed mutagenesis, were necessary to determine which cysteine residues are most important for RNase T activity.
Figure 3:
Site-directed mutagenesis and expression
of mutant RNase T protein. A, schematic representation of the
mutation sites and amino acid substitutions in RNase T. B,
Western blotting of wild type and mutant RNase T. Five µg of
sonicated cell extracts from CA244IT
containing the indicated rnt genes on pBS(+) were
separated on 12% SDS-PAGE and transferred and treated with RNase T
antibody as described under ``Experimental Procedures.'' In lane 1, 30 ng of purified wild type RNase T were loaded as a
marker. Lane 2 was loaded with 5 µg of extract from
CA244I
T
containing only the vector
pBS(+). The positions of the protein size standards are shown on
the left.
In most cases, the replacement of a cysteine residue by
another amino acid had little effect on the amount of RNase T protein
present in the mutant clone as compared to the wild type clone. This
was determined by immunoblotting of extracts from cells containing the
single-copy plasmid (not shown) or the multicopy plasmid (Fig. 3B). However, considerably less RNase T protein
was repeatedly observed with the C168D mutant (10%) (lane
14), and a small decrease was observed with the C112S mutant with
the single copy plasmid (data not shown). No RNase T protein is present
in the host cell used (lane 2).
RNase T activity also can be
estimated in vivo. We have previously shown that strains
deficient in tRNA nucleotidyltransferase are very sensitive to the
level of RNase T activity because RNase T leads to the accumulation of
3`-defective tRNAs that cannot be repaired (3, 6) .
Therefore, transformation of the altered rnt genes into a
CCA strain should affect cell growth such that clones
with higher RNase T activity would grow more slowly. Accordingly,
single copy plasmids containing each of the substituted rnt genes were transformed individually into strain
CA244CCA
T
, and their relative
growth rates were determined based on the time needed to form visible
colonies.
As shown in Table 2, introduction of the vector into
a RNase T cell leads to good growth with colonies
appearing within 24 h (designated by +++), whereas
transformation with the wild type rnt gene leads to few
transformants that appear only after 80 h of incubation (designated by
±). In fact, growth of this latter clone was actually somewhat
slower than that due to a chromosomal copy of the rnt
gene because RNase T activity from the
plasmid was about twice that obtained from the chromosomal rnt
gene (data not shown). In all cases,
growth of the mutant rnt clones correlated well with their
RNase T activity measured in vitro (Table 2). Cells with
high RNase T activity grew poorly, and as the level of RNase T
decreased, growth improved. When the mutant rnt genes were
expressed instead from a multicopy plasmid, almost all of them failed
to grow (Table 2) indicating that the level of RNase T exceeded
what a CCA
cell could tolerate. Only clones
expressing C168N or C168D were able to grow under these conditions,
indicating that these cysteine substitutions almost obliterated RNase T
activity.
The close agreement between the in vitro and in vivo RNase T activities strongly supports the conclusion that the cysteines at positions 112 and 168 are important for RNase T activity, whereas those at the other two positions are not. Thus, modification of residues 112 and 168 by DTNB may account for the loss of RNase T activity observed upon treatment with this reagent. Interestingly, however, the substitutions at positions 112 and 168 behave very differently. At position 112, the serine substitution displays more than twice the activity of the alanine substitution (considering the lowered RNase T protein for C112S), whereas at position 168 alanine is approximately 10-fold more active than serine.
It should also be noted that the clone with the C168S substitution
actually grows even more rapidly than the cell with the vector alone (Table 2), with visible colonies appearing after 18 rather than
24 h. We attribute this difference to the fact that RNase T cells grow more slowly than wild type(6) , and that a
small amount of RNase T may help to overcome that growth defect, yet
still be low enough not to have a significant negative effect on the
growth of CCA
cells.
To
ensure that the K increase observed in the C168S
clone was really due to the mutant enzyme, and not to any problems due
to using extracts, the mutant RNase T was also purified to homogeneity
( Fig. 1and Table 3). The specific activity and overall
purification were apparently much lower for the mutant than for the
wild type enzyme, but this was due to considerable inactivation of the
mutant protein during the purification procedure. As a consequence, V
values for this enzyme are not meaningful.
However, the apparent K
values for the homogeneous
wild type and mutant enzymes were 3.0 and 41 µM,
respectively, in close agreement with those determined in extracts (Table 2).
To examine whether the C168S mutant is affected in
tRNA substrate binding, we have directly measured the binding of
substrate to each of the purified enzymes by determining the
fluorescence quenching of increasing concentrations of E. coli tRNA-C-C-A. These data, determined at two temperatures, are
presented in Table 4. There is very little difference in tRNA
affinity between the C168S mutant and wild type RNase T suggesting that
Cys-168 is not required for binding of substrate. Thus, the lowered
activity of the C168S mutant is not a consequence of a change in
binding affinity. Likewise, the increased K of the
C168S protein is not due to altered substrate binding.
Figure 4: Temperature sensitivity of C168 mutants. Sonicated cell extracts (A) or purified RNase T (B) were preincubated at 37 °C in RNase T assay buffer. Samples were withdrawn at different times and assayed at 16 °C to determine residual RNase T activity which is expressed as a percentage of the activity at zero time. The protein concentration in the preincubation mixture was 0.11 mg/ml in A and 0.035 mg/ml in B. E. coli tRNA was added at 2 mg/ml in the reactions where indicated.
Temperature sensitivity of the mutant was also observed when the purified wild type and C168S RNase T proteins were incubated at 37 °C. C168S RNase T is much more thermosensitive than the wild type enzyme (Fig. 4B). However, the thermosensitivity of the mutant protein can be significantly alleviated by the presence of tRNA (Fig. 4B). This is consistent with the observation that the C168S derivative is somewhat more stable in a cell extract than in purified form.
The temperature sensitivity of the various RNase T
derivatives obviously contributes to their lowered activity when
assayed at 37 °C. The influence of temperature was clearly shown
when the activity of the C168S enzyme was compared to that of the wild
type RNase T at different temperatures (Fig. 5). The relative
activity of the mutant RNase T decreases progressively from 65%
at 16 °C to
5% at 44 °C. These data show that replacement
of the cysteine residue at position 168 dramatically affects the
thermostablity of the mutant enzyme during the assay, and consequently,
the activity of RNase T.
Figure 5: Relative activity of wild type and C168S RNase T at different temperatures. The two purified RNase T preparations (15 ng of each) were assayed at the indicated temperatures under standard conditions for 5 min. The specific activity of wild type RNase T was set at 100% at each temperature.
Although cysteine residues are known to participate in
protein-RNA interactions through Michael adduct formation (23, 24, 25) or through metal
binding(26, 27) , these residues have not previously
been implicated in RNase activity. Here we show that 2 of the 4
cysteine residues of RNase T play an important role in the activity of
this enzyme. Cys-168, in particular, has a dramatic influence on RNase
T stability and hence, on RNase T activity, and functions by virtue of
the hydrophobic properties of the cysteine side chain. We show in a
companion study (28) that this residue is important for
dimer formation by RNase T. The second cysteine
residue, Cys-112, has less of an effect on RNase T activity, but in
this case, the nucleophilic properties of the -SH group appear to
be paramount. However, it is unlikely that this residue participates
directly in catalysis because even with an alanine substitution, at
least 20% of RNase T activity remains.
It is an interesting question as to why position 168 evolved to be a cysteine residue when its hydrophobic properties seem most important. In fact, valine in this position leads to a slightly more active enzyme. On the other hand, the valine derivative is more thermosensitive. Clearly, other factors such as side chain size or hydrogen bonding must also contribute to the structural properties required at this position. It is also possible that transient intersubunit disulfide formation involving Cys-168 could contribute to stability. Structural analysis by x-ray crystallography or NMR spectroscopy will be needed to resolve this question. It is interesting that a cysteine residue in a DNA binding domain, that of the Myb protein, also appears to function by virtue of its hydrophobicity (29) .
A puzzling observation with regard to
the substitutions at position 168 is that as the residue at this
position becomes less hydrophobic, the K value for
tRNA increases. The fluorescence quenching experiments clearly showed
that this is not an effect on substrate binding, so that some other
factor must be involved. Inasmuch as these amino acid substitutions
render RNase T temperature sensitive, and the tRNA substrate serves to
protect the enzyme against inactivation, we suspect that we may simply
be observing increased stability of the mutant RNase Ts as the tRNA
substrate concentration is increased. What appears to be increasing
activity with increasing substrate concentration may only represent
more active enzyme present during the assay. In fact, increasing the
tRNA concentration was found to lead to increasing thermostability of
the C168S RNase T. (
)