The role of individual cysteine residues in the activity of Escherichia coli RNase T.

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 3Ј 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 3Ј processing of tRNA precursors, being most effective in removal of the residue immediately downstream of the -CCA sequence (4,5). 1 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 3Ј 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 ␣ 2 dimer of approximately 47 kDa. The sequence of the rnt gene encoding RNase T has also been elucidated (9). RNase T is a 3Ј35Ј exoribonuclease that initiates attack at a free 3Ј 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.
Purification of RNase T-Strain CAN20-12E PH Ϫ /pUC18 rnt was used for overexpression of wild type RNase T. The purification of RNase T from this strain followed the established procedure (8) with some modifications. The purified protein was concentrated to ϳ 1 mg/ml and stored at Ϫ20°C or Ϫ70°C.
Preparation of Antibody against RNase T-Two 12-month-old New Zealand White female rabbits were injected three times with purified RNase T (total of ϳ300 g of protein for each rabbit) and Freund adjuvants as described elsewhere (16). The antisera were tested against purified RNase T using dot-blots and the peroxidase system for detection. Cross-reacting antibodies were removed from the antiserum by treatment with dried CA244I Ϫ T Ϫ cells containing either a single copy or multicopy plasmid vector (10). Titration of the antiserum against RNase T indicated at least a 10 5 increase in RNase T-specific antibody over that of the preimmune serum.
Recombinant DNA Techniques-Isolation of double-stranded and single-stranded DNA, restriction enzyme digestion, ligation, transformation, and DNA sequencing were carried out using established procedures (10).
Protein Analysis-Protein was measured by a colorimetric method or by A 280 absorbance (17,18). Standard procedures for SDS-polyacrylamide gel electrophoresis and gel staining (19), and immunoblotting and signal detection (20) were followed.
Modification of RNase T with DTNB-Purified RNase T was dialyzed overnight at 4°C against 1000 volumes of 0.1 M NaPO 4 , pH 7.3, 1 mM EDTA, 0.1 mM phenylmethylsulfonyl fluoride, 10% glycerol with two changes. During dialysis N 2 was bubbled through the dialysis fluid. Modification of RNase T was performed in a volume of 400 l containing 0.1 M NaPO 4 , pH 7.3 (purged with N 2 ), RNase T (3.2 M), and DTNB (250 M). The DTNB was prepared as a 10 mM solution and diluted into the reaction mixture. The reaction was carried out in a cuvette and placed in the chamber of a spectrophotometer in which the temperature was kept at 26°C. A blank reaction was also done in which the final dialysis fluid substituted for RNase T. The difference at 412 nm between the sample and the blank was taken as the value due to sulfhydryl modification. Samples of 5 l were taken at different time points and immediately diluted at least 100-fold into an ice-cold solution containing 20 mM glycine-NaOH, pH 8.9, 0.1 mM EDTA, pH 8.0, 0.1 mM phenylmethylsulfonyl fluoride, and 0.15 mg/ml bovine serum albumin. RNase T activity of the diluted material was measured as described above, but in the absence of DTT.
Fluorescence Quenching-The intrinsic tryptophan fluorescence of purified RNase T was measured with a Spex Spectrometer model 1681. Measurements were carried out in 2 ml of solution containing glycine-NaOH (20 mM, pH 8.9) and DTT (5 mM). After equilibration to the desired temperature, the protein was added at 0.13 M and the emitted light at 345-370 nm over that of the buffer was determined. Excitation was at 290 nm. Quenching of fluorescence was measured by adding increasing concentrations of E. coli tRNA. Values were corrected for the fluorescence decrease that occurred during scanning and for the fluorescence of tRNA. Dissociation constants were determined from double reciprocal plots of the fluorescence decrease versus the tRNA concentration.
Site-directed Mutagenesis-This was carried out by the method of Kunkel (10). Single-stranded uracil-containing DNA from pBS(ϩ)-rnt was prepared by infection of strain CJ236 containing this phagemid with phage R408. This DNA was used as the template for synthesizing the mutant strand of DNA. After annealing of the appropriate 5Јphosphorylated mutagenic oligonucleotide, the strand was extended and circularized by Sequenase 2.0 and T 4 DNA ligase in the presence of ATP and dNTPs, and then transformed into strain UT481. Mutant clones were selected randomly and confirmed by DNA sequencing.

Purification of Wild Type
RNase T-As a first step for studying the importance of cysteine residues of RNase T, a procedure was developed for purifying large amounts of the protein. A cell overexpressing RNase T due to a multicopy plasmid carrying the rnt gene (9) was used as the starting material. The purification procedure followed that previously reported for the endogenous enzyme (8), but one chromatographic step was eliminated. The DTT concentration was increased to 5 mM during the purification and in the assay, and this led to significant stabilization of RNase T. Thus, the final specific activity of the preparation described here is 50% higher than that described previously, even though both preparations are homogeneous. The homogeneity of the current preparation was demonstrated both by SDS-polyacrylamide gel electrophoresis (Fig. 1, lane 1) and by amino acid composition, which was in close agreement with that predicted from the DNA sequence (data not shown) (9). A summary of the purification procedure is presented in Table I. As observed earlier (8), removal of inhibitors in the S100 fraction leads to a large increase in activity after the first column step. Approximately 25 mg of homogeneous RNase T was obtained from 150 g of wet E. coli cells. The purified protein was used to generate an RNase T-specific antibody as described in "Experimental Procedures." Modification of RNase T by DTNB-The role of cysteine residues in RNase T activity was assessed by treatment of the purified enzyme with the sulfhydryl modifying agent DTNB. This reagent reacts with free sulfhydryls to form a disulfide with the concomitant stoichiometric release of a colored product that can be measured at 412 nm (e ϭ 14,150) (21). At each time point of the measurement, the degree of modification by DTNB and the level of RNase T activity were determined. Fig. 2 shows 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.
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 (Ͻ1 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.
Site-directed Mutagenesis of RNase T-Conversion of each of the 4 cysteine residues to other amino acids was performed with the phagemid pBS(ϩ)-rnt as described in "Experimental Procedures." The changes made are shown schematically in Fig. 3A. The nucleotide replacement(s) for each mutant were confirmed by DNA sequencing. In addition, to ensure that no changes occurred in nucleotides in other parts of the rnt gene, the mutant clones were checked by DNA sequencing of the whole gene and/or by recloning of the mutated fragments into the wild type gene. Cloning of the mutant rnt genes into the single copy plasmid pOU61 was confirmed by DNA restriction fragment analysis.
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 (ϳ10%) (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).
Cys-112 and Cys-168 Substitutions Affect RNase T Activity-We initially examined the effect on RNase T activity of a serine or alanine substitution at each cysteine residue. A serine substitution would be expected to retain some of the nucleophilicity of the -SH group, whereas this would be completely lost with an alanine substitution. The results are summarized in Table II. In vitro measurements of RNase T activity were carried out in most cases with sonicated cell extracts from strain CA244I Ϫ T Ϫ containing either the wild type or mutant rnt gene on the single copy plasmid pOU61. However, for more accurate determination of the relative activities of C168S, C168N and C168D, extracts from cells containing multicopy plasmids were used. This assay is specific for RNase T. With vector alone, less than 1% of wild type activity is observed. As compared to the wild type gene, the serine mutant at position 168 (C168S) displayed a Ͼ95% decrease in RNase T activity; however, the alanine substitution was lowered only ϳ65%. This observation suggests that although Cys-168 is important for RNase T activity, it is most likely not the nucleophilicity of the -SH group that is the major factor. Substitutions of alanine and serine at position 112 also affect RNase T activity, although in the latter case much of the reduction in activity is due to a reduced amount of RNase T protein. Substitution by serine and alanine at positions 11 and 95 had little or no effect on RNase T activity (Table II).
RNase T activity also can be estimated in vivo. We have  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 412 using a molar extinction coefficient of 14,150. The amount of RNase T protein was determined colorimetrically and by A 280 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.
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 3Ј-defective tRNAs that cannot be repaired (3,6). Therefore, transformation of the altered rnt genes into a CCA Ϫ 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 Ϫ T Ϫ , and their relative growth rates were determined based on the time needed to form visible colonies.
As shown in Table II, introduction of the vector into a RNase T Ϫ cell leads to good growth with colonies appearing within 24 h (designated by ϩϩϩ), whereas transformation with the wild type rnt gene leads to few transformants that appear only after 80 h of incubation (designated by Ϯ). In fact, growth of this latter clone was actually somewhat slower than that due to a chromosomal copy of the rnt ϩ gene because RNase T activity from the plasmid was about twice that obtained from the chromosomal rnt ϩ gene (data not shown). In all cases, growth of the mutant rnt clones correlated well with their RNase T activity measured in vitro (Table II). 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 II) indicating that the level of RNase T exceeded what a CCA Ϫ 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 10fold 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 II), with visible colonies appearing after 18 rather than 24 h. We attribute this difference to the fact that RNase T Ϫ 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 Ϫ cells.
Kinetic Parameters of the Cysteine Substitution Mutants-To further explore the effects that the cysteine substitutions have on RNase T catalytic properties, the K m and V max values were determined for each of the mutants (Table II). The V max values were corrected for the amount of RNase T present as determined by immunoblotting. As shown in Table II, the wild type enzyme has a K m value of 3.9 M and a V max of 3.6 unit/mg. Alteration of the cysteine residue at position 168 led to increasing K m values as the residue was changed to valine, alanine and serine. On the other hand, the changes in V max were less dramatic. Alterations at position 112 led to relatively small changes in K m or V max , and there was essentially no change in these parameters with the changes at the other cysteine positions.
To ensure that the K m 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. 1 and Table III). 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 max values for this enzyme are not meaningful. However, the apparent K m values for the homogeneous wild type and mutant enzymes were 3.0 and 41 M, respectively, in close agreement with those determined in extracts (Table II).
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 IV. 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 m of the C168S protein is not due to altered substrate binding.
Hydrophobicity at Position 168 Influences RNase T Activity-The finding that the C168A derivative of RNase T is close to 10-fold more active than the C168S mutant demonstrated that it is not the nucleophilic properties of the cysteinyl -SH group that are of importance. Rather, it appeared that the hydrophobic properties of the -CH 2 SH moiety might be the determining factor. This conclusion was supported by the observation that a valine mutant is even more active than the alanine mutant, and as active as the wild type enzyme (Table II). Moreover, the more polar amino acids, asparagine and aspartic acid, show very little RNase T activity. In fact, there is a very strong correlation between RNase T activity and the hydrophobicity values of the different amino acids placed at position 168. The activity of the various RNase T molecules is in the order Val (1.640) Ϸ Cys (0.987) Ͼ Ala (0.702) Ͼ Ͼ Ser (Ϫ0.453) Ͼ Ͼ Asn (Ϫ1.003) Ϸ Asp (Ϫ1.935) (based on a combination of the in vitro and in vivo assays) and follows the hydrophobicity values (22) shown in parentheses very closely. These data strongly suggest that the role of cysteine at position 168 is to contribute a hydrophobic side chain.
Mutations at Position 168 Render RNase T Temperaturesensitive-We noted earlier that RNase T with a C168S change loses considerable activity during purification. This suggested that alterations at position 168 may lead to inactivation of RNase T under mild conditions. To test this, cell extracts from the wild type, and the C168V, C168A, and C168S clones were preincubated at 37°C, and at different time points samples were assayed for RNase T activity. As shown in Fig. 4A, wild type RNase T loses 25% of its activity after 5 min of incubation, indicating some degree of thermosensitivity. However, the mutants are inactivated considerably more rapidly. The serine mutant is extremely sensitive to incubation at 37°C, with a half time of inactivation of Ͻ1 min. The thermostability of the various derivatives at position 168 are in the order of Cys Ͼ Val Ͼ Ala Ͼ Ser, in close agreement with the order for activity. The inactivated proteins are not degraded during preincubation, as measured by immunoblotting (data not shown).
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 ϳ 65% at 16°C to ϳ 5% at 44°C. These data show that replacement of the cysteine residue at position 168 dramati- b Activity was assayed under standard conditions using sonicated extracts of CA244I Ϫ T Ϫ cells containing rnt on a single copy plasmid except for C168S, C168N, and C168D in which the multicopy plasmid was used. Results are expressed as a percentage of wild type activity with each plasmid. Values shown are the average of at least two independent experiments, each carried out in duplicate. The very low activity found with the vector alone (1%) has been subtracted from each value.
c Measured with sonicated extracts of CA244I Ϫ T Ϫ cells containing rnt on a multicopy plasmid. Vmax values were normalized to correct for small differences in the amount of RNase T protein determined by immunoblotting.

TABLE III
Purification of RNase T from E. coli strain CA244I Ϫ T Ϫ /pBS(ϩ)C168S Two and one-half g of wet cells were used for the purification.
Step FIG. 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.
cally affects the thermostablity of the mutant enzyme during the assay, and consequently, the activity of RNase T. DISCUSSION 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 ␣ 2 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 m 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. 3