The structural basis for the resistance of Escherichia coli formylmethionyl transfer ribonucleic acid to cleavage by Escherichia coli peptidyl transfer ribonucleic acid hydrolase.

Escherichia coli formylmethionly-tRNA-tMet is unique among N-acylaminoacyl-tRNAs in its resistance to cleavage by peptidyl-tRNA hydrolase. Chemical modification of tRNA-fMet with sodium bisulfite converts fMet-tRNA-fMet into a good substrate for the hydrolase. The products of the enzymatic cleavage are free tRNA-fMet and formylmethionine. Bisulfite treatment produces cytidine to uridine base changes at several sites in the tRNA structure. One of these modifications results in formation of a new hydrogen-bonded base pair at the end of the acceptor stem of tRNA-fMet. We have shown that this modification is responsible for the observed change in biological activity. Enzymatic cleavage appears to be facilitated by the presence of a 5-terminal phosphate at the end of a fully base-paired acceptor stem, because removal of the 5-phosphate group from N-acetylphenylalanyl-tRNA-Phe or bisulfite-modified fMet-tRNA-FMet reduced the rate of hydrolysis of these substrates. The unpaired base at the 5 terminus of unmodified fMet-tRNA-fMet appears to reduce susceptibility of the tRNA to hydrolytic attack both by positioning the 5-phosphate in an unfavorable orientation and by directly interfering with enzymatic binding. The unusual structure of the acceptor stem of this E. coli tRNA thus plays a critical role in maintaining the viability of the organism by preventing enzymatic cleavage of the fMet group from the bacterial initiator tRNA.

From the Department of Developmental Biology and Cancer, Division of Biology, Albert Einstein College of Medicine, Bronx, New York 10461 SUMMARY Escherichia coli formylmethionyl-tRNAfMet is unique among N-acylaminoacyl-tRNAs in its resistance to cleavage by peptidyl-tRNA hydrolase.
Chemical modification of tRNAfMet with sodium bisulfite converts fMet-tRNAfMet into a good substrate for the hydrolase.
The products of the enzymatic cleavage are free tRNAfMet and formylmethionine.
Bisulfite treatment produces cytidine to uridine base changes at several sites in the tRNA structure.
One of these modifications results in formation of a new hydrogen-bonded base pair at the end of the acceptor stem of tRNAfMet. We have shown that this modification is responsible for the observed change in biological activity.
Enzymatic cleavage appears to be facilitated by the presence of a S/-terminal phosphate at the end of a fully basepaired acceptor stem, because removal of the 5'-phosphate group from N-acetylphenylalanyl-tRNAPhe or bisulfite-modified fMet-tRNAfMet reduced the rate of hydrolysis of these substrates.
The unpaired base at the 5' terminus of unmodified fMet-tRNAfMet appears to reduce susceptibility of the tRNA to hydrolytic attack both by positioning the 5'phosphate in an unfavorable orientation and by directly interfering with enzymatic binding.
The unusual structure of the acceptor stem of this E. co2i tRNA thus plays a critical role in maintaining the viability of the organism by preventing enzymatic cleavage of the fMet group from the bacterial initiator tRNA. Peptidyl-tRNA hydrolase catalyzes the hydrolysis of N-acylaminoacyl-tRNAs and peptidyl-tRNAs to free tRNA and N-acyl amino acid or peptide (l-7).
Unsubstituted aminoacyl-tRNAs * This work was supported by research grants from the National Institutes of Health, United States Public Health Service (GM-16995), and the American Cancer Society .
This paper is the seventh in a series. For the preceding paper see Ref. 24. $ Research Career Development Awardee (Grant GM-25161) from the National Institute of General Medical Sciences.
are resistant to attack. The enzyme has been found in a wide variety of species, including Escherichia coli, yeast, wheat germ, and mammalian systems (l-lo). E. coli peptidyl-tRNA hydrolase has been highly purified (11) and has been shown to be an essential enzyme for normal protein metabolism (12,13). Several groups have reported that N-substituted derivatives of the E. coli initiator tRNA are unique in their resistance to cleavage by peptidyl-tRNA hydrolase (2,3,5,14,15). In this report, we show that the presence of an unpaired base at the 5' terminus of tRNAfMet is the structural feature responsible for the resistance of fMet-tRNA fMet to hydrolysis by the enzyme. Unlabeled amino acids were detected by ninhydrin. N-blocked amino acids, N-hydroxysuccinimide, and N-hydroxysuccinimide acetic acid ester were detected as described by Lapidot et al. (16). When radioactive compounds were chromatographed, the paper was cut into 25 strins (1.5 cm) and was counted in a liquid scintillation counter using' 10 ml of a toluene 2,5-diphenyloxazole (PPO)-1,4-bis [2-(5.phenyloxazolyl The product (44 ARM)) was recovered by ethanol precipitation, was washed twice with l-ml portions of ethanol, and was dissolved and stored in 5 mM potassium acetate, pH 4.6, at -20".
An aliquot was treated with 0.5 M NHaOH for 1 hour at 37" and was analyzed by paper chromatography.
All of the radioactivity migrated as N-acetyl[14C]phenylalanine and none could be detected in the position of the free amino acid.
Terminal 5'.phosphate groups were removed from tRNA samples by treatment with E. coli alkaline phosphatase at 65" as described previously (24). The dephosphorylated tRNAs contained no chain breaks as determined by chromatography on Senhadex G-100 at 55" in 10 mM sodium nhosnhate. DH 7.5. and 0.i M NaCl (25,26) and accepted the same amount of'amino acid as the corresponding untreated tRNAs. N-Substituted derivatives of the dephosphorylated aminoacyl-tRNAs were prepared as described above for the untreated tRNAs. Purification of Peptidyl-tRNA Hydrolase-Peptidyl-tRNA hydrolase was purified by a modification of the method of Kijssel (11).
A high speed supernatant (S-150) was prepared from 28 g of E. coli MRE 600 cells as described elsewhere (24) and was added directly to a column (2.4 X 60 cm) of DEAE-cellulose which had been equilibrated with standard buffer containing 10 rnM Tris-HCI, pH 7.3, 10 mM MgC12, 10 mM 2-mercaptoethanol, and 0.1 mM EDTA at 4'. The column was eluted with standard buffer and fractions (7 ml) were collected at a flow rate of 0.5 ml per min. The absorbance at 280 nm was measured and the fractions of the first broad peak were combined (91 ml). This material is referred to as "partially purified enzyme." The hydrolase was further purified by application of t,he DEAE-fraction to a CM-cellulose column (1.3 X 36 cm; NHa+ form) and elution with standard buffer at a flow rate of 0.5 ml per min until the absorbance dropped to 0.1 Atso. Elution with standard buffer containing 0.3 M KC1 was started and l-ml fractions were collected. Peptidyl-tRNA hydrolase activity was assayed by incubating 50 ~1 of each fraction with 10 pmol of N-acetyl-[14C]Phe-tRNArhe and measuring the decrease in trichloroacetic acid-insoluble radioactivity in 20 min at 37". The bulk of the hydrolytic activity was found in Fractions 30 to 32 which were pooled and dialyzed twice against 2 liters of standard buffer for 1 hour each. The sample was added to a 5-ml column of CM-cellulose and the column was washed with 15 ml of standard buffer. Elution with standard buffer containing 0.3 M KC1 was started and 0.5.ml fractions were collected and assayed as above.
The hydrolytic activity was concentrated in Fractions 12 and 13, which were dialyzed against 2 liters of standard buffer for 2 hours, divided into small aliquots, frozen, and stored at -20".
Protein was assayed by the method of Bucher (27)  fMet-tRNAfMet and deacylated tRNAmet. Samples were mixed with 100 A?* of crude carrier tRNA in 1 ml of starting buffer and were applied to a column (1 X 24 cm) of benzoylated DEAE-cellulose equilibrated at 4". Elution was carried out with solutions containing 10 mM MgC12 and 10 mM sodium acetate, pH 4.5, using a linear gradient from 0.4 to 2 M NaCl over 600 ml. Fractions (4 ml) were collected at a flow rate of 15 ml per hour. Aliquots (0.5 ml) were diluted with 0.5 ml of water and were counted in 10 ml of Aquasol (New England Nuclear Corp.).
Fractions were pooled, desalted by dialysis, and concentrated by ethanol precipitation. 6'-l'erminal Nucleotide Analyses-32P-Labeled tRNAs were hydrolyzed to nucleotides by incubation with 0.3 N KOH at 37" for 18 hours.
The released V-nucleoside diphosphates were analyzed as described previously (24,30). Products of Cleavage of BisulJite-modi$ed fMet-tRNAfMe t by Peptidyl-tRNA Hydrolase-In order to determine whether the cleavage of bisulfite-modified fMet-tRNAfMet occurred in a manner analogous to that reported for hydrolysis of other N-substituted aminoacyl-tRNAs, the products resulting from treatment of the modified tRNA with peptidyl-tRNA hydrolase were analyzed.

BisulJile-modi$ed
The data in Fig. 3 show that one product of the reaction is free tRNAfMet.
Bisulfite-modified fMet-tRNAfMet which had been enzymatically hydrolyzed to the extent of approximately 50% could be recharged with methionine to the same level as a control sample incubated in the absence of hydrolase (Fig. 3) At the end of the experiment, paper discs containing the acid-precipitated tRNA were cut into small pieces and were incubated in 0.5 N NH40H at 37" for 1 hour.
The released radioactivity was then analyzed by paper chromatography.
tRNA was found at the origin and the remaining radioactivity migrated with the same mobility as an authentic fMet marker. A control sample incubated with an equivalent amount of enzyme buffer showed very little nonenzymatic hydrolysis. These results exclude the possibility of hydrolysis of bisulfitemodified fMet-tRNAfMet by a nucleolytic cleavage of the modified 3'-terminal C-C-A-OH sequence and show that peptidyl-tRNA hydrolase attacks the modified initiator tRNA in the same manner that it attacks other N-substituted aminoacyl-tRNAs. Fully Base-Paired Acceptor Stem is Required for Eficient Cleavage of Bisuljite-modi$ed fMet-tRNA*Met by Peptidyl-tRNA Hydrolase-One or more of the cytidine to uridine base changes in the structure of bisulfite-modified fMet-tRNAfMet alters the molecule in such a way that it becomes a good substrate for cleavage by peptidyl-tRNA hydrolase.
In order to determine whether the base change at the 5' terminus is responsible for the change in biological activity of the molecule, enzymatic hydrolysis was carried out for various times and the 5'-terminal nucleotides in the remaining fMet-tRNAfMet were analyzed (Table I). The deacylated and formylated tRNAs were separated by gradient elution from benzoylated DEAE-cellulose at acid pH (29). Fig. 5 Table I were carried out as follows: tRNAfMet was treated with sodium bisulfite under conditions leading to partial modification of each of the sites indicated on Fig. 1  pooled using W radioactivity.
Prior to treatment with hydrolase the fMet-tRNAfMct fraction contained 5'.terminal uridine and cytidine in a ratio of about 0.5: 1 (Table I).
Enzymatic cleavage was found to result in a rapid decrease in the amount of 5'.terminal uridine in t,he remaining fMet-tRNAfMct, indicating preferential attack on molecules containing the fully base-paired acceptor stem. After 17% of the total 321'-labeled fMet-tRNAfMet had been hydrolyzed, approximately 4370 of the tRNA containing 5'.t,erminal uridine had been cleaved, whereas only about 3 to 47, of the molecules having a 5'-terminal cytidine had been att,acked.
Aft,er 40% of the total 32P-labeled fMet-tRNAfMet had been hydrolyzed, essentially all of the tRNA containing 5'.terminal uridine had been cleaved, whereas less than 2O$& of the tRNA having a 5'.terminal cytidine had been attacked.
In order to determine whether the modification at Cr was solely responsible for the observed change in biological activity, we compared the initial rates of cleavage of fMet-tRNAfMet molecules modified with bisulfite for various times. Fig. 6 shows that a linear first order plot is obtained for the rate of enzymatic cleavage as a function of t,he ext,ent of cytidine to uridine conversion at Cr. This result rules out the possibility of a two-hit mechanism in which modifications at both Cr and at another site are required to convert fMet-tRNA fMet into an active substrat.e for the hydrolase.
Similar plots of rate versus mole fraction of cytidine at Cr6, Cn, or CT5 gave curves which deviated significantly from linearity (not shown). Although we cannot exclude the possibility of a small contribution (either positive or negative) to the rate of cleavage resulting from these other modifications, the major change in the susceptibility of E. coli fMet-tRNAfMct to attack by peptidyl-tRNA hydrolase following bisulfite treatment is accounted for by the cytidine to uridine base change at the 5' terminus.
Role of  Cleavage by Peptidyl-tRNA Hydrolase-In order to characterize further t'he structural requirements for cleavage of N-substituted aminoacyl-tRNAs by peptidyl-tRNA hydrolase, we have compared the rates of hydrolysis of substrat,es containing a 5'.phosphate with those containing a 5'-OH group (Fig. 7). Removal of the 5'-phosphate from NAcPhe-tRNAPh" was found to reduce drastically the rate of hydrolysis of this tRNA by the hydrolase (Fig. i'A).
Removal of the 5'-phosphate from partially bisulfite-modified fMet-tRNAfMet (70% 5'.uridine, 30% 5'cytidine) had a less dramatic effect but still caused substantial reduction in hydrolytic activity (Fig. 7B). Unmodified fMet-tRNAfMet having a 5'.OH group was cleaved at the same low rate as the tRNA with a 5'.terminal phosphate (Fig. 7C). In control experiments, the dephosphorylated N-substituted AA-tRNAs were found to be as active as the corresponding untreated tRNAs when assayed for initiation of polypeptide chains in an in vitro protein-synthesizing system,2 indicating that the removal of phosphate groups had not caused nonspecific loss of biological activity.

DISCUSSION
The exact role of peptidyl-tRNA hydrolase in viva is not known; however, it is believed to hydrolyze peptidyl-tRNA molecules carrying incomplete proteins which have been prematurely released from ribosomes (32). This processing of free oligopeptidyl-tRNAs regenerates free t,RNA and is required for normal protein metabolism (12, 13). E. coli fMet-tRNAfMet is the only N-substituted aminoacyl-tRNA which has been found to be resistant to attack by peptidyl-tRNX hydrolase (2,3,5,14,15). Inasmuch as formylation is required for initiation of protein synthesis in prokaryotes (33) resistance of tRNAfMet to this enzyme is essential for the survival of bacterial organisms.
Previous studies have shown that it is the tRNA moiety which is responsible for the resistance of N-substituted derivatives of the initiator tRNA to hydrolysis. fMet-, N-acetyl-Met-, and Gly-Gly-Met-derivatives of the noninitiator methionine tRNA, tRNAmMet, are readily cleaved under conditions where little hydrolysis of the corresponding tRNAfMet compounds is observed (11,14,15). The data presented here shows that the unpaired nucleotide at the 5' terminus of the E. coli initiator tRNA is the structural feature responsible for its unique resistance to enzymatic hydrolysis.
The bisulfite modification produces a Cl --f U1 base change and results in formation of a normal U1. Ay3 base pair at the end of the acceptor stem.
This change in local secondary structure also alters the spatial orientation of the 5'-terminal phosphate group. Previous studies from this laboratory have shown that the presence of the 5'.phosphate at the end of a fully base-paired helix is essential for the formation of stable complexes between the bacterial elongation factor Tu and aminoacyl-tRNAs (24). The data presented in this paper suggest an additional role for the 5'-phosphate of the tRNA moiety in the interaction of N-substituted aminoacyl-tRNAs with peptidyl-tRNA hydrolase.
The decrease in susceptibility to hydrolysis of NAcPhe-tRNAPhe and bisulfite-modified fMet-tRNAfMet on removal of the 5'-terminal phosphate group is in keeping with data from other laboratories showing that 3'-terminal N-substituted aminoacyl-oligonucleotides are poor substrates for the enzyme (1,2,5,7).
The difference in the effect of dephosphorylation on hydrolysis of NAcPhe-tRNAPhe and bisulfite-modified fMet-tRNAfMct may reflect the difference in affinity of the hydrolase for the N-blocking group on each tRNA.
The nature of the peptide portion has been found to influence the efficiency of hydrolysis of peptidyl-tRNAs, those containing two pept,ide bonds being cleaved more readily than those containing only one peptide bond (2,23). Formylated derivatives of aminoacyl-tRNAs also have been reported to be hydrolyzed faster than the corresponding N-acetyl compounds (3,23). Binding to the 5'-phosphate of the tRNA may play a relatively more important role in stabilizing the interaction of the enzyme with poorer substrates such as NAcPhe-tRNAPh".
Removal of the 5'-phosphate from unmodified fMet-tRNAfMet was found to have no effect on the rate of hydrolysis of this tRNA.
Thus, the position of the 5'.phosphate cannot be solely responsible for the unique resistance of tRNAfMet to hydrolysis. Modified fMet-tRNAfMet having a 5'-OH is cleaved at least twice as fast as unmodified fMet-tRNAfMet (either 5'-OH or 5'-P), indicating that the unpaired base itself, even without the terminal phosphate, interferes with the interaction between peptidyl-tRNA hydrolase and the initiator tRNA. Our previous studies 011 the structural requirements for recognition of aminoacyl-tRNAs by elonga.tion factor Tu showed that a cytidine to uridine base change in the common 3'9erminal C-C-A-OH sequence reduced the affinity of AAtRNA for this protein (24). The bisulfite-modified fMet-tRNAfMet used in the present studies contained 70 to 80% uridine at position 75 in the C&&-A-OH sequence but was cleaved by purified peptidyl-tRNA hydrolase at a rate comparable to that reported for unmodified 4,,Met (15). Thus, the CT5 ---f U75 modification appears to have little or DO effect on the rate of hydrolysis of fMet-tRNAfMet when experiments are carried out in the presence of excess enzyme.
The structural features which appear to be important for efficient hydrolysis of tRNA derivatives by peptidyl-tRNA hydrolase are an amino acid in the L configuration (2)) the presence of at least 1 peptide bond (l-4), and a tRNA having a fully base-paired acceptor stem terminated by a 5'.phosphate group.