©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
The Leucyl/Phenylalanyl-tRNA-Protein Transferase
OVEREXPRESSION AND CHARACTERIZATION OF SUBSTRATE RECOGNITION, DOMAIN STRUCTURE, AND SECONDARY STRUCTURE (*)

(Received for publication, May 2, 1995; and in revised form, June 13, 1995)

Georgi Abramochkin Thomas E. Shrader (§)

From the Departments of Biochemistry and Anatomy and Structural Biology, Albert Einstein College of Medicine, Bronx, New York 10461

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Previous work has shown that, in the bacterium Escherichia coli, the aat gene is essential for the degradation of proteins bearing amino-terminal Arg and Lys residues via the N-end rule pathway of protein degradation. We now show that the aat gene encodes directly the leucyl/phenylalanyl-tRNA-protein transferase (L/F-transferase). This enzyme catalyzes the transfer of Leu, Phe, and, less efficiently, Met and Trp, from aminoacyl-tRNAs, to the amino terminus of acceptor proteins. We have used the cloned aat gene to overexpress and purify an affinity tagged L/F-transferase. The recombinant L/F-transferase is as active as the previously purified wild type enzyme and contains no detectable RNA component. We have used the recombinant enzyme to demonstrate that both the solubility and substrate specificity, for aminoacyl-tRNA substrates, of the L/F-transferase are dependent on ionic strength conditions and that the modified nucleotides found in natural tRNAs are not essential for recognition by the enzyme. Limited digestion of the L/F-transferase with trypsin removes the proline rich NH(2) terminus of the enzyme identifying a globular core, and circular dichroism demonstrates that the L/F-transferase is predominantly alpha-helical. Finally, a region of sequence conservation between the L/F-transferase and the NH(2)-terminal protein acetylases has been identified.


INTRODUCTION

The leucyl/phenylalanyl-tRNA-protein transferase (L/F-transferase) (^1)catalyzes a peptidyltransferase reaction that is very similar to the reaction catalyzed on the large subunit of the ribosome. The L/F-transferase catalyzes the transfer of Leu, Phe, and, less efficiently, Met and Trp, from aminoacyl-tRNAs to the amino terminus of acceptor proteins (Kaji et al., 1965; Leibowitz and Soffer, 1971a). All known acceptor proteins or peptides contain the basic amino-terminal residues Arg or Lys and are predominantly unstructured at their NH(2) termini (Soffer, 1973). The L/F-transferase catalyzes a true peptidyltransferase reaction, making it unique among those enzymes that catalyze non-ribosomal peptide bond synthesis in Escherichia coli cells. Previously characterized enzymes activate the transferred amino acid as an aminoacyl-enzyme thioester (Lipmann, 1971) or an aminoacyl-phosphate (Wright and Walsh, 1992) prior to peptide bond formation. The L/F-transferase contains only 234 amino acid residues (Shrader et al., 1993), and the purified enzyme lacks any detectable RNA component (see below), making the L/F-transferase a tractable system for in-depth mechanistic dissection of aminoacyl-tRNA recognition and peptidyltransferase reactions.

Substrate modification by the L/F-transferase serves as a powerful degradation signal, identifying the enzyme as a component of the N-end rule pathway (Varshavsky, 1992). It has been shown previously that E. coli cells degrade a beta-galactosidase variant, engineered to expose amino-terminal Arg (Arg-beta-gal), only after modification to Leu-Arg-beta-galactosidase (Shrader et al., 1993; Tobias et al., 1991). The addition of an amino-terminal degradation signal is a simple yet unusually powerful signal for protein turnover. Mutant cells that lack L/F-transferase activity degrade unmodified Arg-beta-gal 100-1,000 times more slowly than wild type cells. The ATP-dependent Clp (Ti) protease (Hwang et al., 1988; Katayama et al., 1988) mediates degradation of these proteins (Tobias et al., 1991). Inclusion of L/F-transferase in the N-end rule pathway provided a functional assay for L/F-transferase activity that allowed the cloning of a gene, termed aat (aminoacyl transferase), essential for L/F-transferase-dependent degradation (Shrader et al., 1993). The aat gene was presumed to encode directly the L/F-transferase, but the lack of significant sequence similarity with any known enzyme left viable other possibilities. In this paper we demonstrate that the aat gene directly encodes the L/F-transferase.

The aat gene lies at the end of a three gene operon whose other members are homologous to the P-glycoproteins responsible for multidrug resistance in mammalian cells (Shrader et al., 1993). These multidrug resistance homologs have been identified as essential for E. coli cells to exit from the stationary phase (Siegele and Kolter, 1993) and are also required for the proper expression of cytochrome d, although they do not encode subunits of this enzyme (Poole et al., 1993). Cytochrome d is a terminal oxidase in the E. coli respiration chain. Correspondingly, these genes have been named surB/cydD and cydC. The chromosomal position of the aat gene remains an intriguing clue to the cellular function of the L/F-transferase.

Previous attempts to purify L/F-transferase from cells expressing natural abundance levels of the enzyme suggest that L/F-transferase is present at very low levels (Leibowitz and Soffer, 1970). To prepare quantities of the enzyme for subsequent characterization, we have constructed overexpression systems for two different affinity-tagged L/F-transferases. The overexpressed enzymes show moderate solubility in vivo and under most conditions in vitro. However, preparations of the soluble fraction of hexa-His-L/F-transferase yield milligram quantities of active enzyme. We present an initial characterization of this recombinant L/F-transferase and demonstrate that the introduced changes do not change the important qualities of the enzyme. In addition, we have demonstrated that unmodified tRNAs are substrates for the enzyme and determined the domain structure and approximate secondary structural content of the L/F-transferase.


EXPERIMENTAL PROCEDURES

Construction of His(6)-L/F-transferase and GST-L/F-transferase Fusion Proteins

L/F-transferase enzymes fused to, NH(2)-terminal, glutathione S-transferase (GST-aat) and hexa-histidine (His(6)-aat) affinity domains were constructed from a single DNA fragment. The DNA fragment was generated using the polymerase chain reaction (PCR, using Perkin-Elmer reagents and the manufacturer's suggested reaction conditions). PCR primers were designed which result in a BamHI fragment encoding the full-length L/F-transferase enzyme. The oligonucleotides used in the PCR reaction were as follows: N-terminal = 5`-GGCGGATCCATGCGCCTGGTTCAGCTT-3`; C-terminal = 5`-GCCGGATCCGGGGAGAAATGTGCCG-3`. The resulting PCR product allows for in-frame fusions with six NH(2)-terminal His residues when inserted into the BamHI site of expression plasmid pQE-8 (Qiagen). The same fragment results in an in-frame fusion with an NH(2)-terminal glutathione S-transferase domain when inserted into the BamHI site of expression plasmid pGST-2T (Pharmacia Biotech Inc.). Each of the expression vectors allows for regulated expression of fusion proteins and employs a hybrid promoter inducible with isopropyl-beta-D-thiogalactopyranoside (IPTG).

In Vivo Characterization of GST-L/F-transferase and His(6)-L/F-transferase Enzymes

The cellular levels of beta-galactosidase were determined using the chromogenic substrate 2-nitrophenyl-beta-D-galactopyranoside (ONPG). Cells containing pUBP1-X-beta-gal plasmids (Shrader et al., 1993; Tobias et al., 1991) encoding Arg-beta-gal or Met-beta-gal were transformed with a second compatible plasmid encoding either wild type L/F-transferase (Shrader et al., 1993) or the GST-L/F-transferase fusion protein. Transformants were grown overnight to saturation and then allowed to stand at room temperature for an additional 24 h. This second incubation results in greater reproducibility for this assay. One ml of cells was pelleted in a microcentrifuge and resuspended in 50 ml of 25% sucrose, 0.25 M Tris buffer (pH 8.0). To this mixture 10 µl of lysozyme (10 mg/ml) was added followed by incubation for 15 min on ice. The resulting protoplasts were lysed by the addition of 0.15 ml of 75 mM EDTA, 0.33 M NaCl, and incubation on ice for 5 min, followed by the addition of 10 µl of 1% Triton X-100. Supernatant from the lysed cells was prepared by centrifugation at 12,000 g for 15 min at 4 °C. After centrifugation, the insoluble cell debris was removed from the supernatant fraction. ONPG assays were performed by incubating 10 µl of the above lysate in 650 µl of Z buffer (60 mM Na(2)HPO(4), 40 mM NaH(2)PO(4), 10 mM KCl, 1.0 mM MgSO(4), and 50 mM 2-mercaptoethanol) at room temperature. One-hundred µl of 10 mM ONPG was added to this reaction and color allowed to develop. The reaction was timed and stopped by the addition of 1 M Na(2)CO(3) and the OD value of the final reaction mixture recorded. These values were normalized by dividing by the concentration of cellular protein as determined by the Bio-Rad protein detection reagent. All values were standardized by setting the value of wild type cells expressing stable Met-beta-gal to 250 arbitrary units. These units are not equivalent to Miller units (Miller, 1972). Stable X-beta-gals yield approximately 15 Miller units in the MC1061 strain used as wild type in these studies.

Purification of His(6)-L/F-transferase

The overexpression of the His(6)-L/F-transferase was achieved by transforming MC1061 (hsdR mcrB araD139 Delta(araABC-leu)7679 galU galK rpsL thi) pREP4 cells with plasmid pHis6-aat and growing the transformants overnight in the presence of 100 mg/ml ampicillin and 25 mg/ml kanamycin. Fresh overnight cultures were diluted 100-fold and grown in fresh Luria broth until the culture's A value reached 0.4. IPTG was added to a final concentration of 20 µg/ml and the cultures incubated for an additional 4 h at 37 °C. Cells were pelleted by centrifugation and resuspended in 100 mM KCl, 1.0 mM imidazole, 5 mM phenylmethylsulfonyl fluoride, 5 mM 2-mercaptoethanol, 50 mM Tris (pH 8.0). Cells were disrupted by two rounds of hydrodynamic sheering employing a French pressure cell (Aminco) operated at 1,500 p.s.i. and the cell debris removed by centrifugation at 15,000 g for 60 min. The supernatant fraction was incubated for 1 h with 1-2 ml of Ni-NTA-agarose (Qiagen)/liter of original cell culture with stirring at 4 °C. The Ni-NTA-agarose was subsequently washed with 400 ml of the above buffer containing 15 mM imidazole. Protein was eluted from the Ni-NTA-agarose resin with the above buffer containing 300 mM imidazole. This fraction was concentrated to 2-3 ml and applied to a Sephadex G-200 column equilibrated with 120 mM (NH(4))(2)SO(4), 50 mM Tris (pH 8.0). Fractions were collected and characterized by SDS-polyacrylamide gel electrophoresis. Fractions containing the L/F-transferase were pooled and concentrated to 1-4 mg/ml using a Centriprep concentrator (Amicon). Glycerol was added to a final concentration of 10% and the concentrated enzyme solution was frozen at -85 °C in aliquots.

Purification of Leucyl-tRNA Synthetase

The leucyl-tRNA synthetase was partially purified from E. coli cells overexpressing the enzyme. Strain MC1061 was transformed with plasmid pLEU1 (Hartlein and Madern, 1987) using standard transformation protocols, and transformed cells were grown overnight with selection for the plasmid. Cells were pelleted by centrifugation at 6,000 g for 10 min and resuspended in lysis buffer (50 mM Tris (pH 7.5), 10 mM MgCl(2), 0.5 mM EDTA, 10% glycerol) and disrupted as described above. Forty ml of cleared lysate was applied to an 80-ml DEAE-Sepharose column that had been equilibrated with lysis buffer. The column-bound protein was washed overnight with lysis buffer containing 50 mM KCl and eluted using a KCl gradient (50-300 mM). The broad protein containing peak was further fractionated by precipitation with solid ammonium sulfate (75% final concentration). The ammonium sulfate pellet was resuspended in 10 ml of lysis buffer with 1.0 mM phenylmethylsulfonyl fluoride and frozen at -85 °C in aliquots. This frozen preparation is not pure, as judged by electrophoresis, but retains high leucyl-tRNA synthetase activity and lacks detectable ribonuclease contamination.

tRNA Synthesis and Purification

Natural tRNAs were purchased from Subriden RNA. Unmodified tRNAs were transcribed using standard procedures with minor modifications (Sampson and Uhlenbeck, 1988). Plasmid-borne tRNA genes were digested with BstNI restriction endonuclease (New England Biolabs) and the enzyme removed by phenol/chloroform extraction. Transcription reactions include: 40 mM Tris (pH 8.0), 20 mM MgCl(2), 25 mM NaCl, 2.0 mM spermidine, 5 mM dithiothreitol, 16 mM each NTP, ± 40 mM GMP, 80 units of RNasin (Promega), and purified T7 RNA polymerase. Transcription reactions were incubated 8-10 h at 37 °C. Heterogeneous tRNA transcripts were separated from unincorporated nucleotides and pyrophosphate, using an NAP-5 column (Pharmacia), followed by ethanol precipitation. Full-length tRNA transcripts were isolated from incorrectly sized transcripts by electrophoresis on 10% acrylamide gels containing 50% urea. The full-length tRNA transcripts were identified by their UV shadow and eluted from the gel slice by soaking overnight at 37 °C. The resulting tRNA transcripts were concentrated by precipitation with 2 volumes of ethanol in the presence of 0.2 M NaCl.

Aminoacyl-tRNA Synthesis and Isolation

Aminoacyl-tRNAs were generated using partially purified leucyl tRNA synthetase (described above) and purified tRNAs or unmodified tRNAs produced by in vitro transcription. Charging reactions were carried out in 30 mM Hepes (pH 7.4), 15 mM MgCl(2), 25 mM KCl, 2.0 mM dithiothreitol, 2.0 mM ATP, 20 µM Leu. Aminoacyl-tRNAs were separated from the leucyl-tRNA synthetase by phenol/chloroform extractions, using phenol equilibrated at pH 5.0, and concentrated by precipitation as described above.

L/F-transferase Activity Assays

One unit of L/F-transferase is defined as the amount of the enzyme required to transfer 1 nmol of radiolabeled amino acid to alpha-casein in 1 min at 37 °C. The standard reaction conditions, defined in the laboratory of R. Soffer, employ a 75-µl reaction containing 1.8 µM purified aminoacyl-tRNA, 4 µM alpha-casein, 200 mM KCl, 50 mM Tris (pH 8.0) (Leibowitz and Soffer, 1970). Qualitative reactions to determine the specificity of the L/F-transferase toward aminoacyl-tRNA substrates, employ a mixture of crude aminoacyl-tRNA synthetases (Sigma). All reactions contain 50 mM Tris (pH 8.0), 10 mM MgCl(2), 10 mM 2-mercaptoethanol, 12 µM alpha-casein, 10 mM ATP, 3-4 A units of E. coli tRNA (Sigma), 500 units of E. coli aminoacyl-tRNA synthetases (Sigma), and purified L/F-transferase. Reactions also contain either: 0.0 M KCl, 0.1 M KCl, 0.5 M KCl or 0.1 M (NH(2))(2)SO(4). Incubations are for 30 min at 37 °C.

Absorption Spectra, Circular Dichroism, and Estimation of L/F-transferase Secondary Structural Content

Protein concentrations of L/F-transferase stock solutions were determined from their A values using the equation: 1.0 mg/ml protein = (5,700n + 1300n)/m(r). In this equation, n and n represent the number of Trp and Tyr residues (7 for each), and m(r) is the molecular weight of the protein (27,998 for His(6)-L/F-transferase) (Cantor and Schimmel, 1980). Circular dichroism was performed on a Jasco J-720 instrument. All measurements were performed at ambient temperature. Samples were prepared from a concentrated stock solution of purified L/F-transferase that was diluted into water immediately prior to the experiment. This dilution was necessitated by the interference of the storage buffer during data collection below 220 nm. A 10-fold dilution of the enzyme was found to be sufficient to allow data to be collected to 190 nm. Secondary structure estimation was performed using the SSE-338 program that compares the sample's CD spectrum with those of 15 reference proteins (Yang et al., 1986). The program estimates the fraction of the sample's residues in each of four conformations: alpha-helix, beta-sheet, beta-turn, and random coil.

Limited Proteolysis

Limited trypsin and chymotrypsin digestions were carried out in 50 mM Tris buffer (pH 7.9). Approximately 50 µg of purified L/F-transferase was incubated with 2 mg of the protease at 37 °C in a total volume of 250 µl. At selected time points during the digestion, 50-µl aliquots were withdrawn, and digestion halted by boiling in 2.0% SDS, 100 mM 2-mercaptoethanol, or precipitation with trichloroacetic acid (10% final concentration).

N-terminal Sequence Determination

The GroEL polypeptide identified during Ni-NTA-agarose purification of the His(6)-L/F-transferase and the stable tryptic fragment of the L/F-transferase were identified by direct NH(2)-terminal sequence determination at the Albert Einstein College of Medicine microchemistry facility. In each case the polypeptide was transferred from an SDS-polyacrylamide gel to ProBlott membrane (Applied Biosystems), followed by 10 cycles of automated Edman degradation. Transfers employed a Trans-Blott Cell (Bio-Rad) operated at 40 volts for 2 h in transfer buffer (10 mM CAPS (pH 11.0), 10% methanol). The polypeptide of interest was identified by staining the ProBlott membrane with Ponceau S (Sigma) and excised from the ProBlott membrane.


RESULTS

Construction and Characterization in Vivo of L/F-transferase Fusion Proteins

In order to obtain milligram quantities of the L/F-transferase enzyme for detailed mechanistic characterization, we used the cloned aat gene to construct two different overexpression systems for the enzyme. The requirement for overexpression prior to enzyme purification was suggested by the previous demonstration that the L/F-transferase is present at very low levels in wild type E. coli cells. Soffer and co-workers (Leibowitz and Soffer, 1970) estimated that a 20,000-fold purification was required to isolate the enzyme from natural abundance sources. In order to aid in the subsequent purification of the overexpressed enzyme, we have employed overexpression systems which result in the synthesis of L/F-transferases bearing NH(2)-terminal affinity domains. Recombinant enzymes employing both glutathione S-transferase (Smith and Johnson, 1988) and hexa-His (Smith et al., 1988) affinity domains have been constructed. The choice of the NH(2) terminus as the fusion junction was suggested by our previous demonstration that the wild type L/F-transferase accepts considerable modification of this region without loss of activity (Shrader et al., 1993). When assayed for their ability to stimulate the degradation of Arg-beta-gal, L/F-transferase mutants lacking up to 31 NH(2)-terminal residues remain partially active in vivo.

The affinity modified L/F-transferase enzymes containing the NH(2)-terminal glutathione S-transferase (encoded by the GST-aat allele) or hexa-His (His(6)-aat) affinity domains were constructed from a single PCR-derived DNA fragment encoding the entire L/F-transferase. This DNA fragment results in in-frame fusions, with the DNA sequences encoding either affinity domain, when inserted into the BamHI restriction site of commercially available expression vectors (see ``Experimental Procedures''). We reasoned that the His(6)-aat-encoded enzyme, which is less drastically modified, would be more suitable for subsequent characterization. However, we also reasoned that if the GST-aat encoded enzyme retained L/F-transferase activity, it would provide confidence that N-terminal modifications to the enzyme do not alter its important characteristics. Data demonstrating that the GST-aat encoded enzyme retains L/F-transferase activity and thereby compliments TS351 cells (MC1061 aat::minitet) (Shrader et al., 1993) for their inability to degrade Arg-beta-gal are shown in Fig. 1. Expression of stable X-beta-gals in E. coli cells results in higher steady state levels of beta-galactosidase than expression of short-lived X-beta-gals (Tobias et al., 1991). Wild type E. coli cells are unable to degrade Met-beta-gal and aat, or ClpA mutants are unable to degrade Met-beta-gal or Arg-beta-gal. Complementation of TS351 cells for their defect in Arg-beta-gal degradation by the plasmid borne GST-aat allele results in an approximate 10-fold reduction in the steady state level of Arg-beta-gal, as measured using ONPG assays (see ``Experimental Procedures''). Complimenting L/F-transferase activity is seen in the absence of full induction of the GST-aat expression system and probably results from incomplete repression of the powerful hybrid promoter upstream of the GST open reading frame. Complementation is more complete from the GST-aat fusion construct than from an alternate L/F-transferase construct (pAat) that expresses the wild type enzyme from cryptic promoter elements located internal to the surB gene immediately upstream of the aat gene (Shrader et al., 1993). These results demonstrate than the addition of large NH(2)-terminal extensions to the L/F-transferase result in, at most, partial loss of activity.


Figure 1: In vivo activity of a GST-L/F-transferase fusion protein. ONPG assays were used to monitor the steady state levels of X-beta-gal enzymes in wild type and mutant E. coli cells. X-beta-gals are beta-galactosidase enzymes engineered to contain the NH(2)-terminal residue X. Met-beta-gal is stable, irrespective of cellular L/F-transferase activity, whereas Arg-beta-gal is degraded only in cells expressing the L/F-transferase and the ClpAP protease. The steady state value of Met-beta-gal is normalized to 250 units. Samples: TS357, MC1061 clpA::minitet cells expressing Arg-beta-gal; TS351, MC1061 aat::minitet cells expressing Arg-beta-gal; M-betagal, MC1061 cells expressing stable Met-beta-gal; pAat, MC1061 aat::minitet (pAS45 (aat)) cells expressing Arg-beta-gal; Gst-Aat, MC1061 aat::minitet (pGST-aat (see methods)) cells expressing Arg-beta-gal; R-betagal, MC1061 cells expressing unstable Arg-beta-gal. Assay results from four isolates of each clone are shown.



Induction of expression of both the GST-aat and His(6)-aat alleles results in the synthesis of the fusion proteins to levels constituting a substantial fraction of total cellular protein as shown in Fig. 2. Induction is only moderately deleterious to cells as wild type growth rates are observed for two to three generation times after the addition of IPTG. Subsequent fractionation of the induced cells demonstrates that 50-90% of the synthesized enzyme is not soluble. This lack of in vivo solubility of the L/F-transferase in its natural host is curious. Both fusions show the same characteristics implicating the L/F-transferase moiety as responsible for fusion protein insolubility. Based on previous reports that the L/F-transferase purifies over several chromatographic steps with a bound tRNA, we interpret our solubility results to suggest that the L/F-transferase exists in cells predominantly as a complex with aminoacyl-tRNAs and that the nucleic acid is partially responsible for the in vivo solubility of the complex. Interestingly, the fraction of soluble protein is substantially greater for the GST-aat expression system than for the His(6)-aat expression system. This suggests that the soluble 26 K GST domain aids in the solubilization of the L/F-transferase enzyme in vivo and indicates that fusions to very large, soluble domains may further increase the yield of active enzyme. We argue below that the insolubility of the L/F-transferase results from a disordered NH(2)-terminal domain and that conditions that increase enzyme stability in vitro result in substantial increases in the enzyme's solubility.


Figure 2: Cellular fractionation of L/F-transferase fusion proteins. Coomassie-stained 12% SDS gel of cells and cell fractions of MC1061 cells expressing the His(6)-aat and GST-aat genes. Lanes: U, total cellular protein from uninduced cells; I, total cellular protein from induced cells; S, soluble protein from induced cells; P, insoluble pellet protein from induced cells.



Purification and Characterization of His(6)-L/F-transferase

Because the His(6)-aat-encoded enzyme contains only a 12-residue NH(2)-terminal extension, we have chosen to use this fusion for further characterization. This fusion protein is as active as the previously purified wild type enzyme (described below) and will be referred to as the L/F-transferase throughout this manuscript. The L/F-transferase has been partially purified using Ni-NTA-agarose based on the affinity of the NH(2)-terminal His residues for nickel (Smith et al., 1988). The resulting enzyme preparation is >70% pure (Fig. 3, lanes Ni and NC) and active in the transfer of leucine and phenylalanine to the NH(2) terminus of alpha-casein. The higher molecular weight contaminants found in partially purified L/F-transferase were removed by gel filtration (Fig. 3, lane GF). The final enzyme preparation contains only small amounts of low molecular weight contaminants. The gel filtration step was also used to transfer the enzyme to 120 mM ammonium sulfate. The introduction of this salt becomes essential in subsequent concentration steps. Purified L/F-transferase is soluble to <0.2 mg/ml in 100 mM KCl, whereas replacement of these monovalent ions with ammonium sulfate allows concentration of the enzyme to at least 4 mg/ml without detectable aggregation or precipitation. The same requirement for ammonium sulfate during L/F-transferase concentration was detected by Soffer and co-workers (Leibowitz and Soffer, 1970) during their purification of the wild type L/F-transferase.


Figure 3: Purification of the L/F-transferase. Coomassie-stained 10% SDS gel monitoring the purification of the L/F-transferase. Lanes: C, total cellular protein; P, insoluble proteins from cell pellet; S, soluble proteins; F, nonbinding flow-through from Ni-NTA-agarose affinity purification step; Ni, protein eluted from Ni-NTA-agarose; NC, concentrated Ni sample used for gel filtration; GF, final L/F-transferase enzyme after gel filtration and concentration to 4 mg/ml.



Activity data for the purified L/F-transferase is shown in Table 1. The recombinant enzyme transfers both Leu and Phe from their cognate aminoacyl-tRNAs to the acceptor protein alpha-casein. In each case, the level of radiolabeled amino acid incorporated into protein (hot trichloroacetic acid-precipitable counts) is >5-fold higher than the steady state level of amino acid available as aminoacyl-tRNA (cold trichloroacetic acid-hot trichloroacetic acid), demonstrating multiple rounds of tRNA charging and transfer of the amino acid to alpha-casein. This result also demonstrates that the aat gene directly encodes the L/F-transferase. We had previously shown that the aat gene is required for L/F-transferase activity, so this is an expected result. However the lack of sequence homology between the gene encoding the L/F-transferase and the yeast gene encoding the physiologically analogous R-transferase (Balz et al., 1990) left possible more indirect roles of the aat gene in L/F-transferase expression. The lack of homology between the two NH(2)-terminal aminoacyl transferases remains a surprise.



As expected from the work of previous laboratories, the transfer of both Ala and Arg by the L/F-transferase is barely detectable (Table 1). However, significant levels of both Thr and Val are incorporated into a hot trichloroacetic acid-stable form, and this incorporation is dependent on the presence of alpha-casein. This suggests that the L/F-transferase is somewhat less specific than previously reported with respect to its choice of aminoacyl-tRNA substrates. Interestingly, raising the ionic strength of the reaction to either 0.5 M KCl or 0.1 M (NH(4))(2)SO(4) increases the specificity of the enzyme with respect to aminoacyl-tRNA recognition. Ionic strength changes do not significantly alter the incorporation of Leu into alpha-casein, suggesting that the specificity of the enzyme is being altered rather than overall enzyme activity. Increased ionic strength also prevents the purified enzyme from aggregating (see above). We attribute the increase in substrate specificity and decreased aggregation of the L/F-transferase at relatively high ionic strengths to an increase in the order of the NH(2)-terminal domain of the enzyme (see below).

The assays described above to determine the substrate specificity of the His(6)-L/F-transferase fusion protein are qualitative in nature. We have also compared the specific activity of our preparation with that of the purified wild type enzyme (Leibowitz and Soffer, 1970). The Soffer laboratory defined one unit of L/F-transferase activity as the amount of enzyme that transfers 1 nmol of amino acid to the acceptor protein alpha-casein in 1 min under defined reaction conditions (see ``Experimental Procedures''). Using this assay, the purified wild type enzyme contains 60 units of activity/mg of protein for the transfer of both Leu and Phe (Leibowitz and Soffer, 1970). Using these assay conditions, our purified enzyme displays 330 units of activity/mg of protein for the transfer of Leu from natural tRNA(CAG) and 250 units of activity for the transfer of Leu from T7 RNA polymerase-transcribed tRNA(CAG). The small difference in specific activity, between natural and unmodified tRNAs, in this L/F-transferase assay may be misleading. Unmodified tRNAs have lower stability than the fully modified tRNAs produced in vivo (Peterson et al., 1993). This may result in some unfolding of the T7-transcribed tRNAs with concomitant loss of activity as a substrate for the L/F-transferase.

The most abundant co-purifying polypeptide in our partially purified L/F-transferase preparation is a polypeptide with an approximate molecular mass of 65 kDa visible in lane NC of Fig. 3. This protein binds to the Ni-NTA-agarose column only in the presence of the L/F-transferase (data not shown). Because this 65-kDa polypeptide might have represented a L/F-transferase substrate or interacting polypeptide, we have determined its NH(2)-terminal sequence. The NH(2)-terminal sequence of this polypeptide: A-A-K-D-(V/K)-(L/F)-I-(H/K/G)-N-D identifies it as GroEL (NH(2)-terminal AAKDVKFGND). The interaction of the L/F-transferase with this molecular chaperonin is provocative in light of the enzyme's preference for unstructured substrates (Soffer, 1973). The possibility that natural substrates of the L/F-transferase are presented to the enzyme, in extended form, by the cellular folding machinery must now be considered. A role for the molecular chaperones in protein degradation has been demonstrated previously in both bacterial and eukaryotic cells (Straus et al., 1988, Sherman and Goldberg, 1992). A second potential role of the GroEL chaperonin in the cellular activity of the L/F-transferase is suggested by the enzyme's in vivo insolubility in the absence of bound tRNA (see above). The chaperonin may be required to maintain solubility of the enzyme during the period of tRNA aminoacyl-tRNA exchange after substrate modification.

Domain Structure of the L/F-transferase

In order to begin to characterize the structure of the L/F-transferase, we have performed limited proteolytic digestions of the purified enzyme. A time course for trypsin digestion is shown in Fig. 4. Within 5 min of the start of the digestion, a stable 15-kDa fragment becomes visible and persists for at least 60 min. Amino-terminal sequence analysis of this fragment yields the sequence F-H-K-R-S-P-Y-R-V-T. This identifies the NH(2) terminus of the trypsin-stable fragment as Phe-81, in the numbering system of the wild type enzyme (Shrader et al., 1993). The electrophoretic mobility of the stable fragment suggests that its COOH terminus is near the COOH terminus of the intact protein. The most likely termini are at Arg-215 (calculated fragment mass of 15,312 daltons) or Arg-218 (15,648 daltons). No other stable L/F-transferase-derived fragments are seen during the digestion. Digestions with chymotrypsin result in a stable fragment with an electrophoretic mobility similar to the fragment generated by trypsin (data not shown). Due to the changes in the solubility and enzymatic properties of the L/F-transferase at increased ionic strength, we have performed trypsin digestions in the presence of increasing amounts of ammonium sulfate. These digestions show qualitatively the same time course as the digestion in 0.1 M KCl (data not shown). Digestions in the presence of up to 0.5 M ammonium sulfate have been performed; the overall rate of digestion slows with increased levels of ammonium sulfate, but no new stable fragments are observed (data not shown). This suggests that the structural changes in the enzyme at increased ionic strength are subtle and do not involve large domain rearrangements. Correspondingly, the implied flexibility of the NH(2)-terminal region of the L/F-transferase probably does not correspond to a complete lack of structure. Rather, this region of the protein undergoes some modest breathing motions which may be required during substrate binding. These motions probably result in aggregation at high enzyme concentrations as aggregation is minimized under ionic strength conditions, which increase the stability of the folded form of proteins (Creighton, 1993).


Figure 4: Limited proteolysis of the L/F-transferase. Silver-stained 15% SDS gels were used to monitor the time course of trypsin digestion of the purified L/F-transferase. An arrow indicates the stable core of the enzyme. The NH(2)-terminal sequence of this fragment corresponds to Phe-81 of the native L/F-transferase. Lane markers: numeric values correspond to the duration of incubation of L/F-transferase with trypsin: L, lysozyme (M(r) = 14,300); I, soybean trypsin inhibitor (M(r) = 21,500); A, bovine serum albumin; T, trypsin enzyme used in digestion reactions; U, undigested L/F-transferase.



Absorption and Circular Dichroism Spectra for the L/F-transferase

The absorption spectrum of purified L/F-transferase is similar to that of other proteins lacking organic co-factors. The A/A ratio is 1.8 and reflects the high content of Trp and Tyr in the enzyme. The high content of aromatic amino acids results in the expected strong fluorescence signal for the purified enzyme. However, the fluorescence signal is not altered measurably by the addition of ammonium sulfate, the ionic strength conditions that reduce aggregation, underscoring the subtlety of the changes in enzyme structure induced by altered ionic conditions (data not shown). The measured A/A value is not consistent with a protein that contains a significant nucleic acid component. In addition, the purified enzyme preparation contains no material that stains with ethidium bromide (data not shown). We conclude that the L/F-transferase catalyzes a peptidyltransferase reaction without the aid of an RNA component. The absorbance at 280 nm also reveals that the L/F-transferase is anomalous in its dye binding properties. For example, a purified enzyme sample whose calculated concentration is 3.7 mg/ml, based on its A value, has a measured concentration of 11.7 mg/ml based on its binding to coomassie brilliant blue. This anomaly probably reflects the high levels of Arg and Lys residues found in the enzyme as these residues interact most strongly with this dye (Congdon et al., 1993).

The far-UV circular dichroism spectrum of the L/F-transferase is shown in Fig. 5. Visual inspection of the CD spectrum suggest an overall similarity to the spectrum of an alpha-helix, however the characteristic inflection at 215 nm is not observed. Secondary structure calculations suggest values of 45-55% alpha-helical residues and approximately 25% each of residues in random coil and beta-turn conformations. No beta-sheet structure is detected. Based on the high concentration of Pro residues in the NH(2)-terminal 80 amino acids and the protease sensitivity of this region, this suggests that the majority of the residues that comprise the protease sensitive NH(2)-terminal domain are in random coil conformation. Correspondingly, this data indicates that the protease resistant COOH-terminal domain is predominantly alpha-helical. Overexpression and purification of the globular domain of the L/F-transferase are in progress. A comparison of the CD spectrum of the isolated globular domain with the spectrum of the intact protein should allow definitive identification of the alpha-helical regions of this enzyme.


Figure 5: Circular dichroism spectrum of purified L/F-transferase. The circular dichroism spectrum of the purified L/F-transferase suggests that the enzyme is strongly alpha-helical. Quantitative curve fitting yields values of 47% alpha-helix, 25% random coil, and 28% turn. The observed spectrum (solid line), the spectrum calculated based on the above percentages (dashed line), and the difference spectrum (dotted line) are shown.




DISCUSSION

The ability to generate milligram quantities of active L/F-transferase has allowed us to employ biochemical methods in conjunction with genetic approaches to separate the regions of the enzyme responsible for substrate recognition and catalysis. A schematic of the primary sequence of the L/F-transferase, depicting our current understanding of the enzyme, is shown in Fig. 6. A striking aspect of this figure is the strong correlation between protease sensitivity and the distribution of Pro residues. The protease-sensitive NH(2)-terminal region of the enzyme contains 10 proline residues, whereas inspection of the primary sequence of the globular COOH-terminal region reveals a continuous stretch of 116 residues completely lacking proline. The proline distribution is consistent with our assignment of the alpha-helical residues, identified using circular dichroism, with the COOH-terminal globular region of the L/F-transferase.


Figure 6: Schematic of the L/F-transferase enzyme. The complete amino acid sequence of the His(6)-L/F-transferase is shown. Previous genetic data characterizing the non-essential portion of the enzyme and the NH(2) terminus of the trypsin-resistant core are indicated. The non-uniform proline distribution is highlighted by small circles. Also indicated are the abundant Cys residues (underlined) and a histidine-rich cluster (double underlined). Experiments are in progress to determine whether these residues are involved in metal binding.



Our working model for the L/F-transferase invokes the protease-sensitive N-terminal domain in substrate recognition. This is suggested by the increase in substrate specificity by the L/F-transferase under conditions that reduce aggregation (see above). The requirement for a flexible substrate recognition domain is also suggested by the wide range of acceptor peptide and aminoacyl-tRNA substrates utilized by this enzyme. Acceptor protein recognition by the L/F-transferase is degenerate with amino-terminal Arg and Lys residues being recognized (Leibowitz and Soffer, 1971b). Arg and Lys each contain basic side chains but are not isosteric, and it is difficult to envision a tight binding pocket with this degenerate specificity. In addition, peptide binding experiments demonstrate that residues beyond the NH(2) terminus further modulate binding (Soffer, 1973).

We have demonstrated that recognition of aminoacyl-tRNAs by the L/F-transferase does not strongly depend on the modified nucleotides found in cellular tRNAs. This result is important for two reasons. First, the lack of crucial contacts between the L/F-transferase and the modified nucleotides found in tRNAs supports previous results implicating the tRNA acceptor helix in L/F-transferase bullet aminoacyl-tRNA recognition (Scarpulla et al., 1976). There are no modified nucleotides in the acceptor helix (Clark et al., 1995). Second, our ability to utilize T7 RNA polymerase to generate mutant tRNAs in vitro will allow us to determine the importance of individual nucleotides for recognition by the L/F-transferase. The L/F-transferase uses a subset of cellular aminoacyl-tRNAs as substrates. This level of specificity is intermediate between enzymes that recognize a single tRNA (the aminoacyl-tRNA synthetases) and enzymes that recognize many or all aminoacyl-tRNAs (elongation factor TU and the ribosome). This subtle discrimination is based on both the amino acid and tRNA moieties (Leibowitz and Soffer, 1971b). In an in vitro reaction using purified components, the L/F-transferase is 30 times more efficient catalyzing the transfer of Met from the Met elongator tRNA (tRNA) to casein than it is catalyzing the transfer of Met from the initiator tRNA (tRNA) to casein (Scarpulla et al., 1976). Initiator tRNAs differ from the family of elongator tRNAs by a unique non-Watson-Crick C:A base pair at the start of the acceptor helix (RajBhandary, 1994). Elongation factor TU also differentiates against the initiator tRNA on the basis of this C:A base pair (RajBhandary, 1994). There are no nucleotides unique to the family of tRNAs active as L/F-transferase substrates. This suggests that nucleic acid recognition is more dependent on shape than sequence and implicates the junction between single-stranded and double-stranded RNA as a recognition determinant.

The recognition of the aminoacyl moiety of aminoacyl-tRNAs is intriguing and suggestive of a flexible enzyme structure. The L/F-transferase differentiates between Leu, Val, and Ile but accepts Met, Phe, and Trp (Kaji et al., 1965; Scarpulla et al., 1976). This recognition suggests that a large hydrophobic side chain is recognized but that an unbranched beta-carbon is also required. The corresponding recognition cavity, hydrophobic with a restricted opening, is difficult to imagine without invoking a flexible enzyme that becomes more structured in the presence of substrates or folds around the amino acid side chain. Studies to monitor increases in secondary structure of the L/F-transferase in response to aminoacyl-tRNA are in progress. Several authors have identified beta-sheet structures that interact with RNA (Oubridge et al., 1994). The lack of detectable beta-structure in the free L/F-transferase should make detection of induced beta-sheet formation more straightforward. In addition we are purifying truncated L/F-transferase enzymes lacking their NH(2)-terminal 33 (active in vivo) and 80 residues (the globular core). A comparison of the overall enzymatic activity of these enzyme and their free and induced secondary structure contents should allow definitive inclusion or exclusion of the disordered NH(2)-terminal region of the enzyme in aminoacyl-tRNA recognition.

We have shown that the L/F-transferase catalyzes a peptidyltransferase reaction without a detectable RNA component. In this respect, the L/F-transferase reaction may differ from the peptidyltransferase reaction catalyzed on the large subunit of the ribosome. This central reaction in translation can be isolated from the complex overall process, and a model peptidyltransferase reaction consists of peptide bond formation between the amino group of puromycin and the carbonyl carbon of formyl-Met donated by a CAACCA-(fMet) ester (Monroe and Marker, 1967). This reaction, along with affinity labeling of ribosomes, employing modified Phe-tRNAs, identified proteins L2, L15, and L16 along with the 23 S ribosomal RNA as essential for the reaction (Noller et al., 1992). Interestingly, the minimal complex, active in the model peptidyltransferase reaction, is extremely resistant to proteases and removal of protein by phenol extraction (Noller, 1993). This has resulted in a search for the true catalytic core of the peptidyltransferase reaction. To paraphrase H. Noller, the ``protein-centric'' view of the peptidyltransferase reaction is that the proteins provide the catalytic groups but are protected by the RNA. Conversely, the ``ribo-centric'' view is that the proteins form a scaffold essential for the proper conformation of a catalytic RNA (Noller, 1993). Understanding the L/F-transferase reaction mechanism in detail may clarify the essential features required for enzymes that catalyze peptidyltransferase reactions.

Using the sequence data banks, we have identified a region of sequence similarity between the L/F-transferase and the RimL NH(2)-terminal acetylase (Tanaka et al., 1989). We have tentatively assigned this region of the L/F-transferase enzyme a role in binding and, perhaps, activating the NH(2) terminus of acceptor peptides. This assignment is based on the participation of a substrate NH(2)-terminal amino group in each reaction, and the conserved cluster of acidic residues found in the conserved region of each enzyme. The conserved region is depicted in Fig. 7and was identified based on a region of strong sequence similarity between the L/F-transferase and bacterial enzymes that acetylate the NH(2) terminus of ribosomal proteins. Interestingly, this same region has been identified by previous authors as the region most closely conserved between the NH(2)-terminal acetylases (Tanaka et al., 1989). The acetylases catalyze a reaction that is similar to that catalyzed by the L/F-transferase and are thought to use an acetyl group activated as acetyl-CoA and the NH(2) terminus of ribosomal proteins as substrates. Note: the suggestion of substrate recognition motifs in the globular domain is not inconsistent with our model invoking a flexible NH(2)-terminal region in the recognition of the family of L/F-transferase substrates, as a free alpha-amino group is common to all acceptor peptide substrates and, therefore, its recognition would not require enzyme flexibility. Finally, the observed sequence conservation between the L/F-transferase and a family of NH(2)-terminal acetylases (Tanaka et al., 1989) provides an additional evolutionary link between the similar activation schemes that have long been noted for the building blocks of protein and lipid synthesis (Lipmann, 1971).


Figure 7: Sequence similarities between the L/F-transferase and the NH(2)-terminal acetylases. Translations of the genes encoding three ribosomal protein NH(2)-terminal acetylases aligned with the aat-encoded L/F-transferase. The regions of the NH(2)-terminal acetylases shown has been identified previously by Isono and co-workers (Tanaka et al., 1989) as the region of strongest sequence conservation between the acetylases. The optimal alignment reported by these authors has been retained. Regions of sequence similarity between the RimL protein and the L/F-transferase are underlined. The regions of the proteins shown are: RimI, residues 78-98; rimJ, 72-92; rimL, 68-92; L/F-transferase, 120-144.




FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 718-430-2892 (or 2893); Fax: 718-892-0703; shrader{at}aecom.yu.edu.

(^1)
The abbreviations used are: L/F-transferase, leucyl/phenylalanyl-tRNA-protein transferase; beta-gal, beta-galactosidase; PCR, polymerase chain reaction; IPTG, isopropyl-beta-D-thiogalactopyranoside; ONPG, 2-nitrophenyl-beta-D-galactopyranoside; NTA, nitrilotriacetic acid.


ACKNOWLEDGEMENTS

We thank Heike Pilka for T7 RNA polymerase and valuable advice on working with tRNA, M. Hartlein for the pLEU1 plasmid, and Guisseppi Tocchini-Valentine for tRNA clones and help with the purification of the leucyl tNRA synthesis enzyme. In addition, we thank Y. Shi and E. Nieves of the Albert Einstein College of Medicine microchemistry facility for NH(2)-terminal sequence analysis and assistance with circular dichroism data collection and analysis.


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