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Cleavage of the N-terminal formymlethionine residue from a bacteriophage coat protein in vitro
J. Mol. Bid. (1973) 79, 631637
Cleavage of the N-terminal Formymlethionine Residue from a Bacteriophage Coat Protein in vitro L. ANDREW BALL? AND PAVLKAESBEFCQ Biophysics Laboratory and Biochemistry Department University of Wisconsin, Madison, Wise. 53706, U.S.A. (Received 8 March 1973, and in revised form 11 June 1973) Studies were made of the N-terminal formylmethionine content of nascent and complete coat protein of bacteriophage Q/I synthesized in an Eachetichiu coli cellfree system. Under normal conditions of cell-free protein synthesis the formylmethionine residue was retained by all the nascent chains but by only about 50% of the completed coat protein molecules. If 2-mercaptoethanol was omitted from the cell-free system, the formyhnethionine residue ~88 cleaved during the course of peptide chain elongation. All nascent peptides which contained fewer than 40& 5 ammo acids retained the formylmethionine residue. Thereafter, the proportion of nascent peptides lacking the residue increased with peptide length to about 70% for nearly full length nascent peptides and complete released coat protein molecules.
1. Introduction Protein synthesis in Escherichia coli is initiated by the incorporation of a formylmethionine residue as the N-terminal ammo acid of each nascent peptide chain (Clark & Marcker, 1966; Adams & Capecchi, 1966; Webster et al., 1966; Capecchi, 1966). Mature E. coli proteins have various amino acids at their N termini (Waller, 1963) as a result of the post-translational cleavage of the initiating fMet residue. This occurs by deformylation (Adams, 1968; Takeda & Webster, 1968; Livingstone t Leder, 1969) which is followed, in the case of 55% of the E. coli proteins, by aminopeptidase cleavage of the methionine residue. Action of the deformylase, which, in vitro, is a very unstable enzyme strongly inhibited by thiol reagents (Adams, 1968) is essential for subsequent aminopeptidase cleavage (Takeda & Webster, 1968). The coat protein of bacteriophage f2 undergoes both reactions during its synthesis on the ribosome; in phage-infected cells, nascent coat peptides lose their N-terminal methionine when they are 40 to 60 amino acids long (Housman et al., 1972). However, during the synthesis of f2 coat protein in vitro a large proportion of molecules retain their fMet even after release from the ribosome (Lodish & Robertson, 1969). This can be ascribed at least in part to inhibition of the deformylase by the thiol reagents which are included routinely in cell-free protein synthesizing systems. The coat protein of bacteriophage Qj also largely retains its N-terminal fMet residue when synthesized in vitro (Osborn et al., 1970). Since the protein contains no internal methionine residues (Konigsberg et al., 1970) it provides an ideal system for 7 Present address: National Institute England.
for Medical Research, Mill Hill, London NW7 IAA, 531
532
L.
A. BALL
AND
P. KAESBERG
quantitative studies of fMet cleavage. We have therefore investigated the fate of the fMet residue during synthesis of Qfl coat protein under various conditions in vitro, in an attempt to clarify this process of post-translational modification.
2. Materials and Methods All the materials 1973).
and methods used have been described previously
(Ball & Kaesberg,
3. Remits Extent of fomylmethionine
cleavage in vitro
The products of cell-free protein synthesis directed by Q/?RNA and doubly labeled with [35S]methionine and [3H]lysine were analyzed by electrophoresis on 10% polyacrylamide gels (Fig. 1). The three proteins synthesized in vitro correspond to polypeptides 1 (a subunit of the viral RNA replicase), llb (a minor component of the virion), and 111 (the major coat protein (Jockusch et al., 1970)). All three contained 35Slabel which, in the case of the coat protein, indicated the presence of an N-terminal fMet residue on some of the molecules. However, it is not possible to calculate the exact methionine content of the coat protein without knowledge of the relative specific activities of the two isotopes used in the cell-free system. This can be measured from the ratio of 3H to 35S in the nascent coat peptides. Each nascent peptide can contain a maximum of one methionine residue, but its content of the other amino
Distance migrated (mm)
Pm. 1. Sodium dodecyl sulf8te/polyscrylamide gel electrophoresis of the proteins synthesized by Qfi RNA in a cell-free system. The proteins were doubly labeled with [93]methionine (-----) ) (26 @/ml) for 30 min. After the treatment described (68 @i/ml) 8nd [3H]lysine ( previously (B811 & Keesberg, 1973) sn amount corresponding to 26 ~1 of the origin81 reection mixture was spplied to 8 10% polyaorylamide gal, 80 mm in length, containing 0.1 o/0 sodium dodecyl sulfate and 6 M-urea, Electrophoresis was at 20 V and 3.6 mA/gel for 17 h.
fMET
CLEAVAGE
IN
VITRO
833
acid depends on its length. For example, peptides up to 23 residues long (including the fMet) till contain no proline since the 6rst proline residue occurs at position 23 in the chain (Konigsberg et al., 1970). Those 24 to 23 long will contain one proline, those 29 to 42, two prolines and so on. Thus the ratio of [3H]proline to [35S]methionine will increase in unit steps as the peptide chain grows, the height of these steps indicating the relative specific activities of the labeled amino acids used in the cell-free system. Any cleavage of the fMet will result in peptides with anomalously high ratios of proline to methionine. Pulse-chase experiments showed that during the first four minutes of cell-free protein synthesis more than 90% of the translation directed by Qj3 RNA occurs from the coat protein gene (unpublished observations). The products of a short in vitro synthesis directed by Q/I RNA were labeled with [35S]methionine and [3H]proline. One fraction was precipitated with 10% trichloroacetic acid in preparation for gel electrophoresis ; another was subjected to sucrose density gradient centrifugation as described in a previous paper (Ball C Kaesberg, 1973). The ribosome-bound nascent peptides (70 S) and the released chains (< 10 S) were precipitated separately in preparation for gel electrophoresis. After hydrolysis of peptidyl-tRNA at pH 11.0, the products were subjected to electrophoresis on highly cross-linked 12.5% polyacrylamide gels in the presence of O*1o/osodium dodecyl sulfate (Swank ik Munkres, 1971). These gels resolve peptides according to their molecular weight in the range 2000 t#o 20,000, and are therefore well suited to the study of the nascent peptides of Qj3 coat protein (molecular weight 14,050). The gels were calibrated with the peptides resulting from cyanogen bromide cleavage of lysozyme, myoglobin and cytochrome c, and the calibration was refined using the N-terminal peptides of Q,fl coat protein as described in Figure 3 of Ball & Kaesberg (1973). Figure 2 shows the ratio of proiine to methionine across the region of the gels containing the coat protein and its nascent peptides. The ratio for the products not subjected to sucrose gradient fractionation (Fig. 2(a)) rises a series of steps with increasing molecular weight of the nascent peptides. The steps which indicate entry of the first six (of the total of eight) proline residues are clearly defined, and the ratio of 3H to 35Sat each stage corresponds exactly to that predicted from the nominal specific activities and measured counting efficiencies of the labeled amino acids. This suggests that there was a negligible pool of unlabeled amino acids in the cell-free system during these short reaction times. In the region of the gel which contains the complete coat protein, the ratio of proline to methionine rises sharply to a value of 13.5f0.5, corresponding to 41&3”,/0 f Met cleavage. Fractionation of the products into nascent and complete chains shows that almost all this fMet cleavage occurs after termination of the coat protein and its release from the ribosome. The ratio of proline to methionine in the nascent chains rises in eight steps to a value of 8.5f0.5 ((6f6)% fMet cleavage) for the complete peptides still bound to ribosomes (Fig. 2(b)). On the other hand, the released coat protein has lost 45% of its fMet which fully accounts for the cleavage observed in Figure 2(a). These results show that not only is the E. cc.%cell-free system partially deficient in the enzyme(s) responsible for fMet removal, but also that the limited cleavage which is observed occurs only after chain termination. This contrasts with the situation in f2 phage-infected cells, where nascent coat peptides lose their terminal fMet residues when they are forty to sixty amino acids long (Housman et al., 1972). 3:I
L. A. BALL
534
AND
P. KAESBERG
Takeda & Webster (1968) showed that modification of the N termini of E. coli proteins by aminopeptidase cleavage of the fMet residue was dependent on prior removal of the formyl group. The deformylase responsible for this reaction in E. coli is strongly inhibited by thiol reagents such as 2-mercaptoethanol (Adams, 1968) which are routinely included during the preparation and reaction of cell-free protein synthesizing systems. It is possible, therefore, that the deficient removal of fMet ~YL vitro is due to the presence of thiol reagents. Cell-free extracts of E. coli which were prepared and assayed in the absence of thiol reagents were capable of active protein synthesis in response to Q/I RNA. Initially, t.heir activity was about 50% of that of routine S30 extracts (prepared, stored and assayed in the presence of 10 mm-2-mercaptoethanol) but they lost their activity
1
28 ,
23
I lN&#b 0 ’ I I I I %.& i
2
30
40
50
60
70
80
90
Distance migrated (mm)
FIQ. 2. Ratio of sH to ?S label across the gel regions containing Qp coat protein and incomplete coat peptides. QB RNA-directed products were doubly labeled in vitro with [s6S]methionine (68 &i/ml) and [sH]proline (25 &i/ml) for 3 min. The molar ratio of proline to methionine was calculated from the specific activities and counting efficiencies of the isotopically labelled amino acids used in the cell-free system. (a) Total products. After the treatment described previously, an amount corresponding to 100 c;l of the original reaction mixture was applied to a 12.5% polyacrylamide gel, 110 mm in length. (b) Fractionated products. -a-•--, Ribosome-bound peptides ; --O--O--, released peptides. Samples (150 ~1) of the untreated reaction mixture were applied to two 6% to 20% sucrose density gradients and centrifuged at 36,000 revs/min for 2 h as described previously (Ball & Kaesberg, 1973). Fractions containing the ribosome-bound peptides (-70 S) and the released chains (< 10 S) were pooled separately and precipitated with 10% trichloroacetic acid, After treatment, amounts corresponding to 160 ~1 of the original reaction mixture were applied to 125% polyaorylamide gels, 110 mm in length. Electrophoresis was at 100 V 8nd 4.0 mA/gel for 13 h. The solid arrows indicate the steps which correspond to the entry of individual proline residues into the growing peptide chain. Their numbers refer to the amino acid positions (Konigsberg et al., 1970). The dotted arrows indicate the position of the complete coat protein.
fMET
IN
CLEAVAGE
VITRO
535
more rapidly during storage at -70°C. The proteins made by the two types of system in response to Qfl RNA were labeled with [14C] and [3H]lysine, respectively, and analyzed by co-electrophoresis on a 10% polyacrylamide gel. There was no detectable difference between the products, showing that 2-mercaptoethanol was not essential for accurate cell-free protein synthesis. The products of a short in vitro synthesis directed by Qfi RNA in these two systems were labeled with [35S]methionine and [3H]lysine. After hydrolysis of peptidyl-tRNA at pH 11.0, the products were subjected to electrophoresis on 125% polyacrylamide gels. Figure 3 shows the ratio of lysine to methionine across the gel regions which contain the coat protein and its nascent peptides. The products made in the presence of 2-mercaptoethanol have a ratio of lysine to methionine which rises in a series of steps with increasing molecular weight of the nascent peptides. As shown in Figure 2(a), fMet cleavage occurs only from the peptides which migrate with complete coat protein molecules. The products made in the absence of 2-mercaptoethanol show very different ratios of lysine to methionine (Fig. 3). For most of the nascent peptides, the ratio is anoma-
Peptide size (armno acid residues)
I
131 I
100 I
00 I
60 I
40 I
30 I
25 -
5 5 B
2o
1
15-
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40
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60
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Distance
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Fm. 3. Ratio of 3H and 35S label across the gel regions containing Qfl coat protein and inoomplete coat peptides. -e-O--, Products made in the presence of 10 mm-2-mercaptoethanol; --O--O--, products made in the absence of 2-mercaptoethanol. QB RNA-directed products were doubly labeled in vitro with [35S]methionine (08 @i/ml) and C3H]lysine (25 &i/ml) for 3 min. The molar ratio of lysine to methionine was calculated from the specific activities and counting efficiencies of the isotopically labeled amino acids used in the cell-free system. After the treatment described previously, amounts corresponding to 60 d of the original reaction mixtures were applied to 12.5% polyacrylamide gels, 110 mm in length. Electrophoresis was at 100 V and 4.0 mA/gel for 15 h. The dotted arrow indiaates the position of complete coat protein, established by ooelectrophoresis in a parallel gel. The upper horizontal scale shows the molecular weight calibration of the gels determined as described in Fig. 3 of Ball & Kaesberg (1973).
L. A. BALL
536
AND
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lously high, indicating fMet cleavage. Only those peptides with R, values greater than 2.0 (relative to Q/3coat protein) fully retain their N-terminal methionine residues. Accurate calibration of the gels by the method described previously (Ball 6 Kaesberg, 1973) shows that this R, value corresponds to a peptide containing 40-&5 amino acids. In the absence of 2-mercaptoethanol, when nascent peptides reach this size in vitro, a substantial proteolytic cleavage of their terminal methionine residues occurs (Fig. 4).
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9-4 -... Lx . 0 0 I I 0
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.-.-.
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120 131
Nascent pepttde chain length (ammo acid residues)
FIG. 4. The rel8tion between the chain length of the nascent C&3 coat peptides made in vitro 8nd the cleav8ge of their N-tannin81 methionine residues (calcultlted from the dat8 shown in Fig. 3). (0) Peptides synthesized in the presence of 10 mM-%mercsptoeth&nol; (0) peptides synthesized in the absence of %merceptoethanol.
4. Discussion In the experiments described above, calculation of the methionine content of Q/I coat protein and its nascent peptides depends critically on the specific radioactivities of the labeled amino acids used in the cell-free system. The height of the individual steps in the molar ratios which correspond to the entry of single proline or lysine residues (Figs 2 and 3) provides an internal check on the accuracy of this calculation. We can therefore conclude that the N-terminal methionine is cleaved from a significant proportion of the Q/I coat protein molecules made in an E. coli cell-free system. This contrasts with the generally accepted view, based on the translation of f 2 RNA, that proteins made in vitro fully retain their terminal methionine residues (Lengyel & Sdll, 1969; Lodish $ Robertson, 1969). This disagreement may reflect differences in the cell-free systems, or differences between the translation of Qfi and f2 RNAs. In the presence of 2-mercaptoethanol, significant methionine cleavage occurs only after peptide chain termination, whereas in the absence of thiol reagents cleavage occurs during elongation. This suggests that 2-mercaptoethanol inhibits an enzyme which is necessary for early methionine removal-probably the peptide deformylase (Adams, 1968). The lack of cleavage from nascent peptides in the presence of
fMET
CLEAVAGE
IN
VITRO
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2-mercaptoethanol probably reflects the slow reaction rate of the inhibited enzyme rather than a change in its substrate specificity. In the absence of 2-mercaptoethanol, nascent peptides become substrates for the aminopeptidase when they are about 40 amino acids long. This resembles the situation in f2 phage-infected cells, where cleavage occurs when the nascent coat peptides contain 40 to 60 residues (Housman et al., 1972). Similarly, removal of the N-terminal methionine from rabbit globin in vivo occurs when the nascent peptides are about 30 (Yoshida et al., 1970) or 40 to 50 residues in length (Wilson & Dintzis, 1970). Moreover, reticulocyte ribosomes protect chains of up to 35 amino acids from digestion by trypsin or pronase (Rich et al., 1966). Since the aminopeptidase which is probably responsible for methionine cleavage in E. coli is not bound to ribosomes (Brown & Krall, 1971), it seems likely that similar shielding by bacterial ribosomes protects the fMet residues of nascent phage coat peptides until they are about 40 amino acids long. We thank Wendy Good and Steve Kagen for invaluable technical assistance. This work was supported by grants from the Institute of Allergy and Infectious Diseases, National Institutes of Health, and from the Biology Division of the U.S. Atomic Energy Commission. REFERENCES Adams, J. M. (1968). J. Mol. Biol. 33, 571-589. Adams, J. M. & Capecchi, M. R. (1966). Proc. Nat. Ad. Sci., U.S.A. 55, 147-155. Ball, L. A. & Kaesberg, P. (1973). J. MOE. BioE. 74, 547-562. Brown, J. L. & Krall, J. F. (1971). Biochem. Biophys. Res. Commun. 42, 390-397. Capecchi, M. R. (1966). Proc. Nat. Acad. Sci., U.S.A. 55, 1517-1524. Clark, B. F. C. & Marcker, K. A. (1966). J. Mol. BioZ. 17, 394-406. Housman, D., Gillespie, D. & Lodish, H. F. (1972). J. Mol. B&Z. 65, 163-166. Jockusch, H., Ball, L. A. & Kaesberg, P. (1970). v’irology, 42, 401-414. Konigsberg, W., Maita, T., Katze, J. & Weber, K. (1970). Nature (London), 227, 271-273. Lengyel, P. & 5611, D. (1969). Bacterial. Rev. 33, 264-301. Livingstone, D. M. & Leder, P. (1969). Biochemktry, 8, 435-443. Lodish, H. F. & Robertson, H. D. (1969). J. Mol. BioZ. 45, 9-22. Osborn, M., Weber, K. & Lodish, H. F. (1970). Biochem. Biophys. Res. Commun. 41, 748-756. Rich, A., Eikenberry, E. F. & Malkin, L. I. (1966). Cold Spring Harbor Symp. Quant. BioZ. 31, 303-310. Swank, R. T. & Munkres, K. D. (1971). Anal. Biochem. 39, 462-477. Takeda, M. & Webster, R. E. (1968). Proc. Nat. Acad. Sci., U.S.A. 60, 1487-1494. Waller, J.-P. (1963). J. Mol. BioZ. 7, 483-496. Webster, R. E., Engelhardt, D. L. & Zinder, N. D. (1966). Proc. Nat. Acad. Sci., U.S.A. 55, 155-161. Wilson, D. B. & Dint&, H. M. (1970). PTOC. Nat. Acad. Sci., U.S.A. 66, 1282-1289. Yoshida, A., Watanabe, S. &Morris, J. (1970). Proc. Nat. Acud. Sci., U.S.A. 67, 1600-1607.
Cleavage of the N-terminal Formymlethionine Residue from a Bacteriophage Coat Protein in vitro L. ANDREW BALL? AND PAVLKAESBEFCQ Biophysics Laboratory and Biochemistry Department University of Wisconsin, Madison, Wise. 53706, U.S.A. (Received 8 March 1973, and in revised form 11 June 1973) Studies were made of the N-terminal formylmethionine content of nascent and complete coat protein of bacteriophage Q/I synthesized in an Eachetichiu coli cellfree system. Under normal conditions of cell-free protein synthesis the formylmethionine residue was retained by all the nascent chains but by only about 50% of the completed coat protein molecules. If 2-mercaptoethanol was omitted from the cell-free system, the formyhnethionine residue ~88 cleaved during the course of peptide chain elongation. All nascent peptides which contained fewer than 40& 5 ammo acids retained the formylmethionine residue. Thereafter, the proportion of nascent peptides lacking the residue increased with peptide length to about 70% for nearly full length nascent peptides and complete released coat protein molecules.
1. Introduction Protein synthesis in Escherichia coli is initiated by the incorporation of a formylmethionine residue as the N-terminal ammo acid of each nascent peptide chain (Clark & Marcker, 1966; Adams & Capecchi, 1966; Webster et al., 1966; Capecchi, 1966). Mature E. coli proteins have various amino acids at their N termini (Waller, 1963) as a result of the post-translational cleavage of the initiating fMet residue. This occurs by deformylation (Adams, 1968; Takeda & Webster, 1968; Livingstone t Leder, 1969) which is followed, in the case of 55% of the E. coli proteins, by aminopeptidase cleavage of the methionine residue. Action of the deformylase, which, in vitro, is a very unstable enzyme strongly inhibited by thiol reagents (Adams, 1968) is essential for subsequent aminopeptidase cleavage (Takeda & Webster, 1968). The coat protein of bacteriophage f2 undergoes both reactions during its synthesis on the ribosome; in phage-infected cells, nascent coat peptides lose their N-terminal methionine when they are 40 to 60 amino acids long (Housman et al., 1972). However, during the synthesis of f2 coat protein in vitro a large proportion of molecules retain their fMet even after release from the ribosome (Lodish & Robertson, 1969). This can be ascribed at least in part to inhibition of the deformylase by the thiol reagents which are included routinely in cell-free protein synthesizing systems. The coat protein of bacteriophage Qj also largely retains its N-terminal fMet residue when synthesized in vitro (Osborn et al., 1970). Since the protein contains no internal methionine residues (Konigsberg et al., 1970) it provides an ideal system for 7 Present address: National Institute England.
for Medical Research, Mill Hill, London NW7 IAA, 531
532
L.
A. BALL
AND
P. KAESBERG
quantitative studies of fMet cleavage. We have therefore investigated the fate of the fMet residue during synthesis of Qfl coat protein under various conditions in vitro, in an attempt to clarify this process of post-translational modification.
2. Materials and Methods All the materials 1973).
and methods used have been described previously
(Ball & Kaesberg,
3. Remits Extent of fomylmethionine
cleavage in vitro
The products of cell-free protein synthesis directed by Q/?RNA and doubly labeled with [35S]methionine and [3H]lysine were analyzed by electrophoresis on 10% polyacrylamide gels (Fig. 1). The three proteins synthesized in vitro correspond to polypeptides 1 (a subunit of the viral RNA replicase), llb (a minor component of the virion), and 111 (the major coat protein (Jockusch et al., 1970)). All three contained 35Slabel which, in the case of the coat protein, indicated the presence of an N-terminal fMet residue on some of the molecules. However, it is not possible to calculate the exact methionine content of the coat protein without knowledge of the relative specific activities of the two isotopes used in the cell-free system. This can be measured from the ratio of 3H to 35S in the nascent coat peptides. Each nascent peptide can contain a maximum of one methionine residue, but its content of the other amino
Distance migrated (mm)
Pm. 1. Sodium dodecyl sulf8te/polyscrylamide gel electrophoresis of the proteins synthesized by Qfi RNA in a cell-free system. The proteins were doubly labeled with [93]methionine (-----) ) (26 @/ml) for 30 min. After the treatment described (68 @i/ml) 8nd [3H]lysine ( previously (B811 & Keesberg, 1973) sn amount corresponding to 26 ~1 of the origin81 reection mixture was spplied to 8 10% polyaorylamide gal, 80 mm in length, containing 0.1 o/0 sodium dodecyl sulfate and 6 M-urea, Electrophoresis was at 20 V and 3.6 mA/gel for 17 h.
fMET
CLEAVAGE
IN
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833
acid depends on its length. For example, peptides up to 23 residues long (including the fMet) till contain no proline since the 6rst proline residue occurs at position 23 in the chain (Konigsberg et al., 1970). Those 24 to 23 long will contain one proline, those 29 to 42, two prolines and so on. Thus the ratio of [3H]proline to [35S]methionine will increase in unit steps as the peptide chain grows, the height of these steps indicating the relative specific activities of the labeled amino acids used in the cell-free system. Any cleavage of the fMet will result in peptides with anomalously high ratios of proline to methionine. Pulse-chase experiments showed that during the first four minutes of cell-free protein synthesis more than 90% of the translation directed by Qj3 RNA occurs from the coat protein gene (unpublished observations). The products of a short in vitro synthesis directed by Q/I RNA were labeled with [35S]methionine and [3H]proline. One fraction was precipitated with 10% trichloroacetic acid in preparation for gel electrophoresis ; another was subjected to sucrose density gradient centrifugation as described in a previous paper (Ball C Kaesberg, 1973). The ribosome-bound nascent peptides (70 S) and the released chains (< 10 S) were precipitated separately in preparation for gel electrophoresis. After hydrolysis of peptidyl-tRNA at pH 11.0, the products were subjected to electrophoresis on highly cross-linked 12.5% polyacrylamide gels in the presence of O*1o/osodium dodecyl sulfate (Swank ik Munkres, 1971). These gels resolve peptides according to their molecular weight in the range 2000 t#o 20,000, and are therefore well suited to the study of the nascent peptides of Qj3 coat protein (molecular weight 14,050). The gels were calibrated with the peptides resulting from cyanogen bromide cleavage of lysozyme, myoglobin and cytochrome c, and the calibration was refined using the N-terminal peptides of Q,fl coat protein as described in Figure 3 of Ball & Kaesberg (1973). Figure 2 shows the ratio of proiine to methionine across the region of the gels containing the coat protein and its nascent peptides. The ratio for the products not subjected to sucrose gradient fractionation (Fig. 2(a)) rises a series of steps with increasing molecular weight of the nascent peptides. The steps which indicate entry of the first six (of the total of eight) proline residues are clearly defined, and the ratio of 3H to 35Sat each stage corresponds exactly to that predicted from the nominal specific activities and measured counting efficiencies of the labeled amino acids. This suggests that there was a negligible pool of unlabeled amino acids in the cell-free system during these short reaction times. In the region of the gel which contains the complete coat protein, the ratio of proline to methionine rises sharply to a value of 13.5f0.5, corresponding to 41&3”,/0 f Met cleavage. Fractionation of the products into nascent and complete chains shows that almost all this fMet cleavage occurs after termination of the coat protein and its release from the ribosome. The ratio of proline to methionine in the nascent chains rises in eight steps to a value of 8.5f0.5 ((6f6)% fMet cleavage) for the complete peptides still bound to ribosomes (Fig. 2(b)). On the other hand, the released coat protein has lost 45% of its fMet which fully accounts for the cleavage observed in Figure 2(a). These results show that not only is the E. cc.%cell-free system partially deficient in the enzyme(s) responsible for fMet removal, but also that the limited cleavage which is observed occurs only after chain termination. This contrasts with the situation in f2 phage-infected cells, where nascent coat peptides lose their terminal fMet residues when they are forty to sixty amino acids long (Housman et al., 1972). 3:I
L. A. BALL
534
AND
P. KAESBERG
Takeda & Webster (1968) showed that modification of the N termini of E. coli proteins by aminopeptidase cleavage of the fMet residue was dependent on prior removal of the formyl group. The deformylase responsible for this reaction in E. coli is strongly inhibited by thiol reagents such as 2-mercaptoethanol (Adams, 1968) which are routinely included during the preparation and reaction of cell-free protein synthesizing systems. It is possible, therefore, that the deficient removal of fMet ~YL vitro is due to the presence of thiol reagents. Cell-free extracts of E. coli which were prepared and assayed in the absence of thiol reagents were capable of active protein synthesis in response to Q/I RNA. Initially, t.heir activity was about 50% of that of routine S30 extracts (prepared, stored and assayed in the presence of 10 mm-2-mercaptoethanol) but they lost their activity
1
28 ,
23
I lN&#b 0 ’ I I I I %.& i
2
30
40
50
60
70
80
90
Distance migrated (mm)
FIQ. 2. Ratio of sH to ?S label across the gel regions containing Qp coat protein and incomplete coat peptides. QB RNA-directed products were doubly labeled in vitro with [s6S]methionine (68 &i/ml) and [sH]proline (25 &i/ml) for 3 min. The molar ratio of proline to methionine was calculated from the specific activities and counting efficiencies of the isotopically labelled amino acids used in the cell-free system. (a) Total products. After the treatment described previously, an amount corresponding to 100 c;l of the original reaction mixture was applied to a 12.5% polyacrylamide gel, 110 mm in length. (b) Fractionated products. -a-•--, Ribosome-bound peptides ; --O--O--, released peptides. Samples (150 ~1) of the untreated reaction mixture were applied to two 6% to 20% sucrose density gradients and centrifuged at 36,000 revs/min for 2 h as described previously (Ball & Kaesberg, 1973). Fractions containing the ribosome-bound peptides (-70 S) and the released chains (< 10 S) were pooled separately and precipitated with 10% trichloroacetic acid, After treatment, amounts corresponding to 160 ~1 of the original reaction mixture were applied to 125% polyaorylamide gels, 110 mm in length. Electrophoresis was at 100 V 8nd 4.0 mA/gel for 13 h. The solid arrows indicate the steps which correspond to the entry of individual proline residues into the growing peptide chain. Their numbers refer to the amino acid positions (Konigsberg et al., 1970). The dotted arrows indicate the position of the complete coat protein.
fMET
IN
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VITRO
535
more rapidly during storage at -70°C. The proteins made by the two types of system in response to Qfl RNA were labeled with [14C] and [3H]lysine, respectively, and analyzed by co-electrophoresis on a 10% polyacrylamide gel. There was no detectable difference between the products, showing that 2-mercaptoethanol was not essential for accurate cell-free protein synthesis. The products of a short in vitro synthesis directed by Qfi RNA in these two systems were labeled with [35S]methionine and [3H]lysine. After hydrolysis of peptidyl-tRNA at pH 11.0, the products were subjected to electrophoresis on 125% polyacrylamide gels. Figure 3 shows the ratio of lysine to methionine across the gel regions which contain the coat protein and its nascent peptides. The products made in the presence of 2-mercaptoethanol have a ratio of lysine to methionine which rises in a series of steps with increasing molecular weight of the nascent peptides. As shown in Figure 2(a), fMet cleavage occurs only from the peptides which migrate with complete coat protein molecules. The products made in the absence of 2-mercaptoethanol show very different ratios of lysine to methionine (Fig. 3). For most of the nascent peptides, the ratio is anoma-
Peptide size (armno acid residues)
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Distance
migrated (mm)
Fm. 3. Ratio of 3H and 35S label across the gel regions containing Qfl coat protein and inoomplete coat peptides. -e-O--, Products made in the presence of 10 mm-2-mercaptoethanol; --O--O--, products made in the absence of 2-mercaptoethanol. QB RNA-directed products were doubly labeled in vitro with [35S]methionine (08 @i/ml) and C3H]lysine (25 &i/ml) for 3 min. The molar ratio of lysine to methionine was calculated from the specific activities and counting efficiencies of the isotopically labeled amino acids used in the cell-free system. After the treatment described previously, amounts corresponding to 60 d of the original reaction mixtures were applied to 12.5% polyacrylamide gels, 110 mm in length. Electrophoresis was at 100 V and 4.0 mA/gel for 15 h. The dotted arrow indiaates the position of complete coat protein, established by ooelectrophoresis in a parallel gel. The upper horizontal scale shows the molecular weight calibration of the gels determined as described in Fig. 3 of Ball & Kaesberg (1973).
L. A. BALL
536
AND
P. KAESBERG
lously high, indicating fMet cleavage. Only those peptides with R, values greater than 2.0 (relative to Q/3coat protein) fully retain their N-terminal methionine residues. Accurate calibration of the gels by the method described previously (Ball 6 Kaesberg, 1973) shows that this R, value corresponds to a peptide containing 40-&5 amino acids. In the absence of 2-mercaptoethanol, when nascent peptides reach this size in vitro, a substantial proteolytic cleavage of their terminal methionine residues occurs (Fig. 4).
I
0
9-4 -... Lx . 0 0 I I 0
20
40
. ...-.-.-.-.
-0-e
I
I
I
60
80
100
.-.-.
I
I
120 131
Nascent pepttde chain length (ammo acid residues)
FIG. 4. The rel8tion between the chain length of the nascent C&3 coat peptides made in vitro 8nd the cleav8ge of their N-tannin81 methionine residues (calcultlted from the dat8 shown in Fig. 3). (0) Peptides synthesized in the presence of 10 mM-%mercsptoeth&nol; (0) peptides synthesized in the absence of %merceptoethanol.
4. Discussion In the experiments described above, calculation of the methionine content of Q/I coat protein and its nascent peptides depends critically on the specific radioactivities of the labeled amino acids used in the cell-free system. The height of the individual steps in the molar ratios which correspond to the entry of single proline or lysine residues (Figs 2 and 3) provides an internal check on the accuracy of this calculation. We can therefore conclude that the N-terminal methionine is cleaved from a significant proportion of the Q/I coat protein molecules made in an E. coli cell-free system. This contrasts with the generally accepted view, based on the translation of f 2 RNA, that proteins made in vitro fully retain their terminal methionine residues (Lengyel & Sdll, 1969; Lodish $ Robertson, 1969). This disagreement may reflect differences in the cell-free systems, or differences between the translation of Qfi and f2 RNAs. In the presence of 2-mercaptoethanol, significant methionine cleavage occurs only after peptide chain termination, whereas in the absence of thiol reagents cleavage occurs during elongation. This suggests that 2-mercaptoethanol inhibits an enzyme which is necessary for early methionine removal-probably the peptide deformylase (Adams, 1968). The lack of cleavage from nascent peptides in the presence of
fMET
CLEAVAGE
IN
VITRO
537
2-mercaptoethanol probably reflects the slow reaction rate of the inhibited enzyme rather than a change in its substrate specificity. In the absence of 2-mercaptoethanol, nascent peptides become substrates for the aminopeptidase when they are about 40 amino acids long. This resembles the situation in f2 phage-infected cells, where cleavage occurs when the nascent coat peptides contain 40 to 60 residues (Housman et al., 1972). Similarly, removal of the N-terminal methionine from rabbit globin in vivo occurs when the nascent peptides are about 30 (Yoshida et al., 1970) or 40 to 50 residues in length (Wilson & Dintzis, 1970). Moreover, reticulocyte ribosomes protect chains of up to 35 amino acids from digestion by trypsin or pronase (Rich et al., 1966). Since the aminopeptidase which is probably responsible for methionine cleavage in E. coli is not bound to ribosomes (Brown & Krall, 1971), it seems likely that similar shielding by bacterial ribosomes protects the fMet residues of nascent phage coat peptides until they are about 40 amino acids long. We thank Wendy Good and Steve Kagen for invaluable technical assistance. This work was supported by grants from the Institute of Allergy and Infectious Diseases, National Institutes of Health, and from the Biology Division of the U.S. Atomic Energy Commission. REFERENCES Adams, J. M. (1968). J. Mol. Biol. 33, 571-589. Adams, J. M. & Capecchi, M. R. (1966). Proc. Nat. Ad. Sci., U.S.A. 55, 147-155. Ball, L. A. & Kaesberg, P. (1973). J. MOE. BioE. 74, 547-562. Brown, J. L. & Krall, J. F. (1971). Biochem. Biophys. Res. Commun. 42, 390-397. Capecchi, M. R. (1966). Proc. Nat. Acad. Sci., U.S.A. 55, 1517-1524. Clark, B. F. C. & Marcker, K. A. (1966). J. Mol. BioZ. 17, 394-406. Housman, D., Gillespie, D. & Lodish, H. F. (1972). J. Mol. B&Z. 65, 163-166. Jockusch, H., Ball, L. A. & Kaesberg, P. (1970). v’irology, 42, 401-414. Konigsberg, W., Maita, T., Katze, J. & Weber, K. (1970). Nature (London), 227, 271-273. Lengyel, P. & 5611, D. (1969). Bacterial. Rev. 33, 264-301. Livingstone, D. M. & Leder, P. (1969). Biochemktry, 8, 435-443. Lodish, H. F. & Robertson, H. D. (1969). J. Mol. BioZ. 45, 9-22. Osborn, M., Weber, K. & Lodish, H. F. (1970). Biochem. Biophys. Res. Commun. 41, 748-756. Rich, A., Eikenberry, E. F. & Malkin, L. I. (1966). Cold Spring Harbor Symp. Quant. BioZ. 31, 303-310. Swank, R. T. & Munkres, K. D. (1971). Anal. Biochem. 39, 462-477. Takeda, M. & Webster, R. E. (1968). Proc. Nat. Acad. Sci., U.S.A. 60, 1487-1494. Waller, J.-P. (1963). J. Mol. BioZ. 7, 483-496. Webster, R. E., Engelhardt, D. L. & Zinder, N. D. (1966). Proc. Nat. Acad. Sci., U.S.A. 55, 155-161. Wilson, D. B. & Dint&, H. M. (1970). PTOC. Nat. Acad. Sci., U.S.A. 66, 1282-1289. Yoshida, A., Watanabe, S. &Morris, J. (1970). Proc. Nat. Acud. Sci., U.S.A. 67, 1600-1607.