Vol. 127, No. 2

JOURNAL OF BACTERIOLOGY, Aug. 1976, p. 923-933 Copyright ©D 1976 American Society for Microbiology

Printed in U.S.A.

Genes for the a and ,B Subunits of the Phenylalanyl-Transfer Ribonucleic Acid Synthetase of Escherichia coli M. MARGARET COMER* AND AUGUST BOCK Lehrstuhl fur Mikrobiologie, Universitat Regensburg, D-8400 Regensburg, Federal Republic of Germany

Received for publication 10 May 1976

The phenylalanyl-transfer ribonucleic acid synthetase of Escherichia coli is a tetramer that contains two different kinds of polypeptide chains. To locate the genes for the two polypeptides, we analyzed temperature-sensitive mutants with defective phenylalanyl-transfer ribonucleic acid synthetases to see which subwas altered. The method was in vitro complementation; mutant cell exwere mixed with purified separated a or /8 subunits of the wild-type enzyme to generate an active hybrid enzyme. With three mutants, enzyme activity appeared when a was added, but not when was added: these are, therefore, assumed to carry lesions in the gene for the a subunit. Two other mutants gave the opposite response and are presumably mutants. Enzyme activity is also generated when a and /8 mutant extracts are mixed, but not when two a or two /8 mutant extracts are mixed. The inactive mutant enzymes appear to be dissociated, as judged by their sedimentation in sucrose density gradients, but the dissociation may be only partial. The active enzyme generated by complementation occurred in two forms, one that resembled the native wild-type enzyme and one that sedimented more slowly. Both a and /8 mutants are capable of generating the native form, although a mutants require prior urea denaturation of the defective enzyme. With the mutants thus characterized, the genes for the a and subunits (designated pheS and pheT, respectively) were mapped. The gene order, as determined by transduction, is aroDpps-pheT-pheS. The pheS and pheT genes are close together and may be

unit

tracts

immediately adjacent.

Although the aminoacyl-transfer ribonucleic acid (tRNA) synthetases all catalyze essentially the same reaction, they differ greatly in their molecular weights and types of subunit structure. In Escherichia coli these enzymes are mostly monomers or aggregates of identical subunits (12); three, however, contain two kinds of polypeptide chains. In the latter cases, it is of interest to know whether the genes for the two proteins are transcribed together and regulated as an operon or whether they are completely independent. One of these enzymes, the dimer glutamyltRNA synthetase, is dependent upon no less than three genes, which lie in widely separated places on the bacterial chromosome (14). The two genes for the tetrameric enzyme glycyltRNA synthetase lie in more or less the same place (6, 19), but detailed genetic studies have not been reported. We have studied the third of these enzymes, phenylalanyl-tRNA synthetase. It is a tetramer with a molecular weight of 267,000 and the subunit composition a2932, where a and ,B have molecular weights of 39,000

and 94,000, respectively (5, 9). Two types of mutants have been isolated that are altered in this enzyme, temperature-sensitive mutants and mutants resistant to the analogue p-fluorophenylalanine (11, 18, 22). All of these mutations have been found to lie at about 33 min (3, 22). Two of the p-fluorophenylalanine-resistant mutants have been identified as having altered a subunits (11), but it has not previously been known whether any of the other mutations affected the /8 subunit. In the present work we have identified the defective subunit of the phenylalanyl-tRNA synthetase in several of these temperature-sensitive mutants, and we have used the characterized mutants to determine whether the a and /3 genes are located close together on the bacterial genome. MATERIALS AND METHODS Media. The minimal medium used was salt solution P (7) supplemented with 0.2% (NH4)2SO4 and 0.4% glucose; required amino acids and vitamins were added to a concentration of 40 ,ug and 1 ,ug/ml, 923

J. BACTERIOL.

COMER AND BOCK

924

respectively. Rich medium contained 1% tryptone, 0.5% yeast extract, and 0.2% glucose. Plates contained agar at a concentration of 1.5%. Bacterial strains. Table 1 lists the strains used with their source or derivation. Construction methods included the following. Selection for streptomycin resistance (Strr) was on plates containing 100 ,ug of streptomycin per ml. Selection for p-fluorophenylalanine (pFphe) resistance (and screening for the pheS12 allele when unselected) was on minimal plates containing the D,L form of this analogue at a concentration of 200 ,ug/ml; for aroD6 strains the concentration of L-phenylalanine in these plates was limited to 5 /.Lg/ml. The presence of the pheT354 allele was indicated by failure to grow on rich medium plates incubated at 42°C. The presence of the pps-4 allele was indicated by failure to grow on minimal plates with 0.8% sodium pyruvate as the carbon source. The pheS and pheT markers were checked by assay of the phenylalanyl-tRNA synthetase.

Assay of phenylalanyl-tRNA synthetase. Activity was measured by the attachment of L['4C]phenylalanine to tRNA. Reaction mixtures contained, in a total volume of 0.25 ml: 100 mM

tris(hydroxymethyl)aminomethane - hydrochloride, pH 7.5; 10 mM MgCl2; 10 mM KCl; 2 mM reduced glutathione; 2 mM adenosine triphosphate; 1.6 mg of E. coli tRNA per ml; 20 ,uM L-['4C]phenylalanine, 10 ,uCi/,umol; and the enzyme preparation to be assayed. The mixtures were incubated at 28°C (unless otherwise stated) for 10 min; the reaction was then stopped by chilling and precipitation with 2 ml of 10% trichloroacetic acid. The precipitates were collected on glass-fiber filters and washed with 10% trichloroacetic acid and 70% ethanol; the radioactivity was then measured in a scintillation counter. The background value obtained with no enzyme is subtracted from all data reported. The presence of the phenylalanyl-tRNA synthetase mutations pheS12 (p-fluorophenylalanine resistance) and pheT354 (temperature sensitivity)

TABLE 1. E. coli strains used Genotype

Strain

NP37 NP313

F', F148 his', A(cheB-cheC), cheA+, aroD+lthi-1, his-4, aroD5, proA2, recAl, xyl-5 orxyl-7, nalA12, tsx-i? or tsx-29?, X-, supE44? Hfr (Cavalli), pheS5,a rel-i, tonA22, T2r Hfr (Cavalli), pheS6,a rel-i, tonA22, T2r

NP512

pheS8,a thr-

JP1116 JP5122 K10 AB1360

HfrH, pheT354, thi-, galE-PL5, rel-i F-, pheT353, a arg-, galE-, rel-i Hfr (Cavalli), rel-i, tonA22, T2r F-, thi-i, argE3, his-4, proA2, aroD6, lacYl, galK2, mtl-i, xyl-5, tsx-29, supE44? F-, pheS12,b thi-i, argE3, his-4, proA2, aroD6, lacYI, galK2, mtl-i, xyl-5, tsx-29, supE44? F-, pheSi2,b strA, thi-i, argE3, his-4, proA2, aroD6, tacYI, galK2, mtl-i, xyl-5, tsx-29, supE44? F-, pheS12,b thi-i, argE3, his-4, proA2, lacYI, galK2, mt1-i, xyl-5, tsx-29, supE44? pheT354,a thi-, argE3, proA2, aroD6, strA, probably other parental markers (his+, pheS+)

KLF48/KL159

AB1360-12

MC101 MC102 MC103

MC1440

F-, pheS12,b pheT354,a thi-i, argE3, his-4, proA2, lacYI, galK2, mt1-i, xyl-5, tsx-29, supE44?

MC104

F-, pheSi2,b pheT354,a thi-i, argE3, his-4, proA2, aroD6, tacYI, galK2, mtt-i, xyl-5, tsx-29, supE44?

PA505-1-5

F-, pps-4, metA90, argHI, proA44, aceA4, str9, X-

MC105

F-, pps-4, thi-i, argE3, his-4, proA2, tacYi, galK2,

mt1-i, xyl-5, tsx-29, supE44? MC106 a

b

F-, pheS12,b pheT354, a pps--4, thi-i, argE3, his-4, proA2, lacYI, galK2, mt1-i, xyl-5, tsx-29, supE44?

Temperature-sensitive phenylalanyl-tRNA synthetase. p-Fluorophenylalanine-resistant phenylalanyl-tRNA synthetase.

Source or derivation

K. B. Low via B. J. Bachmann

F. C. Neidhardt (3) F. C. Neidhardt (called EV5 in reference 18) thr- derivative of the Neidhardt strain NP51 (C1-2 in reference 18) R. R. B. Russell (22) R. R. B. Russell (22) Reference 1 A. L. Taylor via B. J. Bachmann (21) Reference 11, 21

Spontaneous Strr mutant of AB1360-12 Spontaneous Aro+ revertant of AB1360-12 Mating: JP1116 x MC101; selection for His+ and Strr Transduction: P1/JP1116 AB1360-12; selection for Aro+ (Table 5) Transduction: P1/MC1440 -AB1360; selection for pFphe resistance H. Kornberg via B. J. Bachmann Transduction: Pl/PA505-15 -. AB1360; selection for Aro+ Transduction: Pl/MC1440 -* MC105; selection for pFphe resistance

VOL. 127, 1976

PHENYLALANYL-tRNA SYNTHETASE GENES

was tested by assay at 28 and 42°C, and at 28°C with DL-['4C]p-fluorophenylalanine instead of L-[14C]-

phenylalanine. Enzyme purification and subunit separation. Wild-type phenylalanyl-tRNA synthetase was purified from strain KLF48/KL159, which was chosen for the purpose of enhancing enzyme yields, since it carries the phenylalanyl-tRNA synthetase genes on both episome and chromosome (20). The subunits were separated as described by Hennecke and Bock (11).

Preparation of mutant cell extracts. Exponentially growing cells were harvested and resuspended in buffer containing 20 mM tris(hydroxymethyl)aminomethane-hydrochloride (pH 7.5), 30 mM NH4Cl, 10 mM MgCl2, 0.2 mM ethylenedinitrilotetraacetic acid, and 6 mM 2-mercaptoethanol (PRS buffer). A 5-,ug amount of deoxyribonuclease was added per ml, and the cells were then disrupted in a French press. Debris was removed by centrifugation at 27,000 x g; the supernatant fluid was then centrifuged for 2 h at 45,000 rpm in a Beckman 75 Ti rotor. The resulting cell extract usually contained 5 to 10 mg of protein per ml, as determined by the Lowry method (15). In vitro complementation. Extracts (50 jig of protein) were mixed with purified a (0.05 ,ug) or /3 (0.125 ,ug) subunits in a total volume of 0.1 ml of PRS buffer; the amounts of subunits are equal in molar terms because of the molecular-weight difference (11). For sucrose gradient centrifugation, the amounts of the reactants were increased, but the proportions were kept the same. The conditions for incubation of these mixtures varied somewhat, and no systematic attempt was made to determine optimal conditions. The variations tried seemed to make no difference except that JP1116 extracts must be exposed to high temperature to inactivate the mutant enzyme. In early experiments (including Tables 2 and 3), the mixtures were incubated at 28°C for 30 min and then at 42°C for 5 min, and finally the enzyme formed was assayed. In later experiments the extracts were usually incubated at 42°C before mixing. Treatment of extracts with urea was for 1 h at room temperature in 4 M urea; the treated extracts were then mixed with subunits and dialyzed overnight against PRS buffer to remove the urea. Sucrose gradient centrifugation. Samples containing 0.5 to 0.9 mg of extract protein in a volume of 0.2 ml were layered on 10-ml sucrose gradients (5 to 20%, in PRS buffer). They were centrifuged at 40,000 rpm for 23 h in a Beckman SW 41 rotor. Then, fractions of 6 drops each were collected, and samples of 50 to 100 ,ul were assayed for enzyme activity. For assays by complementation, 50 ,ul of each fraction was mixed with 0.1 ,ug of a or 0.25 ,4g of /8 in a total volume of 0.1 ml and incubated at 28°C for 30 min; the enzyme activity formed was then assayed. Mapping by transduction. Transduction by phage Plkc was performed as described by Miller (17). For the experiments in Table 5, transductants carrying the donor aroD+ allele were selected on minimal plates containing all required amino acids and vitamins, except for the aromatic ones, and incubated at 30°C. For Table 6, pps+ transductants were selected

925

on minimal plates with sodium pyruvate (0.8%) as the carbon source and incubated at 30°C. The transductants thus selected were purified on the same plates and then tested for the pheS and pheT markers as described above. Transductants in the two least frequent recombinant classes from the Table 5 experiments were examined by enzyme assay to verify their genotypes. In experiment 1 of Table 6, MC105 was used rather than its parent PA505-1-5, because p-fluorophenylalanine resistance and sensitivity is easier to distinguish in the plate test with the AB1360 genetic background.

RESULTS

Identification of mutant subunit. Our procedure was based on the assumption that a mutant with a defective a subunit, for example, would have a normal ,3 subunit, and we anticipated that if purified wild-type a was added to a mutant cell extract it might be possible to generate wild-type enzyme activity. This approach was made possible by the availability of techniques for separating the subunits without permanently inactivating them; a and /8 are inactive alone, but enzyme activity can be regenerated when they are mixed (11). Three of the temperature-sensitive strains tested (NP37, NP313, and NP512) lose phenylalanyl-tRNA synthetase activity very rapidly after the cells are broken, and extracts have essentially no enzyme activity at any temperature (18). Another strain (JP1116) proved to be active in vitro at 28°C, but its enzyme is irreversibly inactivated by a brief incubation at 42°C. It was therefore possible to assay for phenylalanyltRNA synthetase activity under conditions where no activity would be detected unless the added wild-type subunits complemented the mutant enzymes in the extracts to form an active hybrid enzyme. Furthermore, for strains NP37, NP313, and NP512 it had already been demonstrated (13) that such an active enzyme can actually be formed when mutant extracts are combined with an unfractionated mixture of wild-type subunits. Table 2 shows the results of experiments in which purified wild-type subunits were added to crude extracts of the temperature-sensitive strains. No prior denaturation of the mutant enzymes to separate the defective from the active subunits proved necessary; activity was generated simply upon mixing. With three of the mutants (NP37, NP313, and NP512), enzyme activity appeared when a was added, but not with /3; we infer that in these strains the temperature-sensitive lesion is in the a subunit. Conversely, mutants JP1116 and JP5122 yielded activity with ,B but not with a; these are, accordingly, assumed to have altered /3 subunits.

926

COMER AND BOCK

TABLE 2. Complementation ofphenylalanyl-tRNA synthetase mutants with purified wild-type enzyme subunits Enzyme activitya Mutant ex- Extract plus Extract plus (3 a tract alone 408 4,772 326 NP37 129 3,869 122 NP313 129 3,981 104 NP512 2,307 260 186 JP1116 2,305 162 142 JP5122 a Expressed as counts per minute of L['4C]phenylalanine attached to tRNA. Enzyme was assayed at 42°C except for NP37 (280C). The a and /8 subunits separately have no detectable activity under these conditions.

Mutant

Since purified subunits can complement the mutant enzymes, it might be expected that a and 8 mutants should complement each other. As Table 3 shows, this is indeed the case. Two extracts yield enzyme activity when mixed if one is derived from a strain classified in the previous experiment as an a mutant and the other is from a , mutant, but no activity appears upon mixing extracts of two a mutants or two 8 mutants. Analysis of complementation process. The next series of experiments was aimed at characterizing the inactive enzyme present in mutant extracts and determining whether the active products of complementation resembled the native wild-type enzyme. The method chosen was centrifugation in sucrose density gradients. Although the , mutant JP1116 is active in vitro at 280C, after centrifugation no enzyme activity could be detected anywhere in the gradient; this mutant enzyme is therefore inactivated in the course of centrifugation. The inactive enzyme material containing the normal a subunit was located by complementation with wild-type 8 subunits. It sediments much more slowly than the native enzyme (Fig. 1A). A rough calculation (16) of the molecular weight of this material, based solely on its mobility relative to that of the native enzyme, suggests that it is on the order of twice the size of the a monomer (molecular weight, 90,000, as opposed to 39,000 for a; 9). It is therefore possible that this inactive enzyme fragment contains more than one subunit. For technical reasons we have unfortunately not been able to determine the sedimentation mobilities of the purified subunits for comparison. When the JP1116 extract was mixed with 8 and the active enzyme thus formed was analyzed by sucrose gradient centrifugation, two

J. BACTERIOL.

components with enzyme activity appeared (Fig. 1B and C). One of these (component II) sedimented in the same position as the native enzyme; JP1116 therefore contains everything necessary to form an apparently wild-type enzyme, except for the p subunit. The identity of the other active component (I) is less obvious. It sedimented in an intermediTABLE 3. Complementation between different mutants Enzyme activitya

a Mutants NP37 NP313 NP512

/ Mutants

a Mutants

Mutant NP37

NP313

_b 92 129

139

NP512 JP1116 JP5122

-

,B Mutants JP1116 5,252 6,473 6,858 JP5122 5,790 7,520 7,457 304 a Counts per minute of L['4C]phenylalanine attached to tRNA by mixed extracts; assayed at 42°C. -, See values for individdal mutant extracts alone in Table 2.

FRACTION NUMBER FIG. 1. Inactive enzyme of a /3 mutant and active complementation products. Extracts were fractionated by sucrose gradient centrifugation; the top ofthe gradient is at the left. (A) JP1116 extract alone, assayed by complementation with purified /3. (B) JP1116 extract complemented with / as in Table 2 before centrifugation; standard assay. (C) Like (B), but with 10 times as much /3. PRS marks the position of purified wild-type phenylalanyl-tRNA synthetase.

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PHENYLALANYL-tRNA SYNTHETASE GENES

ate position between the native enzyme and the inactive a-containing material of JP1116 (Fig.

1A) and has a molecular weight (calculated as above) of roughly 150,000. When the ratio of 18 to extract protein was the same as in the complementation experiments of Table 2, the two components appeared in approximately equal amounts (Fig. 1B). But when the amount of ,B was increased by a factor of 10 (Fig. 1C), most of the activity was in the position of native enzyme; furthermore, the total activity was much higher (note the difference in scales). The missing activity in Fig. 1B can be located by assay of the fractions by complementation with added f8; a third component appeared in the position of the inactive JP1116 material (not shown). It would therefore appear that the amount of 8 was limiting in the experiment of Fig. 1B, and that the formation of the component I active enzyme is favored by such conditions. The a mutant NP37 is analyzed in Fig. 2. For assay of the inactive extract alone, the fractions were complemented with wild-type a; this method presumably locates material containing the normal /8 subunit. A single component is seen (Fig. 2A) that sediments considerably more slowly than the native phenylalanyltRNA synthetase but faster than the corresponding JP1116 product (Fig. 1A). Its molecular weight is roughly 130,000, or one-halfthat of the native enzyme; /8 alone has a molecular weight of 94,000 (9). An immunological assay has also shown that the inactive enzyme material in NP37 is about one-half the size of the wild-type phenylalanyl-tRNA synthetase (2). The active product formed by complementation of NP37 with wild-type a is shown in Fig. 2B. It is not identical to the native enzyme but sedimented slightly faster than the inactive NP37 material shown in Fig. 1A; the difference, although small, was reproducible. Furthermore, when the gradients of Fig. 1B and 2B were centrifuged together, it became apparent that the position of the NP37 complementation product coincided with that of component I of the JP1116 complementation product. No material corresponding to JP1116 component II could be detected, however, even when the amount of was increased (not shown). These results are not unique to the NP37 mutant; when the same experiment was done with NP512, an a mutant derived from a different parent strain (F. C. Neidhardt, personal communication), a peak identical to that of Fig. 2B is obtained (not shown). Despite the fact that the NP37 complementation product and the JP1116 component I product sediment in the same position, they are not a

I

4000

-

I

I

I

I

I

927

I

A. NP37

3000 I-

A

PRS

2000

E

1000

C-) I

P*CD < LL lL

2000

Ba NP37 + (X

1500 1000

500

N

z ll

C. NP37ureav + 1200 900 600 300

I

I I I

I

A

N -~

2 4 6 8 10 12 14 16 18 20 22 24

26

I

-

28

FRACTION NUMBER FIG. 2. Inactive enzyme of an a mutant and active complementation products. Extracts were fractionated by sucrose gradient centrifugation; the top of the gradient is at the left. (A) NP37 extract alone, assayed by complementation with purified (B) NP37 extract complemented with a as in Table 2 before centrifugation; standard assay. (C) Urea-treated NP37 extract complemented with a; standard assay. PRS marks the position of purified wild-type phenylalanyl-tRNA synthetase. a.

exactly identical; a comparison of enzyme activities at low and high temperatures reveals a difference (Table 4). Wild-type phenylalanyltRNA synthetase is more active at 42°C than at 28°C, as are both JP1116 complementation products. The NP37 product, however, displays a certain amount of residual temperature sensitivity (see also reference 13); this fact suggests that a temperature-sensitive subunit is incorporated into the complementation product. Supporting evidence for this hypothesis is presented by the finding (Hennecke and Bock, unpublished data) that, when an NP37 extract was complemented with purified p-fluorophenylalanine-resistant subunits, the resulting active enzyme behaved as if it contained both a temperature-sensitive and a p-fluorophenylalanine-resistant a subunit. Such a result would be explained if, when the NP37 enzyme was inactivated, the temperature-sensitive subunit remained firmly bound to the wild-type ,8 subunit, for instance as an ae8 dimer. The calculated molecular weight of the material in Fig. 2A (130,000) is not inconsistent with that expected for such a dimer (a + /8 = 133,000). a

a

928

COMER AND BOCK

J. BACTERIOL.

TABLE 4. Residual temperature sensitivity of some complementation products Enzyme activitya Enzyme material

28°C

42 °C

42°C 28°C

(%)

Wild-type enzyme 2,552 3,199 +27 Complementation products JP1116 extract + 'ab 2,336 3,445 +47 Component Ic 1,171 1,672 +43 Component Ilc 985 1,345 +37 NP37 extract + ab 3,934 3,481 -12 Single component" 1,925 1,509 -22 (NP37 extract),,,, + a Component IIC 1,063 1,450 +36 +6 JP1116 extract + NP37 5,038 5,328 extractb a Expressed as counts per minute of L['4C]phenylalanine attached to tRNA at the indicated temperature. b Complementation performed as for Tables 2 and 3. ' Fractions from sucrose gradients; see Fig. 1B, 2B, and 2C.

If the NP37 enzyme really contains a single mutation in the a protein and has a wild-type ,3 subunit, then if wild-type a is added everything necessary to form a wild-type enzyme should be present. Since we did not find any upon simply mixing mutant extract and subunits (Fig. 2B), we treated the extract first with urea to dissociate the mutant enzyme and then added a (Fig. 2C). Most of the enzyme activity thus generated sedimented in the position of the native enzyme (component II); this material, furthermore, has the temperature stability of the wild-type enzyme and not the residual temperature sensitivity of the product obtained without urea (Table 4). We interpret these results as indicating that the urea separates the temperature-sensitive a subunit from /3 and thus permits the formation of completely wildtype enzyme. Urea treatment also generated a small amount of a second component (I) that was enzymatically active and sedimented more slowly than the complementation product formed without urea. Its position, in fact, coincides with the peak in Fig. 2A, which represents inactive material that was detected only by complementation with added a. If the latter is ats,8, as speculated above, then we can speculate further that component I is wild-type a,/; this dimer could well combine in the assay tubes to form active a2,/3. Other explanations for this material are possible, however, and it remains unidentified.

In Table 3 it is shown that active enzyme could be generated not only by adding subunits to extracts of mutant strains, but also by mixing extracts of a and ,B mutants. Figure 3 shows the sucrose gradient analysis of the product thus formed. It consisted of a single component that sedimented not in the position of the native enzyme but in the same position as the (NP37 + a) product and the (JP1116 + /3) component I product. The activity of this material at 42°C is shown in Table 4; it seems to have some residual temperature sensitivity, but the result is not as clear as for the (NP37 + a) product. Since treatment of the NP37 extract with urea before addition of a permitted the formation of enzyme resembling wild type, one might expect that mixing urea-treated NP37 extract with JP1116 extract (treated or untreated) would also generate wild-type enzyme; in preliminary experiments we have indeed observed such enzyme material, at least in small amounts. In summary, we conclude that (i) the inactive mutant enzymes are dissociated, but probably not completely to monomers; (ii) the active enzyme formed by the simple mixing of mutant extracts and subunits consists partly or entirely of material that sediments more slowly than the native phenylalanyl-tRNA synthetase; and (iii) it is, nevertheless, evident that the mutants contain one good subunit that can contribute to the formation of enzyme that resembles wild type. Size of the mutant a and 18 polypeptide chains. One possible explanation for the active enzyme that sedimented more slowly than normal is that it contained mutant subunits that were smaller than wild type, e.g., as a result of chain-terminating nonsense mutations. The

0.

I

-U

>. 2000 t

-

I

I

I

JP1116 + NP37

> 1500 I() 1000

PRS

LU 500 z LL

FRAC' 'N

2 4 6 8 10 2 14 16 18 20 22 24 26 28

FRACTION NUMBER

FIG. 3. Active complementation products formed by mixing mutant extracts. NP37 and JP1116 extracts were mixed as in Table 3 and fractionated by sucrose gradient centrifugation; the top of the gradient is at the left. PRS marks the position ofpurified wild-type phenylalanyl-tRNA synthetase.

PHENYLALANYL-tRNA SYNTHETASE GENES

VOL. 127, 1976

929

fact that the mutants are temperature sensitive ping data, the mutations were all originally suggests that the mutations are missense assigned to the same gene (pheS); we propose rather than nonsense but is not conclusive that the pheS designation be retained for the a proof. The possibility has been directly elimi- gene and that the ,B gene be called pheT. Both nated, however, by an analysis of the size of the pheS and pheT map at about 33 min and comutant polypeptide chains. For this purpose, transduce with aroD (32 min); the genes aroD, the mutant enzymes were recovered from cell pps, and pheT are arranged in that order (3, extracts by precipitation with antiserum 22). The experiments in this section were deagainst wild-type phenylalanyl-tRNA synthe- signed to establish the order ofpheS and pheT tase and then analyzed by electrophoresis on in relation to the other genes in the area and to sodium dodecyl sulfate-polyacrylamide gels provide an estimate of how close the two genes (20). Figure 4 shows the results; both the a and are. For this purpose we used three-factor transthe , chains of all mutant enzymes migrated in positions indistinguishable from those of the ductional crosses mediated by phage P1. To wild-type subunits. The mutant subunits, distinguish the a and ,3 genes, we used a ptherefore, are at least approximately the same fluorophenylalanine-resistant mutation previously shown (11) to affect the a subunit size as the wild type. Map location of the a and f3 genes. Previous (pheS12) along with one of the temperaturework with the mutants whose defective sub- sensitive /3 mutations (pheT354; JP1116); the units have now been identified indicates that genotype of the transductants with respect to the a and /8 genes are in roughly the same the phenylalanyl-tRNA synthetase markers region of the map. Because of the similar map- could thus be determined simply by their

..

r

L 8 9 10 11 12 13'14 "I617 18 19 FIG. 4. Electrophoresis of immunoprecipitates of wild-type and mutant phenylalanyl-tRNA synthetases. Enzyme was precipitated from cell extracts with antiserum from rabbits immunized against wild-type phenylalanyl-tRNA synthetase; the washed immunoprecipitates were then solubilized and analyzed on a sodium dodecyl sulfate-polyacrylamide slab gel (20). The figure shows a photograph of the stained gel. Lanes 1 and 3 show the pattern of 15- and 25-,l immunoprecipitates of strain K10, lanes 4 and 6 those of strain NP37, lanes 7 and 9 those ofstrain NP313, lanes 10 and 12 those of strain NP512, lanes 13 and 15 those of strain JP1116, lanes 16 and 18 those ofstrain K10, and lanes 19 and 21 those ofstrain JP5122. Lanes 2, 5, 8, 11, 14, 17, and 20 give the electrophoresis position of the a and 8 subunits of purified phenylalanyl-tRNA synthetase from wild-type K10. H and L mark the heavy and light chains of immunoglobulins, respectively.

930

J. BACTERIOL.

COMER AND BOCK

growth on plates containing p-fluorophenylalanine or incubated at high temperature. In the first set of experiments the donor was aroD+ and the recipient was aroD-; transductants that had acquired the aroD+ allele were selected and then tested for the unselectedpheS and pheT markers. The order of the genes can be inferred from the relative frequencies of the various possible recombinants. As the diagrams in Fig. 5 show, one type can only be generated by multiple recombination, and this type is different for the two possible gene arrangements (A and B); the identity of the least frequent recombinant should thus reveal which arrangement is correct. Moreover, when the donor and recipient are reversed with respect to the pheS and pheT markers (experiment 2),

the predictions for the least frequent recombinants are also reversed. Table 5 shows the results of such transductions. Experiments 1 and 2 are those diagrammed in Fig. 5; experiments 3 and 4 are a similar pair with a different arrangement of alleles. These data show that pheT was cotransduced with aroD at an average frequency of 69%, and pheS was co-transduced with aroD at an average frequency of 67%; we obtained a similar result with a temperature-sensitive pheS mutant (not shown). Earlier published work with the same temperature-sensitive strains has yielded values of 44% (22) for pheT and 33% (3) and 66% (23) for pheS. We do not know why there is so much variation; perhaps the genetic background of the recipient

Experiment 1

Experiment 2

pheTts

aroD

pheS'

aroD'

pheSpfr

pheT

ii I I

A

I

_

II

I ~~~~~I

I

aroD

pheT

pheSpfr

aroD-

pheTts

pheS'

aroD'

pheS

pheTts

aroD

pheSpr

peT

II

B

_

I

~~~~~~~

-

aroD-

pheSpfr

I

I ..

pheT+

aroD-

pteS

pheltS

FIG. 5. Rationale for mapping the and genes. (A) and (B) show the two possible gene arrangements, where pheS designates the gene and pheT the gene. In each diagram the upper line indicates the genotype of the donor in transduction and the lower line that of the recipient. Transductants carrying aroD+ are selected and then tested for the unselected markers; ts indicates a temperature-sensitive mutation and pfr indicates a pfluorophenylalanine-resistant mutation. The broken line indicates the genotype of the transductant expected to be least frequent in each case because of the necessity for multiple recombination. a

a

TABLE 5. Evidence for the gene order aroD-pheT-pheS aroD+ transductants

Expt 1 Expt 2 Expt 3 aroD+ pheT354 pheS+ a aroD+ pheT+ pheS12a aroD+ pheT354 pheS12a aroD6 pheT+ pheS12°

No.

pheT354 pheS+

%

aroD6 pheT354 pheS+b No.

%

aroD6 pheT+ pheS+6

No.

%

Expt 4 aroD+ pheT+ pheS+a

aroD6 pheT354 pheS12b No.

9

36.6 5 1.7 0 0 66.8 631 110 0 60.0 9 3.0 pheT+ pheS12 31.8 0 301 180 212 82 27.4 pheT354 pheS12 0.7 70.6 8 0.8 2 83 69.7 pheT+ pheS+ 0.5 2.7 27.7 209 5 8 a Donor strains in experiments 1 through 4, respectively: JP1116, MC102, MC1440, and K10. b Recipient strains in experiments 1 through 4, respectively: AB1360-12, MC103, AB1360, and MC104.

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PHENYLALANYL-tRNA SYNTHETASE GENES

strain makes a difference. Nevertheless, we can conclude from the similarity of the co-transduction frequencies for pheS and pheT in the experiments reported here that these two genes are close together relative to the distance between them and aroD. It is evident from the data in Fig. 5 that transductants that inherited their pheS allele from one parent and the pheT allele from the other are rare; this result confirms the earlier data placing the two genes on the same side of aroD (at 33 min), since if the order were pheS-aroD-pheT such recombinants could be generated as easily as any others. In experiments 1 and 2 the least frequent transductant types are those shown in the diagrams of Fig. 5 for the gene order A: aroDpheT-pheS. The fact that reversing the parents with respect to the pheS and pheT alleles reversed the transductant frequencies, as expected, implies that the frequency differences are really a function of the gene order and are not the result of some artifactual elevation or depression of the frequencies of certain recombinant types. Experiments 3 and 4 likewise gave the results predicted for the order aroDpheT-pheS. Furthermore, experiment 1 has been repeated with a different pheT mutant (JP5122), with similar results (not shown). Combining this result (aroD-pheT-pheS) with the earlier determination of the gene sequence aroD-pps-pheT (22) gives the overall order aroD-pps-pheT-pheS. Additional evidence for this arrangement was obtained from transductions with pps, pheT, and pheS (Table 6). These experiments are exactly analogous to experiments 3 and 4 of Table 5, except that the selected marker was pps+ instead of aroD+. Predictably, pheT and pheS were co-transduced with pps at a higher average frequency than with aroD: 88% for pheT and 87% for pheS. The possible order pheS-pps-pheT can be excluded, because with such high co-transduction frequencies the least frequent transductant would be that with both phe genes derived from the recipient and only the pps+ from the donor; in fact, this is the second most frequent recombinant class. The remaining two possible orders (pps-pheS-pheT and pps-pheT-pheS) can be distinguished by the argument outlined in Fig. 5. In both experiment 1 and experiment 2 the least frequent recombinant class is that expected for the order pps-pheT-pheS, and reversing the donor and recipient with resp1ect to the phe markers also reversed the recombinant frequencies. The data are, therefore, all completely consistent and indicate the gene arrangement aroD-pps-pheT-pheS.

931

TABLE 6. Evidence for the gene order pps-pheT-pheS ppS+ transductants

Expt 2

Expt 1

pps+ pheT354 pheS12a pps+ pheT+ pheS' pps-4 pheT+ pheS- pps-4 pheT354 pheSl2° No.

No.

0 0 10 2.1 pheT354 pheS+ 5 1.4 1 0.2 pheT+ pheS12 55 15.0 423 89.2 pheT354 pheS12 8.4 308 83.7 40 pheT+ pheS+ I_I_I a Donor strains in experiments 1 and 2, respectively: MC1440 and K10. b Recipient strains in experiments 1 and 2, respectively: MC105 and MC106. _

DISCUSSION In this work we have shown that, of the five temperature-sensitive phenylalanyl-tRNA synthetase mutants studied, three are defective in the a subunit and two in the /8 subunit. The other subunit in each case is normal, as judged from the fact that under the appropriate conditions it can contribute to the formation of hybrid enzyme that resembles wild type (Fig. 1C and 2C; Table 4). The temperature-sensitive mutations seem to weaken the interactions between subunits, since upon centrifugation in sucrose gradients the normal subunits did not appear in the position of the native enzyme but sedimented considerably more slowly (see also reference 2). This dissociation, however, may not proceed all the way to separate monomers. In the case of the a mutant NP37, the evidence suggests that the temperature-sensitive subunit remains bound to the normal 8 and is incorporated with it into active enzyme when wild-type a is added (Table 4). This phenomenon does not occur with the 83 mutant JP1116; its normal a subunit, however, sediments at a position that implies a molecular weight roughly twice that of a alone. As a working hypothesis, we propose that the NP37 enzyme dissociates to ats,3 and the JP1116 enzyme to a2. The molecular weights estimated for these products (see Results) are close to those predicted for such structures from the known molecular weights of the monomers (9); we must emphasize, however, that these estimates were derived from sedimentation distances alone and should not be considered definitive. If the hypothesis is correct, it implies that mutations in different subunits can weaken different interactions holding the tetramer together and that, consequently, there are

932

COMER AND BOCK

J. BACTERIOL.

different ways that a mutant enzyme can dissociate. When the mutant enzymes were mixed with the appropriate wild-type subunits, active hybrid enzyme was formed. As judged by sucrose gradient centrifugation, this active enzyme can occur in two forms: one that sediments as expected for the native wild-type enzyme, and one that sediments more slowly. Our current working hypothesis is that the latter is the trimer a2f3 (although another oligomeric state cannot be excluded) and that the two products are generated as follows: JP1116: a2/32ts NP37:

f3tS + a2 a*4 +

a2tS132

a4,-82

als 2 aaus3

The molecular weight estimated for the more slowly sedimenting active product (150,000) is not inconsistent with that expected for such a trimer (172,000). The hypothesis is further supported by the findings that in the case of JP1116 the formation of this product is favored if , is limiting (Fig. 1B), that the NP37 product has residual temperature sensitivity but neither JP1116 product does (Table 4), and that NP37 yields nothing that sediments like the native enzyme without prior urea denaturation (Fig. 2B). The hypothesis requires that phenylalanyl-tRNA synthetase be active if three of the four subunits are present; a similar active trimer is known to occur in the case of E. coli tryptophan synthetase (4, 8). This enzyme, also an a212 tetramer, dissociates to a and 182 (which retain only trace activity) and reassociates to af32 or a232, where the relative amounts of subunits determine which product is formed (as proposed for JP1116). The a0,32 trimer is half as active as the a2f32 tryptophan synthetase, or equally active per a subunit. The chemical dissociation of phenylalanyltRNA synthetase has been recently investigated by Hanke et al. (10). With their treatments the enzyme dissociated into inactive monomers or dimers and reassociated into active tetramers; they did not observe any other enzyme forms, such as trimers. These results, however, are not at variance with our working hypothesis, since the reconstitution conditions in the two studies differ both qualitatively and quantitatively. They used symmetric reassociation mixtures containing stoichiometric amounts of all the dissociation products of the wild-type enzyme; we, on the other hand, used asymmetric mixtures containing a single wildtype subunit plus a partially dissociated mutant enzyme.

The identification of mutations in both the a and /8 subunits of the phenylalanyl-tRNA synthetase made it possible for us to study the locations of the genes for the two subunits on the E. coli genome; we found that they are arranged in the order aroD-pps-pheT-pheS. The pheS gene was co-transduced with aroD and with pps at very nearly the same frequencies as was pheT; it is, therefore, a possibility that the two genes are immediately adjacent to each other. It will be of interest to test whether they are transcribed together into messenger RNA. ACKNOWLEDGMENTS This work was supported by the Deutsche Forschungsgemeinschaft. We thank B. Bachmann, F. C. Neidhardt, and R. R. B. Russell for providing strains used in this study, and H. Hennecke for the antiserum. LITERATURE CITED 1. Bachmann, B. J. 1972. Pedigrees of some mutant strains of Escherichia coli K-12. Bacteriol. Rev. 36:525-557. 2. Bock, A. 1968. Relation between subunit structure and temperature-sensitivity of mutant phenylalanyl RNA synthetases of Escherichia coli. Eur. J. Biochem. 4:395-400. 3. Bock, A., and F. C. Neidhardt. 1967. Genetic mapping of phenylalanyl-sRNA synthetase in Escherichia coli. Science 157:78-79. 4. Creighton, T. E., and C. Yanofsky. 1966. Association of the a and 2 subunits of the tryptophan synthetase of Escherichia coli. J. Biol. Chem. 241:980-990. 5. Fayat, G., S. Blanquet, P. Dessen, G. Batelier, and J. P. Waller. 1974. The molecular weight and subunit composition of phenylalanyl-tRNA synthetase from Escherichia coli K-12. Biochimie 56:35-41. 6. Folk, W. R., and P. Berg. 1970. Isolation and partial characterization of Escherichia coli mutants with altered glycyl transfer ribonucleic acid synthetases. J. Bacteriol. 102:193-203. 7. Fraenkel, D. G., and F. C. Neidhardt. 1961. Use of chloramphenicol to study control of RNA synthesis in bacteria. Biochim. Biophys. Acta 53:96-110. 8. Goldberg, M. E., T. E. Creighton, R. L. Baldwin, and C. Yanofsky. 1966. Subunit structure of the tryptophan synthetase of Escherichia coli. J. Mol. Biol. 21:71-82. 9. Hanke, T., P. Bartmann, H. Hennecke, H. M. Kosakowski, R. Jaenicke, E. Holler, and A. Bock. 1974. Lphenylalanyl-tRNA synthetase of Escherichia coli K12. A reinvestigation of molecular weight and subunit structure. Eur. J. Biochem. 43:601-607. 10. Hanke, T., P. Bartmann, and E. Holler. 1975. Quaternary structure and catalytic functioning of L-phenylalanine:tRNA ligase of Escherichia coli K10. Eur. J. Biochem. 56:605-615. 11. Hennecke, H., and A. Bock. 1975. Altered a subunits in phenylalanyl-tRNA synthetases from p-fluorophenylalanine-resistant strains of Escherichia coli. Eur. J. Biochem. 55:431-437. 12. Kisselev, L. L., and 0. 0. Favorova. 1974. AminoacyltRNA synthetases: some recent results and achievements. Adv. Enzymol. Relat. Areas Mol. Biol. 40:141238. 13. Kosakowski, M. H. J. E., F. C. Neidhardt, and A.

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16.

17.

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Bock. 1970. Complementation in vitro of phenylalanyl-tRNA synthetases of Escherichia coli. Eur. J. Biochem. 12:74-79. Lapointe, J., and G. Delcuve. 1975. Thermosensitive mutants of Escherichia coli K-12 altered in the catalytic subunit and in a regulatory factor of the glutamyl-transfer ribonucleic acid synthetase. J. Bacteriol. 122:352-358. Lowry, 0. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275. Martin, R. G., and B. N. Ames. 1961. A method for determining the sedimentation behavior of enzymes: application to protein mixtures. J. Biol. Chem. 236:1372-1379. Miller, J. H. 1972. Experiments in molecular genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. Neidhardt, F. C. 1966. Roles of amino acid activating

19. 20.

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23.

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enzymes in cellular physiology. Bacteriol. Rev. 30:701-719. Ostrem, D. L., and P. Berg. 1970. Glycyl-tRNA synthetase: an oligomeric protein containing dissimilar subunits. Proc. Natl. Acad. Sci. U.S.A. 67:1967-1974. Piepersberg, A., H. Hennecke, M. Engelhard, G. Nass, and A. Bock. 1975. Cross-reactivity of phenylalanyltransfer ribonucleic acid ligases from different microorganisms. J. Bacteriol. 124:1482-1488. Pittard, J., and B. J. Wallace. 1966. Distribution and function of genes concerned with aromatic biosynthesis in Escherichia coli. J. Bacteriol. 91:1494-1508. Russell, R. R. B., and A. J. Pittard. 1971. Mutants of Escherichia coli unable to make protein at 42 C. J. Bacteriol. 108:790-798. Vinopal, R. T., and D. G. Fraenkel. 1975. p1 B and ptkC loci of Escherichia coli. J. Bacteriol. 122:11531161.