of neurospora crassa'mistranslation in neurospora 453 tetraacetic acid). after thorough...

AN APPARENT REILATIONSHIP BETWEEN MISTRANSLATION AND AN ALTERED LETJCYL-TRNA SYNTHETASE IN A CONDITIONAL

LETHAL MUTANT OF NEUROSPORA CRASSA'

DEBORAH B. PRINTZ AND S. R. GROSS

Division of G,metics, Department of Biochemistry, Duke Uniuersity, Durham, North Carolina 27706

Rzceived September 27, 1966

A N analysis of the genetic control of the biogenesis of leucine biosynthetic enzymes in Neurospora crussu has established that four cistrons are involved

in the determination o F the structures of the three enzymes specifically responsible for the formation of leucine (GROSS 1965). However, the function of a fifth cistron, leu-5 represent'ed by a single mutant strain, 45208t, remained enigmatic. This strain was isolated by HOULAHAN, BEADLE, and CALHOUN (1949) after ultraviolet irradiation and was classified as an auxotroph with an unknown growth requirement. Subsequently, DUBES ( 1953) determined that 45208t was a temperature sensitive mutant that responded auxotrophically to leucine at low and intermediate temperatures. At 37°C the growth of 45208t was severely restricted regardless of supplementation.

The growth response of 45208t is different from that of temperature sensitive auxotrophs which display, characteristically, an increased dependence on added growth factor as a function of an increase in incubation temperature. Failure of the strain to grow at elevated temperatures, regardless of leucine supplementation, suggested the involvement of the leu-5 cistron in some nondispensable function involving either the control of leucine production or the utilization of leucine in protein synthesis. The results of experiments reported below indicate that a large amount of the protein synthesized during growth of the leu-5 mutant is structurally altered ancl enzymatically defective. The data obtained suggest that the 45208t phenotype results from a decrease in the fidelity with which leucine is incorporated into protein probably as a consequence of the production of a leucyl-tRNA synthetase with altered substrate binding properties.

MATERIALS A N D METHODS

BiologicaL T h e following: strains of Neurospora crmsa have been used: ( 1 ) Std 6a and Std 4A, wild-type strains containing the genetic background of all mutants described except 45208t; (2) Abbott 4A and Lindegi-en 25a, wild-type strains from which mutant 45208t was isolated after ultraviolet irradiation (HOULAHAN, BEADLE and CALHOUN 1949) ; (3) 45208t-515A ( leu-5) , a derivative of the original temperature sensitive, leucine auxotroph isolated after two successive backcrsss2s to Std 4A. This isolate is heterokaryotically compatible with all mutants in the Std

' Research was supported in part by National Science Foundation Grant GB-727 and Public Health Service Grant GiW 07250.

Genetics 5 5 : 451467 March 1967.

452 D. B. PRINTZ A N D S. R. GROSS

background; (4) D221-134a (leu-I) , an absolute leucine auxotroph lacking P-isopropylmalate dehydrogenase; (5) 45208t-3-3A (leu-I; Zeu-5), a double mutant isolated from a cross between D221-1-3a and 4620t-2-15A; (6) FLR9,-%262a (FLR,,) , a trifluoroleucine-resistant leu-4 mutant that produces an a-isopropylmalate synthetase that is insensitive to feedback control (GROSS 1965); (7) 46208t-9-4a (FLR,,; Ieu-5), a double mutant isolated from a cross between FLR9,-2-262a and 45208t-2-15A. Hereafter, strains are referred to according to locus designa- tion of mutant genes.

Crosses were incubated at 22 to 25°C on synthetic crossing medium (WFSTERGAARD and MITCHELL 1947). Ascospores were isolated at random by the sorbose plating method of NEW- MEYER (1954) but were germinated at 30°C subsequent to heat shock.

The synthetic growth medium used throughout was VOGEL’S minimal medium N (VOGEL 1956) containing 1 % sucrose with the following supplements when required: m-leucine, 300 mg/l; L-lysine HC1, 200 mg/l; L-inositd, 20 mg/l; L-isoleucine, 210 mg/l; and L-valine, 90 mg/l. 5’,5’,5’-trifluoroleucine, a leucine antimetabolite, was added, 1 a0 mg/l, where specified.

Mycelial mass was measured by growth in 20 ml synthetic medium inoculated with one drop of a dense conidial suspension ( 0 ~ ~ ~ ~ 1.0) of the appropriate strains. The flasks were incubated at the desired temperature for 72 hours, then the mycelial contents were filtered, dried, and weighed. All determinations were done at least in duplicate.

Linear growth progression was measured by the tube method of RYAN, BEADLE, and TATUM (1943).

Preparation of extracts: Extracts for all enzyme assays, except for aminoacyl-tRNA synthetases, were prepared as follows: mycelia were grown with aeration in one liter of synthetic medium inoculated with 5 ml of dense conidial suspensions and appropriately supplemented with either limiting amounts, 35 to 50 mg/l, o r nonlimiting amounts, 300 mg/l, of DL-leucine. Growth was generally for 24 hours but varied somewhat depending on stain, temperature, and leucine concentration. However, in order to make up for the slow rate of germination of leu-5 mutants, especially on limiting leucine at 34”C, leu-5 mutants usually were preincubated for 12 hours at 30°C prior to the 24 hours growth period. The amount of growth during preincubation did not contribute significantly to the total m a s of mycelia obtained. In all cases, attempts were made to harvest cultures after equivalent amounts of growth. Ammonium sulfate precipitates (0 to 75%) of extracts of mycelia were prepared by the method of GROSS (1965).

Extracts for aminoacyl-tRNA synthetase activity were prepared by a modified version of the method of BARNETT (1965). Mycelia were grown and harvested through the first low speed centrifugation step as indicated above with the exception that the buffer used was 0 . 0 2 5 ~ Tris (tris-hydroxymethylaminomethane) -HCl, pH 8.0, containing I O - 3 ~ sodium thioglycollate and 2 x I W M L-leucine. The low speed supernatant was centrifuged at 105,000g for 1 hr and 5 ml 1 . 0 ~ manganese chloride was added per 100 ml of the resulting supernatant to remove nucleic acids. After stirring 20 minutes, the precipitate was removed by centrifugation and solid ammonium sulfate slowly added to 75% saturation. After stirring at least 2 hr, and preferably overnight, the precipitate was collected by centrifugation and either stored at -20°C or immediately back-extracted. For back-extraction, 0 to 75% ammonium sulfate precipitates were SUS- pended in 0.2M potassium phosphate buffer, pH 7.0, containing 1 0 ‘ 3 ~ sodium thioglycollate, 2 X I e 4 ~ L-leucine, and 2 . 2 ~ ammonium sulfate. After stirring 20 minutes, the precipitate was collected by centrifugation and the extraction repeated. The final precipitate was stored at -20°C until just prior to use, when a portion was suspended in 1 ml 0 . 0 5 ~ Tris-HC1 buffer, pH 7.7, containing 0.004.~ magnesium acetate, 0 . 0 1 2 5 ~ potassium chloride, and IG-3~ sodium thioglycollate and passed through a G-25 Sephadex column (1 x I 2 cm) pre-equilibrated with the same buffer. The leucyl-tRNA synthetase is much more stable during purification if it is kept insoluble. Purification by removing protein soluble in 2 . 2 ~ ammonium sulfate resulted in the maximum recovery of the enzyme with minimum contamination by ribonuclease activity.

Preparation of tRNA: Transfer RNA from N . crassu was prepared from mycelia, grown in six liters of synthetic medium under vigorous aeration for 24 hours at 34°C. The mycelia were collected by filtration, washed with distilled water, then suspended in 100 ml/20g wet weight 0 . 0 1 ~ potassium phosphate buffer, pH 7.0, containing 5 x I W M EDTA (ethylenediamine-

MISTRANSLATION I N NEUROSPORA 453

tetraacetic acid). After thorough homogenization in a Lourdes blender, 0.25 volume of 5% sodium lauryl sulfate in 45% ethanol was added and the slurry heated for 7 minutes with con- stant swirling in a b3iling water bath. The first ethanol precipitate, prepared according to the method of OFENGAND, DIECKMANN, and BERG (1961), was collected by centrifugation and then suspended in 0 . 0 5 ~ sodium chloride. After rapid stirring in the cold for 1 hr, insoluble material was removed by centrifugation and 0.5 volume 2 . 0 ~ postassium acetate buffer, pH 5.0, and 2.5 volumes of 95% ethanol were added to the supernatant and the mixture allowed to stand at -20°C overnight. The precipitate formed was collected by centrifugation, brought up in 0.2111 sodium chloride, and applied to a DEAE-cellulose column (2.5 x 20 cm for 200 mg RNA) pre- equilibrated with 0 . 2 ~ sodium chloride. The column was eluted with 0 . 2 ~ sodium chloride until the optical density at 260 mp approached zero, then the tRNA was eluted with 0 . 7 5 ~ sodium chloride and precipitated with 3.0 volumes of 95% ethanol at -20°C overnight. The tRNA precipitate was suspended in 0 . 1 ~ glycine-sgdium hydroxide buffer, pH 10.0, and incubated at 37°C for 1 hr to remove est.erifiod amino acids. An equal volume of 2 . 0 ~ potassium acetate buffer, pH 5.0, was added and the tRNA reprecipitated with 3.0 volumes of 95% ethanol a t -20°C overnight. Before use, this precipitate was suspended in water, 1.4 pmoles magnesium chloride/ mg RNA was added, and the solution dialyzed against 10-3111 sodium acetate buffer, pH 5.0, overnight. The final RNA solutions thus obtained were stored at -20°C.

Transfer RNA was prepared from E. coli B also by the method of OFENGAND, DIECICMANN, and BERG (1961) or purchased from California Corporation for Biochemical Research.

Enzyme assay procedures: Unless otherwise specified, enzyme specific activity assays were performed on ammonium sulfate precipitates ( 0 to 75%), prepared as described, suspended in 0 . 1 ~ phosphate buffer, pH 6.0, containing 2 x 1 0 - 4 ~ L-leucine, and centrifuged 1 hr at 105,OOOg in a Spinco Model L ultracentrifuge.

a-Isopropylmalate synthetase activity was determined by the NEM (N-ethyl-maleimide) assay procedure described by WEBSTER and GROSS (1965). Isopropylmalate isomeras. activity was determined by the method of GROSS, BURNS, and UMBARG~R (1963) ,8-isopropylmalate dehydrogenase activity was determined by the method of GROSS (1965) except that the incubation period was for 5 instead of 1 and 2 minutes.

Glucose-6-phosphate dehydrogenase activity was determined by a modified version of the method of RADHAKRISHNAN (1 960). Ammonium sulfate precipitates were suspended in enough 1 . 0 ~ Tris-HC1 buffer, pH 8.0, to yield a protein concentration of 1 to 2 mg/ml and assayed immediately without ultracentrifugation in reaction mixtures containing 1 pmole glucose-6- phosphate, 0.3 pmoles NA:DP, 1 . 0 ~ Tris-HC1 buffer, pH 8.0, in a total volume of 1.0 ml. The reaction was followed by measuring the initial rate of increase in absorbancy at 340 mp at 30°C.

A modified version of -the method of YANOFSKY (1955) was used to dctermine trypto2han synthetase activity. Ammonium sulfate precipitates were suspended in 0 . 1 ~ sodium phosphate buffer, pH 7.8, containing i2.5 mix reduced glutathione and assayed directly without ultracentrifugation. The assay mixture contained: 1.5 pmoles indole, 44) pmoles L-serine, 20 pg pyridoxal phosphate, 1.0 pmole reduced glutathiono, 60 pmoles sodium phosphate buffer, pH 7.8, and an appropriate am" of enzyme extract in a total volume of 1.0 ml. Incubation was at 37°C for 30 minutes and the reaction then was stopped with 0.2 ml of 5% sodium hydroxide.

NADP-specific glutamic dehydrogenase activity was determined by the method of BARRATT and STRICKLAND (1963). The supernatant solution after ultracentrifugation of 0 to 75% ammmium sulfate precipitates dissolved in 0 . 1 ~ phosphate buffer, pH 6.0, containing 2 x I e 4 ~ L-leucine was diluted in 0 . 2 ~ Tris-HC1 buffer, pH 7.8, and assayed at 34°C in a mixture containing 420 pmoles Tris-HC1 buffer, pH 7.8, 4.50 pmoles ammonium chloride, 33.4 pmoles a-ketoglutarate, and 0.192 pmoles NADPH in a total volume of 3.0 ml.

The activity of 5-dehydroshikimate reductase was measured by the method of GROSS and FFXN (1960). The buffer used to suspend the 0 to 75% ammonium sulfate precipitates was 0 . 2 ~ Tris- HC1, pH 7.8, and activity was measured after ultracentrifugation by determining the initial rate of increase in absorbancy at 340 mp at 34°C in the following mixture: 0.5 pmoles shikimic acid, 5.0 pmoles NADP, 100 #moles Tris-HC1 buffer, pH 7.8 and an appropriate amount of enzyme extract in a total volume of 1.0 ml.

454 D. B. P R I N T Z A N D S. R. GROSS

Alcohol dehydrogenase activity was determined by the method of VALLE and HOCH (1955). The suspending buffer used was 0 . 1 ~ pyrophosphate buffer, pH 8.5. Histidase and fumarase were assayed by the methods of TABOR and MEHLER (1955) and RACKER (1950), respectively. NAD- ase activity was determined by the method of KAPLAN (1955). The reaction between cyanide and NAD before and after 16 minutes of incubation at 37°C was measured at 325 mp.

Aminoacyl-tRNA synthetase activity was determined by a modified version of the method of MANS and NOVELLI (1961). The assay mixture usually contained per ml: 50 pmoles Tris-HC1 buffer, pH 7.2, 18 pmoles magnesium acetate, 0.1 pmoles sodium thioglycollate, 2.5 pmoles adenosine triphosphate, 0.25 pmoles cytidine triphosphate, 0.004 pmoles L-leucine 4, 53H HC1. 5.0 curies/mmole o r an equivalent amount of another labeled amino acid, 500 to 1000 pg tRNA, and 200 t3 700 pg synthetase preparation. Reactions were incubated at either 22", 30", or 37°C. At desired time intervals 0.1 ml aliquots of the reaction mixture were pipetted onto filter paper discs (Whatman #3mm, 2.5 cm diameter), dried for 30 seconds in a stream of warm air and plunged into ice cold 10% trichloroacetic acid. Subsequently, the discs were washed as follows: three times for 10 min each in 5% trichloroacetic acid at room temperature; once for 30 min at 37°C in 2.0 N NaOH: glacial acetic acid: 95% ethanol (1:15:234); once for 30 min at room temperature in 2.0 N NaOH; glacial acetic acid: 95% ethanol: ethyl ether (1:15:234:250); and thrse times for 5 min each in ethyl ether. The discs were then dried and counted in a Packard Tri-Carb liquid scintillation counter.

Amimacyl-tRNA synthetase activity was determined also by a modified version of the method of LOFTFTELD and EICNER (1959). The assay mixture contained: 5.0 pmoles Tris-HC1 buffer, pH 7.2, 1.8 pmoles magnesium acetate, 1.0 x 1 ~ 3 pmoles sodium thioglycollate, 0.25 pmoles adenosine triphosphate, 22.4 pmoles hydroxylamine (adjusted to pH 7.2), 14C L-leucine, 1.732 X lo-* pmoles, 231 pc/gmole, and an appropriate amount of enzyme extract to a total volume of 0.15 ml. After 15 minutes at 37°C the reaction tubes were placed at 55°C for 5 minutes to destroy the enzyme and the contents were evaporated onto a line one inch from the end of by 5 inch strip of Amberlite IR-120 ion exchange paper (sulfonic acid resin, sodium form); 0 . 0 5 ~ sodium phosphate buffer, pH 7.0, was then allowed to rise by capillary through the strip. The strip was dried and the radioactivity of the region around the origin was measured.

Thermal inactivation: Extracts of leu-5 and leu-5 + strains were adjusted to equivalent protein concentrations for all comparative thermal inactivation studies. For each of the enzymes examined, thermal inactivation rates of mixtures of equal amounts of leu-5 and leu-5+ activities also were determined. In all cases mixtures had thermolabilities intermediate to those of the single components.

The determination of specific aetivities: Specific activities for aminoacyl-tRNA synthetases are expressed in terms of counts incorporated per minute per milligram protein in one milliliter reaction volumes. All other specific activities are expressed in terms of units of enzyme activity per milligram protein, a unit of activity equalling that amount of enzyme which catalyzes the formation or disappearance of one nyLmole of product or substrate.

Protein concentration was determined either by the method of WARBURC and CHRISTIAN (1941 ) or the method of LOWRY, ROSEBROUGH, FARR, and RANDALL (1951 ).

Chemical: All chemicals were obtained from standard commercial sources except for 5',5',5'- trifluoroleucine which was synthesized by the method of RENNERT and ANKER (1963).

RESULTS

The characteristic effects of temperature and leucine concentration on the growth of the leu-5 mutant are illustrated in Figure 1, in which the yield of mycelia from the wild type, leu-5, FLR,, and the FLR,,; leu-5 double mutant after 72 hours growth in liquid medium is plotted as a function of leucine concentration. The data presented indicate that the growth of leu-5 at 23" and 34°C is stimu1a:ed appreciably by exogenously supplied leucine. The growth of this

MISTRANSLATION IN NEUROSPORA 455

FIGURE 1 .-Total yieldis of mycelia from wild-type (0): leu-5 (a), FLR,, (a), and FLR,,; leu-5 (S) strains after 72 hours growth in liquid medium as a function of exogenously supplied m-leucine concentration. Growth temperatures were 23°C (-), 34°C ( ), and 37°C (---).

FIGURE 2.--Responses of wild-type, leu-5, leu-I, and leu-1; leu-5 strains to shifts from 23°C (-) to 37°C (- - -) during growth on solid medium supplemented with 15 mg/l DL-

leucine ( ), or 150 mg/l DL-leucine ( X ) .

mutant. however, is strikingly temperature sensitive for at 37"C, irrespective of the presence of leucine, little or no growth is obtained.

The growth responses of FLR,, and the FLR,,; leu-5 double mutant, also illustrated in Figure 1, indicate that the leucine requirement in the double mutant is at least partially separable from the temperature sensitive phenotype. The FLR,, mutation is one in the leu-4 cistron that leads to the production of an a-isopropylmalate synthetase that is insensitive to feedback control ( WEBSTER and GROSS 1965). Relaxation of feedback control results in the production of a large excess of leucine (luring growth. The data presented indicate that the endog- enous production of leucine in the FLR,,; leu-5 double mutant is sufficient to satisfy the leu-5 auxoiropic response but the temperature sensitive lethality is only slightly altered; 39°C is required for complete inhibition of growth instead of 37°C.

Results of experiments involving shifts from 23" to 37°C during growth on solid medium supplemented with leucine are illustrated in Figure 2. The grocwth rate of the wild-type strain is independent of leucine concentration but increases significantly when the temperature is raised from 23" to 37°C. The response of leu-5 to a similar shifi in temperature, however, is complex and related to the concentration of leucine supplied exogenously. On low concentrations of leucine, the growth rate of Zeu-5 after a shift to 37°C immediately starts to decrease and, within 24 hours, growth essentially stops. On higher levels of exogenous leucine, the growth rate of leu-5 after a shift to 37°C increases appreciably for a while then rapidly decreases until growth stops.

456 D. B. PRINTZ A N D S . R. GROSS

m $ +

%

+ A

Y

1

h

+" + f

2 2

-t

t 2

3. 2

2 -A x +

MISTRANSLATION I N NEUROSPORA 45 7

The growth responsles of leu-1 and leu-I; leu-5 to temperature-shifts are also illustrated in Figure 2. leu-I mutants have an absolute requirement for leucine due to a deficiency in P-isopropylmalate dehydrogenase, the enzyme that catalyzes the last reaction in the biosynthetic sequence that is unique to leucine synthesis, the conversion of P-isopropylmalate to a-ketoisocaproate. The growth rate of leu-1 is dependent on exogenous leucine concentration, and increases appreciably when the incubation temperature is raised from 23" to 37°C. The growth response of the leu-I; leu-5 double mutant, however, to a shift in temperature from 23" to 37°C is essentially the same as that of the leu-5 single mutant.

Genetic analysis of r'eu-5 is presented in Table 1. The data obtained, in agree- ment with those of PERKINS and MURRAY ( 1963), indicate that leu-5 is on linkage group V. More specifiically, they locate leu-5 between lys-1 and sp about 7.5 units from iv-I. None of the four leu cistrons that specify the structure of the leucine biosynthetic enzymes is located on linkage group V.

The genetic and physiological properties of the mutant suggest that the leu-5 locus is not involved in the synthesis of one of the leucine biosynthetic enzymes but rather in the specification of some nondispensable function involving leucine subsequent to its synthesis. Support for this notion is derived from the fact that, as indicated in Table 2, extracts of leu-5 contain a nearly normal complement of the leucine biosynthetic enzymes. Furthermore, the data obtained suggest that the control mechanisms governing the synthesis of the leucine biosynthetic enzymes is not seriously affected by the leu-5 mutation. Syntheses of a-isopropylmalate synthetase and isomerase in leu-1 and leu-1; leu-5 are equivalently sensitive to the concentration of leucine supplied even though the specific activity of the isomerase was found to be somewhat lower in leu-1; leu-5.

The participation of leu-5 in some specific nondispensable function is further

TABLE 2

Aiwlysis of the Ieucine biosynthetic enzymes*

Specific activity Growth conditions a-Isopropylmalate isopropylmalate P-Isopropylmalate

Strain (mg/l oL-leucine) synthetaset isomerase dehydrogenase

leu-5 01 300

wild type 0 300

Ieu-l,leu-5 35 300

leu-1 35 300

10.2 k 0.7 7.6 i- 0.3

17.2 f 1.2 12.2 i- 1.2 96.3 51.9

43.6 1 20

54.0 f 9.6 8.0 54.0 t 11.6 5.6 76.4 i- 5.4 12.6 57.0 * 3.0 9.0

310 166 5 63 298

* Enzyme exkarts were prepared from mycelia grown at 23°C. The values for the synthetase and the isomerase of leu-5 and wild-type preparations are averages and calculated standard deviations determined for three independent enzyme preparations; values for the dehydrogenase and the synthetase and isomerase of leu-1 and leu-1, Ieu-5 preparations are averages of two separate determinations on single preparations. The dehydrogenase data are not directly equivalent to those of GROSS (1965) in that 5 rninute assays instead of 1 and 2 minute assays were done. The data obtained for leu-I are comparable to those presented by GROSS (1965).

.;- lo-% r-leucine inhibited the :.ynthetase in all cases.

45 8 D. B. PRINT2 A N D S. R. GROSS

suggested by the fact that leu-5 complements leu-1, leu-2, leu-3, and leu-4 in heterokaryons but, at high temperature, wild-type growth was rarely obtained. The expression of the leu-5 phenotype as a partial dominant was most easily demonstrated using the leu-1; leu-5 double mutant. At 23°C complementation between the double mutant and leu-2,leu-3,1eu-4 or pan-1 was efficient and wild- type growth rate was obtained. At 37"C, however, the growth of the heterokaryons was characteristically erratic and often stopped before the end of the growth tube was reached.

Attention was focused on the possibility of the involvement of a generalized defect in protein synthesis in leu-5 when it was observed that the specific activities of a number of enzymes were especially low in extracts of the leu-1; leu-5 double mutant as compared to extracts of leu-1. A comparison of the specific activities of several enzymes in extracts obtained from mutants grown at 30°C and 34°C on 50 and 300 mg leucine per liter is illustrated in Figures 3 and 4.

I , The data indicate clearly not only thai extracts of leu-5 strains, but also that,

- specific activities in general are low in particularly in the case of tryptophan

SPECIFC ACTIVITY UN1 TS

L-tRNA SYNTHETASE xm3

L-tRNA SYNTHETASEX~O-~

YCROGENASE x 10'

SPECIFIC ACTIVITY UNITS

L-tRNA SYNTHETASExl

FIGURE 3.-The effect of growth temperature on specific activities of enzymes from leu-1 (0) and leu-1; leu-5 (E) strains grown in the presence of 50 mg/l DL-leucine. The values indicated are averages and standard deviations of a minimum of two separate determinations of three independent enzyme preparations.

FIGURE 4.-The effect of leucine concentration on specific activities of enzymes from leu-1 (U), and leu-1; leu-5 (H) strains grown at 34°C. The data presented for leucyl-tRNA synthetase were obtained from wild type (0) and leu-5 (H). The values indicated are averages and calculated standard deviations of a minimum of two separate determinations for three independent enzyme preparations.


synthetase and lysyl-tRNA synthetase, reduced temperature and/or the pro- vision of a high concentration of leucine during growth of leu-I; leu-5 results in a relative increase in the yield of active enzyme. It is important to point out, however, that the growth conditions were chosen in order to optimize the temperature sensitive phenotype without introducing a significant disparity between the growth of the leu-1 single and the leu-1; leu-5 double mutant. The temperature was limited to a range well below that of maximum temperature sensitivity.

In view of the data presented in Figures 3 and 4 it is important to note that the specific activities of several enzymes, fumarase, NADP-glutamic dehydrogenase and histidase were found to be essentially equal in extracts of leu-1 and leu-2; leu-5 strains grown on limiting leucine at 34°C. The leu-5 mutation there- fore does not affect equivalently the synthesis of all enzyme protein. This suggests, in turn, that the decrease in specific activity probably is due to the production of a large amount of “dead” or defective enzyme protein by leu-5 and that the loss of fidelity in the protein synthesizing mechanism must involve some specific. relatively infrequent, error in translation or transcription.

Synthesis of altered enzyme protein was detected by analyses of the rates of thermal inactivation of several of the enzymes that displayed reduced specific activities in extracts of‘ the leu-I; leu-5 double mutant. As illustrated in Figure 5, a considerable fraction of the tryptophan synthetase from the leu-I; leu-5 double mutant is more heat labile than the enzyme obtained from leu-I. It was found further that the amount of the heat labile component present varies as a function of the specific activity of the extracts which, as has been pointed out previously, varies as a function of growth conditions of temperature and supply of exogenous leucine. 5-dehydroshikimate reductase from the leu-I ; leu-5 double mutant also was found to display an altered thermolability. In this case, although the specific activity of the enzyme obtained from leu-I; leu-5 was lower than that obtained from leu-2, the enzyme was more stable to thermal inactivation at 48°C. Analyses of the thermal stabilities of alcohol dehydrogenase and NADase isolated from

2 4 6 8 1012 I4 16 1820 MINUTES AT 5’fC

FIGURE 5.-Kinetics of thermal inactivation of tryptophan synthetase in extracts of leu-1 (0) and leu-I; leu-5 ( ) . Protein concentrations were adjusted to 6 mg/ml prior to heating.


leu-1 and leu-I; leu-5 again indicated that a large fraction of each of these enzymes produced by leu-1; leu-5 was more thermolabile than normal. Although the kinetics of thermal inactivation of each of these enzymes were complex, the data obtained indicated that, as was found for tryptophan synthetase, at least some enzyme protein with nearly normal thermal stability was synthesized. It is important, to point out that in the case of each of the enzymes studied heat inactivation of mixed extracts yielded inactivation curves that corresponded to that expected of mixtures of leu-1 and leu-1; leu-5 extracts thus ruling out extran- eous factors in the enzyme preparations that might specifically affect thermostability. It is also important to note that fumarase, NADP-glutamic dehydrogenase, and histidase, enzymes produced in normal amounts by the leu-I; leu-5 double mutant grown on limiting leucine at 34"C, were found to possess identical thermostability properties regardless of source.

In view of the auxotrophic response of leu-5 to leucine, the temperature sensitive conditional lethality, and the rather prevalent production of enzymatically "dead" and structurally altered enzyme protein, it seemed reasonable to assume that some error in translation involving leucine incorporation into protein was involved. Attention was focused on the formation of leucyl-tRNA and specifically on the properties of the leucyl-tRNA synthetase. Extracts of leu-I; leu-5 were found to contain less leucyl-tRNA synthetase than leu-I even when the double mutant was grown at low temperature and in the presence of a high concentration of leucine. It is perhaps important to note, however, that the specific activity of leucyl-tRNA synthetase in extracts of the leu-5 single mutant, although relatively low when grown under restrictive conditions, is nearly equal to the wild type when grown under relatively permissive conditions.

I

MINUTES AT 42.5-C FIGURE 6.-Kinetics of thermal inactivation

of leucyl-tRNA synthetase in extracts of wild- type (U), leu-5 (m), leu-I (O) , and leu-I; leu-5 ( 0 ) strains. Protein concentrations were adjusted to 3 mg/ml prior to heating.

MISTRANSLATION IN NEUROSPORA 46 1

Figure 6 is a plot of the kinetics of thermal inactivation of the leucyl-tRNA synthetase obtained from the wild type, the leu-l and leu-5 single mutants and the leu-1; leu-5 double mutant. The final rate of thermal inactivation for the leucyl-tRNA synthetase of leu-5-containing strains is at most slightly faster than that of the wild-type or leu-1 strains. However, synthetase activity obtained from leu-5-containing strains displays significant activation after brief exposure to 42.5 OC: This activation, while most marked in enzyme preparations obtained from leu-1; leu-5 is always discernible in extracts of the leu-5 single mutant-no matter what growth conditions are employed. Passage of the enzyme preparation through G-100 Sephadex does not eliminate the activation, and the addition of tRNA to leu-1 or wild-type extracts during the heating period does not produce this stimulation of activity. Furthermore, there is no alteration in the substrate binding constants of the leucyl-tRNA synthetase of leu-5 after thermal “activation”. Despite the thermal activation displayed by the leucyl-tRNA synthetase of leu-5 the mutant enzyme is markedly less stable than the corresponding enzyme obtained from either the wild type or leu-1. After 24 hours at 4”C, 59% activity of the leu-5 leucyl-tRNA synthetase is lost while only 25% of the activity of the wild-type enzyme is lost under comparable conditions. Recovery of activity of the leu-5 enzyme during extraction and fractionation is similarly reduced.

On the assumption that translational errors of leucine codons may result from errors committed by the leucyl-tRNA synthetase, the K, values for leucine and tRNA of the leucyl-tRNA synthetases of wild-type and leu-5 strains were determined. Lineweaver-Burk plots of velocity of the formation of leucyl-tRNA as a function of leucine concentration in the presence of excess tRNA are presented in Figure 7. Several determinations of the K, for leucine derived from Lineweaver-Burk plots of either the velocity of leucyl-tRNA formation or the velocity of the activation step as determined in the presence of hydroxylamine are listed in Table 3. Although it is obvious that the calculated K, for leucine as determined by the hydroxylamine assay of amino acid activation is higher than

FIGURE 7.-Lineweaver-Burk plots of velocity of the formation of leucyl-tRNA by wild- type (0) and leu-5 ( .) leucyl-tRNA synthetase as a function of leucine concentration in i he presence of excess tRNA.


TABLE 3

K , values for leucine for leucyl-tRNA synthetases calculated from Lineweaver-Burk plots of the velocity of l e q I - t R N A formation or the uelocity of the leucine actiuation

reaction in the presence of hydroxylamine

K, leucine

Strain Growth conditions Hydroxamate leucyl-tRNA

f m d l DL-leucine) formation formation

Wild type

leu-5

leu-5

0 1.42 x 1 W 2.13 x 10-7 1.51 x 2.46 x 1@7

2.38 x IO-; 50 5.52 x 10-7

6.74 x 10-7 5.44 x 1 0 - 7

300 2.25 x 1 W 4.18 x IO-: 4.52 x I@; 4.50 x 10-7

3.39 x 10-6

the K, calculated by the formation of leucyl-tRNA, both methods clearly indicate that the leucyl-tRNA synthetase from leu-5 has a significantly higher K, for leucine than does the !wild-type enzyme. Incubation temperature and the amount of leucine supplied to the mutant during growth, although strongly affecting the phenotype of leu-5 had only a slight effect on the affinity of the enzyme for leucine. Reduction of the temperature of the assay from 37" to 32°C had little effect on the difference between the K, values of mutant and wild-type enzymes.

Despite the difference in the leucine binding efficiency displayed by the leucyl- tRNA synthetase from leu-5, magnesium and pH optima as well as the binding constants of the mutant and wild-type enzymes for tRNA are essentially identical. Figure 8 is a Lineweaver-Burk plot of the reaction velocity of the leucyl-tRNA synthetases from leu-5 and the wild-type strain as a function of tRNA concen-

FIGURE 8.-Lineweaver-Burk plots of the reaction velocity of wild-type (0) and leu-5 ( ) leucyl-tRNA synthetases assayed in the presence of excess L-leucine and varying substrate tRNA concentrations.


tration. The data obtained indicate that the K, for the tRNA preparation used is approximately 5.4 x assuming an average molecular weight of tRNA of 25,500 (TISSI~RES 1956).

In order to determine whether, in addition to the lower affinity of the leu-5 leucyl-tRNA synthetase for leucine, translational errors were introduced during protein synthesis because of the charging of non-leucine specific tRNA with leucine, the fractionation of the leucyldH-tRNA synthesized with the leu-5 enzyme (from leu-2; leu-5) and the wild-type enzyme (from leu-2) was compared on MAK columns (YAMANE and SUEOKA 1963). The distributions of leucy13H- tRNA obtained with bloth enzyme preparations ‘were qualitatively similar. Al- though strain independent quantitative differences in peak height were often observed, one major and four relatively minor peaks were usually discernible. The presence of a full complement of amino acids during charging had little or no effect on the distribution obtained. A similar study using the mutant and wild- type enzymes to charge E. coli tRNA also failed to indicate charging of non- leucine specific tRNA.

Ribosome binding studies done according to the method of LEDER and NIREN- BERG (1964) with the major peak, after purification by chromatography on MAK indicated a strong preferential binding to poly UC (5:l) and somewhat less to poly UC ( 1 : 1 ) with very little binding to poly UA or poly UG. The minor peaks significantly bind to ribosomes in the presence of poly UA and poly UG as well as poly UC but absolute coding assignments are difficult because of excessive cross contamination and low specific activities. Nonetheless, the data obtained indicate that the major leucine tRNA probably recognizes a 2U, 1C triplet. The results of E. W. BARNETT (personal communication) suggest that the major peak is probably separable into two or three fractions by counter current distribution all responding to poly UC. The predominant fraction of the leucyl-tRNA of E. coli also responds to poly LJC ( WEISBLUM, GONANO, VON EHRENSTEIN, and BENZER 1965).

The ability of enzyme extracts of leu-5 and the wild-type strains to activate and transfer leucine to tRNA was found to be unaffected by the presence of 1,000-fold excesses of other amino acids. But in order better to detect possible recognition errors committed by the Zeu-5 leucyl-tRNA synthetase, tRNA charged with leucine was treated with periodate and subsequently assayed for the ability to accept leucine, phen:ylalanine, valine, isoleucine and methionine with mutant and wild-type leucyl-tRNA synthetases. Unfortunately, Neurospora leucyl-tRNA seems to be especially sensitive to periodate so that, under conditions necessary to destroy completely uncharged tRNA species, a major portion of the tRNA charged with leucine is also destroyed. At intermediate levels of periodate oxidation of leucyl-tRNA the “noise level” of residual valine, isoleucine and phenylalanine charging is too high to detect subtle differences. It should be pointed out that the sensitivity to periodate is not a general property of Neurospora tRNA, for phenylalanine charged tRNA is resistant to periodate oxidation and can be freed of essentially all leucine specific tRNA.


DISCUSSION

The data presented indicate that the leu-5 cistron determines some process involving leucine utilization rather than synthesis. This is suggested by the following observations: ( 1 ) the leu-5 mutant responds auxotrophically to exogenous leucine only at sublethal temperatures; (2) the temperature sensitivity characteristic of leu-5 is expressed regardless of leucine concentration in the presence or absence of a functional leucine biosynthetic pathway; ( 3 ) leu-5 complements mutations in each of the cistrons known to determine the structures of the leucine biosynthetic enzymes. However, the temperature sensitive phenotype of leu-5 is partially dominant in heterokaryons irrespective of the markers involved; ( 4 ) in leu-5 the leucine biosynthetic enzymes are present and normal regulatory mechanisms for the syntheses of these enzymes are operative.

It is apparent that the leu-5 mutation studied results in a rather widespread effect on protein synthesis. During growth at high temperatures a large fraction of the protein synthesized appears to be structurally altered and enzymatically “dead”. The production of significant amounts of thermolabile tryptophan synthetase, alcohol dehydrogenase, NADase, and a somewhat more thermostable 5-dehydroshikimic reductase in addition to synthesis of several enzymes with normal thermolabilities suggests that the leu-5 phenotype may result from a progressive loss of the fidelity of translation of some specific codon or codons as the temperature is raised.

That it is specifically a leucine codon or codons that is mistranslated is sug gested strongly by the auxotrophic response to added leucine at sublethal temperatures and the partial reversal of defective enzyme production by added leucine. This is most clearly demonstrable for tryptophan synthetase. Not only is the specific activity of this enzyme higher in extracts of the mutant grown in the presence of leucine but also, as has been observed repeatedly, the more leucine that is supplied during the growth of leu-5 the greater is the relative synthesis of enzyme with normal thermostability.

In view of the fact that the leucyl-tRNA synthetase produced by leu-5 has a significantly higher K, for leucine than the corresponding enzyme from the wild- type strain, it is tempting to propose that the alteration in the amino acid binding properties of the enzyme results in amino acid misrecognition at low but biologically significant levels. It does not seem likely, however, that the mere reduction of the affinity of the synthetase for leucine would be sufficient to generate amino acid recognition errors; leucine auxotrophs grown on limiting amounts of leucine do not produce significant amounts of structurally altered enzyme protein. Apparently the amino acid binding specificity of the normal leucyl-tRNA synthetase is sufficiently great at leucine concentrations well below the K, to effectively preclude mischarging of leucine specific tRNA. It seems clear then that if the primary effect of the leu-5 mutation is on leucyl-tRNA synthetase, the change in K, must be a manifestation of some specific alteration in the primary structure of the enzyme, presumably involving the amino acid “binding


site", that results in a small but biologically significant decrease in the fidelity of amino acid recognition.

Although the tempe rature-dependent partial auxotrophy and lethality of Zeu-5 can be accounted for rather readily with the aid of some simple assumptions regarding error frequency as a function of leucine concentration and temperature, it should be emphatically emphasized that direct evidence has not been obtained in support of the notion that the Zeu-5 cistron specifies the structure of the leucyl-tRNA synthetase or, for that matter, that mistranslation of leucine codons results in the observed production of defective enzyme protein. It has been impossible, primarily because of the instability of Neurospora leucine-specific tRNA to periodate oxidation, to demonstrate in uitro, mischarging of leucyl- tRNA; the in uitro synthesis of some specific protein employing tRNA charged with Zeu-5 leucyl-tRN.A synthetase should prove more revealing. It should also be emphasized that because of the relative instability of the leucyl-tRNA synthetase, the kinetic and physical properties of the enzyme have been determined only on relatively crude enzyme preparations. The kinetics of thermal inactivation of the synthetase in such preparations suggest that a single enzyme species is responsible for more than 80% of the synthetase activity. However, it has recently become quite clear that Neurospora extracts, like extracts of E. coli (Yu and RAPPAPORT 1966) ., contain at least one leucyl-tRNA synthetase in addition to the major enzyme species (M. F. TRIMBLE, personal communication). The presence of a plurality of leucyl-tRNA synthetases tends to complicate somewhat the analysis of the causal relation between a structural alteration in a leucine- tRNA synthetase and a decrease in the fidelity of translation. An analysis of the kinetic and structural properties of the purified synthetases and their genetic determination is now underway.

The authors are grateful to MRS. EVELYN GILMORE for her skillful assistance throughout these investigations.

SUMMARY

The mutant 45208t (Zeu-5), a temperature sensitive conditional lethal that is a partial leucine auxotroph at 22 to 30°C, synthesizes all of the leucine biosynthetic enzymes under iessentially normal regulatory control. A large fraction of the protein synthesized by the mutant under growth restrictive conditions, however, is modified structurally. The data strongly suggest that Zeu-5 is uniquely altered so that the fidelity of translation of some specific codon or codons is pro- gressively lowered as ihe incubation temperature is raised during growth. The mutant has been found to produce under both permissive and restrictive growth conditions a leucyl-tRNA synthetase with an increased K, for leucine.

466 D. B. P R I N T Z A N D S . R. GROSS

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