Met-tRNA hydrolase from reticulocytes specific for on 40S ribosomal subunits

ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS 162, %fi-397 (19%)

Met-tRNA Hydrolase from Reticuiocytes Specific for MET-tRNkMti

on 40s Ribosomal Subunits

JERRY MORRISEY AND BOYD HARDESTY

Clayton Foundation Biochemical Institute, Department of Chemistry, The University of Texas, Austin, Texas Y8Yll

Received May 17, 1972; accepted June 19, 1972

A protein fraction removed from rabbit reticulocyte ribosomes by 0.5 M KC1 re- leases methionine from methionyl-tRNAr Met bound to the smaller ribosomal subunit. The active component appears to be a Met-tRNA hydrolase. In the presence of the smaller subunit, the hydrolysis reaction is dependent upon codon-directed binding. Release of methionine from methionyl-tRNAtMet is inhibited by addition of larger ribosomal subunits to the reaction mixture. Both Met-tRNA#et and Met- tRNAfMet are hydrolyzed at low Mg* concentration in the absence of 405 subunits, however, the relative hydrolysis of Met-tRNAnMet is reduced in the presence of subunits. The Met-tRNAfMet binding requirement for hydrolysis in the presence of the subunits appears to reflect binding of the enzyme to the smaller subunit. Little or no hydrolysis is observed with N-acetylmethionyl-tRNAfn”t, Phe-tRNA or other forms of aminoacyl-tRNA tested, either in the presence or absence of 40s subunits. It. is suggested that the reaction described may be related to the accumulation of deacylated tRNArMet on ribosomes isolated from reticulocytes incubated with NaF.

Hoerz and McCarty (1) reported that NaF inhibition of intact rabbit reticulocytes causes an accumulation of polysomes with an extra 40s ribosomal subunit. Culp, Morrisey and Hardesty (2) and Pawelek et al. (3) found an accumulation of tRNArMst on these ribosomes isolated from cells incubated with NaF. A high proportion of this tRNArMet, up to 98 % or more, appears to be in the deacylated form. These NaF ribosomes support in vitro synthesis of globin peptides with valine as the aminoterminal amino acid (4). This synthesis is insensitive to edeine, an antibiotic that blocks formation of an mRNA ~40s subunit. Met-tRNAtMet initiation complex (5). These data have been interpreted to indicate that NaF blocks functional attachment of 605 ribosomal subunits to the initiation complex formed with the small subunit. It is not clear whether or not peptides with aminoterminal valine can be formed in vitro on ribosomes that bear deacylated tRNA*Met without formation of a Met-Val

peptide. Peptide initiation with aminoterminal methionine has been demonstrated in many laboratories and is generally accepted as the initiation pathway in higher organisms. Here we present evidence for an enzyme, tentatively named Met-tRNA hydrolase, with the anticipated properties that might allow it to release methionine from a Met-tRNArMet.40S ribosomal subunit * mRNA initiation complex formed in the presence of NaF.

MATERIALS AND METHODS

Biological Materials and Ap UpG

Description of the techniques and procedures, including preparation of unfractionated tRNA from rabbit liver, ribosomes and enzyme, used with the rabbit reticulocyte cell-free system are presented in a single reference source (6, 7). The salt wash fraction from which “Fraction III” is derived by chromatography on G-290 Sephadex, is prepared from regular reticulocyte ribosomes at a concentration of 20 mg/ml in a solution of 0.25 M sucrose, 0.1 mnx EDTA (pH 7.5) and 1 mM

3%

Copyright @ 1972 by Academic Press, Inc. All rights of reproduction in any form R?SWV~J%

386 MORRISEY AND HARDESTY

dithiothreitol. This solution of ribosomes was brought to 0.5 M KC1 by the cautious addition with stirring of 4.0 11p KC1 then allowed to stand on ice with gentle stirring for 15 min. The ribosomes or subunits were removed from the solution by centrifugation at 50,000 rpm for 2.0 hr (50 rotor, Beckman Instruments, Inc.). Protein was precipitated from the supernatant solution by the addition of 7.0 ml of saturated ammonium sulfate to 3.0 ml of supernatant solution to give a final concentration of 7Oyc of saturation at 0°C. The precipitated protein was collected by centrifugation and resuspended in a volume of solution B (100 mM KCl, 20 mM Tris.HCl, pH 7.5, 5 mrvr

p-mercaptoethanol and 0.1 mM EDTA) equal to the initial volume of the ribosome solution and stored at - 90°C until it was used as the “Salt Wash Fraction.”

Subunits of reticulocyte ribosomes were prepared by the procedure of Falvey and Staehlin (8) used with slight modification as described by Obrig et aE. (5). Met-tRNA and other forms of aminoacyl-tRNA were formed from deacylated rabbit liver tRNA under the conditions described by Culp et al. (2). Met-tRNA formed with methionine labeled with either 1% or generally 3% were used interchangeably. Met-tRNAr”et was separated from Met-tRNA#et by chromatography on BD-cellulose essentially as described by Kerwar, Spears and Weissbach (9). The N- acetylMet-tRNA was produced from unfractionated Met-tRNA by treatment with acetic anhydride as described by Haenni and Chapeville

(10). The polynucleotides used to direct binding of

aminoacyl-tRNA to ribosomes or subunits were from Miles Laboratories, Inc., Elkhart, Indiana. ApUpG was from Miles Laboratories or, for most of the work, was formed from ApU by a modified form of the procedure described by Leder (11). Modifications were suggested by Leder (personal communication) as follows: The ApU concentration in the initial reaction mixture in which ApUpG was formed was increased to 10 rnM and 4500 units of T-I ribonuclease (Worthington Biochemical Corp., Freehold, NJ) were added to this reaction mixture before incubation.

The Standard Met-tRNA Hydrolase Assay The basis for the assay system is similar to

that used by Goldstein, Beaudet and Caskey (12) to study peptide termination except that ApUpG is the only form of mRNA used with 40s ribosomal subunits rather than intact ribosomes. Met-tRNAr”et is nonenzymatically bound with ApUpG at relatively high Mgl+ concentrations to 40s ribosomal subunits during a preliminary incubation and then enzymatic hydrolysis of Met- tRNAr”et is measured as acid-soluble methionine

formed during a second incubation. Each milliliter of the preliminary incubation mixture con- tains 20 mM A-2-hydroxyethylpiperazine-N’- 2’ethanesulfonic acid (HEPES) adjusted to pH 7.2 at room temperature with KOH; 80 mM KCI; 40 mM MgC12, 4 mM dithiothreitol, 50 pmoles Met-tRNArMet (generally %.methionine at 1.0 Ci/mmole); 7.5 AzsO units of 405 ribosomal subunits, and 1.5 A2ea units of ApUpG trinucleotide. This mixture is incubated for 8 min at 37°C then chilled to 0°C. Typical preparations of Met- tRNA? contain about 8% of the total methionine as free methionine soluble in cold 5% trichloroacetic acid before this incubation. About 20% of the total methionine present in the incubation mixture is soluble in cold 5% trichloroacetic acid after this incubation.

After this preliminary incubation, 100 ~1 of the reaction mixture is placed in each of two tubes for each reaction conditions to be tested. To one of the tubes 20 ~1 of fraction III protein, prepared as described later, is added and an equal amount of solution B is added to the other. Unless otherwise indicated the exact amount of fraction III protein used is adjusted by dilution of fraction III with solution B to give 2.0 f 0.2 pmoles of enzymatic hydrolysis. A typical relation between fraction III protein and enzymatic hydrolysis is given in Fig. 1. The final salt concentrations in the second incubation mixture are 33 mM

MgCl2, 83 mM KCl, 17 mM HEPES, 3.3 mM Tris, and 17 PM EDTA. Both tubes are incubated for an additional 3.0 min at 37°C. The tubes are placed in ice and 120 d of 10% cold trichloroacetic acid and then 20 ~1 caseine solution (10 mg caseine/ml of water) are added to each tube. The precipitated material is pelleted by centrifugation at 4°C. From the upper, liquid phase of each sample, 130 ~1 is placed into a liquid scintillation counting vial and counted with 10 ml of a 10% Biosolve (Beckman Instruments, Inc., Palo Alto, Cal.) toluene scintillation fluid (5.0 g 2,5 diphenyloxa- zole per 1 of toluene). Samples were counted at room temperature to a standard error of 2.0%. The 3% is counted with an efficiency of 89% under the conditions used. Enzymatic hydrolysis is calculated as the difference in total soluble methionine formed in the reaction mixtures in the presence and absence of the enzyme fraction.

Binding to 40X Subunits

For some experiments, the amount of Met- tRNA@ bound to 40s ribosomal subunits after the second incubation was determined as material retained on nitrocellulose filters by the procedure described by Nirenberg and Leder (13) used with minor modification. For these determinations, the preliminary and second incubations were carried out as described pre-

SPECIFIC HYDROLYSIS OF Met-tRNA 387

viously. After the second incubation, the reaction diately. Each filter was washed with three sepa- mixture was diluted by the addition of 3.0 ml of rate portions, approximately 3 ml each, of the cold solution containing 20 mM HEPES, pH 7.2, same cold solution then dried at 120°C for 5 min 40 mM MgC12, and 80 mM KC1 then filtered imme- and counted by liquid scintillation. Typical

2.4

0.0 0 5 IO IS 20 25 30 35

pg PROTEIN

I 2 3 4 5 6

TIME (min)

FIG. 1. The effect of fraction III protein and time of incubation on Met-tRNAfMet hydrolysis in the standard assay procedure. A. The times indicated are for incubation at 37°C during the second incubation of the standard assay procedure. Enzymatic hydrolysis was calculated as the difference in the amount of free methionine observed in the presence and absence of fraction III, as described under Methods. B. Fraction III was con- centrated to 1.7 mg protein per milliliter of solution B by vacuum dialysis of the fractions from G-200 Sephadex. Aliquots from this preparation of fraction III were diluted with solution B to give the indicated amounts of protein in 20 ,,J, then used in the standard hydrolysis assay procedure. The 40s ribosomal subunits were omitted from the standard assay reaction mixtures as indicated.


results for binding and hydrolysis with a number of experimental conditions are given in Table I.

Preparation of Fraction III

Fraction III, containing Met-tRNArMet hydrolase activity was prepared routinely by chromatography of the salt wash fraction on G-200 Sephadex as depicted in Fig. 5. Salt wash fraction containing 10 mg of protein in about 2 ml was layered on a 2 x 50 cm column of G-200 Sepha- dex (Pharmacia Fine Chemicals Inc., Piscataway, NJ) thoroughly equilibrated wibh solution B. The column was eluted with soIution B at n flow rate of about I5 ml/hr. Absorption at 280 nm and enzymatic activity was determined on aliquots of 3.0-ml fractions. Met-tRNA?“t hydrolase activity in 40-~1 aliquots of these fractions was determined by the standard assay system as described previously with the following modifications. The second incubation in the presence of Met-tRNA? hydrolase is carried out for 4.0 min at 37°C in- stead of 3.0 min. Also, 140 ~1 rather than 120 ~1 of 10% t,richloroacetic is added to the reaction mixture after incubation to give a final concentration of 5%.

TABLE I

REQUIREMENTS FOR HYDROLYSIS OF

MET-tRNAr”et

Addition in first Fraction Met- Total free Enzymatic incubation” III added tRNAtme’ methionine hydrolysis

bound pm&s

pm&s pmoles

None - 0.02 1.18 0.23 + 0.15 1.41

405 - 0.13 0.81 0.71

+ 0.22 1.52 40s + - 2.12 0.91 2.24

APUPG + 0.43 3.15 60s - 0.20 1.13 0.16

+ 0.28 1.29 40s + 60s - 1.77 0.68 0.19

+ 1.87 0.87 40s + 60s + - 2.51 0.78 0.35

APUPG + 2.23 1.13

0 The components indicated were added before the preliminary incubation. The preliminary incubation mixture of the standard assay procedure was modified by omission of 40s ribosomal subunits and ApUpG except as indicated. Met- tRNAfMet bound to subunits after the second incubation was determined as described under Methods. Total free methionine is material soluble in 5% trichloroacetic acid after the second incubation. Enzymatic hydrolysis is calculated as the difference in total free methionine observed in the presence and absence of fraction III.

Fractions from G-290 Sephadex were also tested for ability to hydrolyze Met-tRNArMrt in the absence of 40s subunits with the results depicted in Fig. 5B. For these assays a solution was prepared that contained the following per milliliter: 20 PM N-2-hydroxyethylpiperazine- N’-2-ethanesulfonic acid (HEPES), pH 7.2, 80 mM KCl, 40 mM MgCl2, 4 mM dithiothreitol, and 50 pmoles Met-tRNArMet (generally 3%. methionine at 1.0 Ci/mmole). These are the concentrations of these components that are used in the first incubation mixture of the standard assay system described previously. To 100~~1 aliquots of this mixture were added 40 yl of the G-200 fractions collected as described previously or solution B as a blank. The 140-~1 reaction mixtures were incubated for 4.0 min at 37°C then 140 pl of 10% trichloroacetic acid and 0.2 mg caseine in 20 ~1 were added at 0°C. The precipitate is pelleted by centrifugation and acid-soluble methionine in 150 ~1 of the supernatant is determined as in the standard assay procedure. En- zymatic hydrolysis is calculated from the increase in acid-soluble methionine observed in fractions containing enzyme as described for the standard assay system.

Fractions from G-200 Sephadex within the volume eluted from about 90-115 ml containing Met-tRNA hydrolase were pooled as shown in Fig. 5B then vacuum dialyzed (14) to a final volume of l-2 ml against solution B containing 5% glycerol. The protein and nucleic acid con- centratious of typical fraction III preparations is about 0.8 mg/ml and 8 pg./ml, respectively, as determined by the method of Warburg and Chris- tian (15). Fraction III was stored at -90°C up to 2 months until it was used. Only preparations of fraction III that liberate 2.0 f 0.2 pmoles of methionine with 10-20 pg of protein were used in this study, as considered in relation to Fig. 1B.

RESULTS

Characterization of the Assay System

The effect of 40s and 60s ribosomal subunits and ApUpG on hydrolysis of Met-tRNAtMet by the hydrolase fraction, fraction III, is indicated in Table I. In the basic assay system used in this study, Met-tRNArMet is bound to reticulocyte 405 ribosomal subunits with ApUpG at 40 m&f MgCl. Use of unfractionated Met-tRNA in the standard hydrolysis assay system results in an appreciably lower proportion of the total free methionine that is related to the activity of Met-tRNAtMet hydrolase because of free methionine from Met- tRNAMMet. Typically, about 2.1 pmoles of


Met-tRNArMet are bound to the 405 subunit during the preliminary incubation. The reaction mixtures are chilled and the hydrolase fraction is added as indicated. Then the mixtures are incubated at 37°C for an additional 3 min. Free, labeled methionine from Met-tRNAfMet is determined in an aliquot of the reaction mixture as radioactive material soluble in 5% trichloroacetic acid at 0°C. Details of the procedure used are given under Methods. For the data of Table I, Met-tRNArMet bound to ribosomal subunits after the second incubation was determined in aliquots of the reaction mixture as material retained on nitrocellulose filters. Met-rRNArMet in appreciable amounts is retained by the filters with reaction mixtures containing 405 subunits and ApUpG.

Appreciable hydrolysis of Met-tRNAtMet as indicated by the total free methionine present in t’he reaction mixtures occurs in the absence of fraction III. The amount of free methionine observed is dependent upon the specific conditions used. Generally, the amount, of free methionine formed in the absence of fraction III is lower under conditions in which appreciable binding of the Met-RNA to ribosomes is observed. This free methionine appears to represent primarily nonenzymatic hydrolysis of Met- tRNAfMet that occurs before and during the two consecutive incubations. It con- stitutes a troublesome blank value that must be considered in evaluating enzymatic hydrolysis. Unless stated otherwise, all values for “enzymatic hydrolysis” presented below represent the difference between total free methionine observed in the presence and absence of fraction III under the particular conditions used as described for the standard assay system. Also, preparations of unfractionated salt wash fraction contain some enzymatic activity for hydrolysis of all forms of aminoacyl-tRNA. Unlike the Met-tRNA hydrolase activity of fraction III characterized in this study, it is not dependent upon binding of aminoacyl- tRNA to the 40s subunit. This hydrolysis can be inhibited by addition of rRNA to the hydrolysis reaction mixture and appears to be due, at least in part, to nonspecific nuclease activity. Nonspecific activity for

hydrolysis is low in fraction III used in this study, but may be relatively high in unfractionated preparations of salt wash protein.

The data of Table I depict net hydrolysis of Met-tRNArMet due to the fraction III. Enzymatic hydrolysis appears to be strongly dependent upon codon-directed binding to the 40s subunit. This specific hydrolysis is inhibited by the addition of 60s ribosomal subunits to the reaction mixture.

The time course for formation of free methionine during the second incubation of the standard assay system is presented in Fig. 1A. Free methionine present in the reaction mixtures after the preliminary incubation has been subtracted from the total free methionine observed. In the presence of fraction III hydrolysis during the second incubation is nearly linear for about 3 min. the time used in the standard assay system. Hydrolysis in the absence of fraction III is linear for at least 10 min. Hydrolysis determined in the standard assay system modified to cont’ain the indicated amount of protein from fraction III is depicted in Fig. 1B. Some variation of hydrolysis activity has been observed for different. preparations of fraction III. Only preparations that liberate about 2.0 pmoles of methionine with lo-20 Ng of protein were used in this study. The exact. amount of protein used in the experiments described later has been adjusted to give 2.0 f 0.2 pmoles of hydrolysis under the standard assay conditions, unless otherwise indicated.

Binding of Met-tRNArMet to 40s subunits in the presence of ApUpG is strongly dependent on Mg2+ concentration in the basic assay system, as shown in Fig. 2A. The curve for hydrolysis by fraction III in the presence of 40s subunits is approximately parallel to the curve for binding over the range of Mg2+ concentrations tested and appears to reflect a requirement for binding of Met-tRNAtMet to ribosomes for enzymatic hydrolysis, under these reaction conditions.

The Mg2+ concentration has a striking effect on enzymatic hydrolysis in the absence of 40s subunits. Under these conditions fraction III dependent hydrolysis is low at high concentrations of Mg”+, but in- creases to relatively high levels at low con-


centrations of Mg”+. Met-tRNA hydrolase activity in the presence of 405 subunits appears to be specific for Met-tRNAtMat, however, in the absence of 405 subunits, Met-tRNAMet is hydrolyzed. These observations are considered later in relation to Tables II and III.

Results for a range of K+ concentration

at 40 rntir Mg2+ are shown in Fig. 2B. Bind- ing of Met-tRNAt Met to 40s subunits at 40 mu Mg2+ decreases slightly between about 80 nnr and 300 rnmr KU. In contrast, a change of nearly tenfold is observed for enzymatic hydrolysis over this K+ concentration range. Although it cannot be excluded that these K+ and Mg2+ effects

I 1 I I

0 60 120 I80 240 300

c 1 KCI * mM FIG. 2. The effect of MgClz and KC1 on binding and hydrolysis of Met-tRNAF. The pre-

liminary incubation mixture of standard assay procedure was modified to contain the indicated concentration of MgCla with 80 mM KCl, or KC1 with 40 mM MgC12. Binding to 40s subunits in the absence of fraction III or enzymatic hydrolysis of Met-tRNAPt was measured after the second incubation, as described under Methods. A-A-Enzymatic hydrolysis without 405 subunits; A--A-enzymatic hydrolysis with 405 subunits; O-O -Met-tRNAP binding with 40s subunits.


TABLE II

AiuIivoAcyL-tRNA SPECIFICITY FOR ENZYMATIC

HYDROLYSIS AT 40 mM MgClz IN THE

PREWNCE OF 40s RIBOSOMAL SUBUNITS

Type tRNA pmoles 40s Enzymatic useda binding hydrolysis

pmoles pmoles

Met-tRNAf”et 5 1.92 2.15 Met-tRNA#et 20 1.50 0.30 N-acetylMet-tRNA 20 1.95 -0.01 Phe-tRNA 5 2.04 0.13 Val-tRNA 20 1.17 0.36 Lys-tRNA 20 1.28 -0.09

a The total amount of aminoacyl-tRNA insoluble in cold 5% trichloroacetic acid added to the preliminary incubation mixture. The standard assay procedure was modified by substitution of the indicated type and amount of tRNA and an appropriate form of mRNA into each 1.0 ml of the preliminary incubation mixture as: ApUpG, 1.5 Azsa units, for Met-tRNAr”et and N-acetylMet- tRNA; poly AGU, 400 pg, for Met-tRNAn”et, Val-tRNA and Lys-tRNA; poly U, 200 pg, for Phe-tRNA. Binding of aminoacyl-tRNA in reaction mixtures lacking fraction III was measured after the second incubation.

might reflect the activity of two or more enzymes in fraction III, they appear to reflect a requirement for simultaneous binding to 405 subunits of both Met- tRNAfMst and a single Met-tRNA hydrolase. Met-tRNA is efficiently bound only at high Mg2+ concentrations. The enzyme may bind efficiently to subunits only at relatively low concentrations of K+ so that enzymatic hydrolysis is reduced for this reason above about 80 m&i KCl. Binding of the enzyme to 405 subunits was tested with the results depicted in Fig. 3. For these experiments fraction III and 40s ribosomal subunits were incubated together in the proportions used in the standard assay system, then layered on a sucrose gradient containing the same salts present in the incubation mixture. After centrifugation, the distribution of Met-tRNA hydrolase activity and 40s ribosomal subunits in the gradient were determined. Met-tRNA hydrolase is efficiently bound to 40s subunits at 80 mM KC1 and 1.2 mnr MgCl,. Hydrolysis at low Mg2+ in the absence of 40s subunits (Fig. 2A) may occur when both enzyme

and Met-tRNA are in solution at low Mg2+ concentration.

The results of similar experiments not depicted here indicate that the enzyme is not bound to 40s subunits in 300 mM KCl. These results appear to support the hypothesis outlined previously, however, they provide no indication of why Met-tRNA hydrolase activity is reduced to low levels at 40 mM Mg2+ in the absence of 40s subunits. Further investigation is required for a firm conclusion about the basis for these effects of K+ and Mg2+ as well as the change in specificity for hydrolysis of Met-tRNAMMet described later.

Aminoacyl-tRNA Specificity

The activity of fraction III for enzymatic hydrolysis of various forms of aminoacyl- tRNA in the presence of 405 subunits is presented in Table II. The standard assay system was modified by the addition of the indicated amount and type of aminoacyl- tRNA with an appropriate form of mRNA to provide codon-directed binding to 405 ribosomal subunits. The levels of aminoacyl-tRNA binding to 40s subunits are

TABLE III

AMrNoAcYn-tRNA SPECIFICITY FOR ENFXMATIC

HYDROLYSIS IN THE ABSENCE OF MgClz AND 40s RIBOSOMAL SUBUNITS

Type tRNA” Enzymatic hydrolysis pmoles

Met-tRNArMet 4.94 Met-tRNAMMet 5.05 N-acetylMet-tRNA -0.32 Phe-tRNA 0.35 Val-tRNA 0.15 Lys-tRNA 0.09 Arg-tRNA 0.02

0 5.0 pmoles of aminoacyl-tRNA insoluble in cold, 5% trichloroacetic acid were added to each reaction mixture. The standard reaction mixture for enzymatic hydrolysis was modified by the omission of MgClz , 405 ribosomal subunits and ApUpG or other forms of mRNA. The reaction mixtures were not preincubated before the addition of 10 pg of fraction III protein. Hydrolysis was determined as the increase in acid soluble amino acid caused by fraction III during 3.0 min of incubation at 37°C.

MORRISEY AND HARDESTY

2.0 3.0 4.0 5.0

Fraction , ml

0 1.0 2.0 3.0 4.0 5.0

Fraction , ml

FIG. 3. Sucrose gradient centrifugation of Met-tRNA hydrolase in the presence or absence of 405 subunits. Fraction III protein, 86 pg in 0.5 ml of solution containing 80 mM KCl, 1.2 mu MgClz, 16 mM Tris-HCl, pH 7.5,2 mM dithiothreitol, 1.6 rnM ,+mercaptoethanol and 1% glycerol, was layered on a 153Oa/, linear sucrose gradient also containing the components cited. Centrifugation was for 2.5 hr at 60,006 rpm. The distribution and concentrations of protein and 40s subunits were determined from the optical density of about 0.5 ml fractions. Enzymatic hydrolysis was determined with 46 d of each fraction assayed in the standard assay system ( -, protein; - - -, 405 ribosomal subunits; -, enzymatic hydrolysis). (A) Fraction III protein centrifuged as described above. (B) Fraction III protein plus 1.25 mg of 405 ribosomal subunits added to give the final 0.5 ml solution were centrifuged and assayed as described above.

indicated. This binding was measured in the absence of fraction III after the second incubation of the standard assay procedure. Only Met-tRNArMet gives appreciable enzymatic hydrolysis under these conditions. It is particularly interesting that Met- tRNAMMet and N-acetylMet-tRNAtMst are not hydrolyzed efficiently even though both are bound to 405 subunits in relatively high amounts with Poly(A,G,U) or ApUpG. Housman et al. (16) reported that initiating methionine is removed from nascent. globin peptides but cannot be removed when its

amino group is blocked as in N-formyl- methionine.

The aminoacyl-tRNA specificity for enzymatic hydrolysis with no added MgClz and in the absence of 405 ribosomal subunits is shown in Table III. In contrast to the specificity for Met-tRNArMet observed at 40 mM MgClz in the presence of 4QS subunits, nearly all of the Met-tRNAM”et added to these reaction mixtures was also hydrolyzed. No appreciable hydrolysis is observed with any of the other forms of aminoacyl-tRNA tested including N-acetyl-


Met-tRNAfMet. These data appear to indicate hydrolase activity specific for Met- tRNArMst rather than Met-tRNAM”et only when the enzyme and tRNA are bound to 40s subunits. They might reflect the requirement for hydrolysis of a specific spatial relation between Met-tRNAtMet and the enzyme that does not occur with bound Met-tRNA#et.

Analysis of the Hydrolysis Products

Electrophoresis and chromatography on paper were used to determine whether the product released from Met-tRNArMet by fraction III was methionine, a related derivative, or possibly a nucleotide frag- ment from tRNAfMst to which methionine remained attached. Electrophoresis was carried out in solvent systems at pH 1.9 and pH 6.0. Descending chromatography was performed in two different solvent

systems. Typical results for one chromatographic system are shown in Fig. 4. In all analytical systems, more than 80 %, usually about 90 %, of the soluble product formed migrated with authentic methionine. Methionine is concluded to be the product released by the action of fraction III in cleaving the ester bond between adenosine and methionine. An additional minor radioactive component, possibly an oxida- tion product of methionine, was observed in most reaction mixtures. A similar component was observed in preparations of 35S .methionine that had not been attached to tRNA or carried through the incubation procedures of the standard assay system.

The other product of the reaction, tRNAf”et was isolated from a standard hydrolysis assay by the phenol extraction procedure previously described (6). The isolated tRNAfMst was tested for methionine

Ninhydrin I I I

a I

Minor

t Methionlne

4

0 5 IO I5 20 25 30

CM FROM ORIGIN

FIG. 4. Chromatography of the hydrolysis product from %.Met-tRNAr”et. The hydrolysis reaction was carried out under the conditions of the standard assay procedure except that 10% trichloroacetic acid was not added. An aliquot of 100 d of the chilled reaction mixture was spotted directly on Whatman 3MM paper. The dried sample was chromato- graphed with a descending solvent of butanol:acetic acid:water, 120:30:50, until the solvent front had moved about 45 cm. Samples of untreated *%.Met-tRNArMst and asS. methionine or unlabeled methionine were run as controls. Unlabeled methionine was detected by ninhydrin with the results indicated. Radioactive material was detected in a double-window gas flow strip counter (Model 330, Vanguard Instrument Co., La Grange, IL).


acceptance capacity as previously outlined Fig. 4 that only methionine is cleaved from (2) and it was found that the tRNAPe* Met-tRNAfMet in the standard hydrolysis retained greater than 90% of the original assay. acceptance capacity (data not shown). ” Thus the tRNArMet appears to be intact as Chromatography and Imxtivation of the

measured bv its abilitv to accent methio- Salt Wash Fraction

nine. This “observation is consistent with The elution profile of material removed the conclusion drawn from the data in from ribosomes with 0.5 I\~I KY, the salt

A

2 1.0 - zi cu - 0.9 - > I- 5 E P ;: 0.5 - 0 F 8

9 HEMOGLOBIN

4

2.7 s

0

Met-tRNA L

-2.1 a z

I

-1.5 2

0.2

55 70 95 100 II5 130 I45

FRACTION IIt

55 70 85 100 II5 130 I45

ELUTION VOLUME (ml)

FIG. 5. Chromatography of the salt wash fraction from ribosomes on G-200 Sephadex. The chromatographic and assay procedures used to determine Met-tRNAf”et hydrolysis are described in Methods for the preparation of fraction III. The 40s ribosomal subunits were added (A-A) or omitted (A-A) from the reaction mixtures as indicated. Met-tRNA synthetase activity (O-O) was determined by the formation of Met-tRNA essentially as described previously (21) except that 0.10 ml from each G-200 fraction was used as an enzyme source.


wash fraction, on G-200 Sephadex is shown in Fig. 5. A large peak of protein and nucleic acid is eluted near the column void volume. Hemoglobin, molecular weight about 64,000, is eluted at about 125 ml. Met-tRNA synthetase activity as indicated by charging of unfractionated tRNA with methionine emerges as a nearly symmetrical peak slighty behind the void volume at about 60 ml. Activity for hydrolysis of Met-tRNArMet in the standard assay system used without 40s ribosomal subunits is seen as a single symmetrical peak near the void volume, as shown in Fig. 5B. Two peaks of activity for hydrolysis in the presence of 40s subunits are observed. A large, nearly symmetrical peak of activity not observed in the absence of 40s subunits is seen under these conditions. The maxi- mum of this peak at an elution volume of about 105 ml indicates a component of a molecular weight in the order of 100,000. This hydrolysis activity appears to be distinct and separable from Met-tRNA synthetase. Fractions eluting between 90 and 115 ml were combined and used as fraction III in this study.

A second, smaller peak of hydrolysis activity in the presence of 40s subunits is observed near the void volume at about 60 ml and appears to be coincident with hydrolysis activity observed in the absence of 40s subunits. This activity appears to be related to nonspecific hydrolysis of aminoacyl-tRNA as described previously under characterization of the assay system. Reduction in the amount of hydrolysis observed with this component in the presence of 40s subunits may be related to binding of Met-t.RNAtMet to the 405 subunit. Met- tRNAfMet bound to subunits may be par- tially protected from hydrolysis.

The activity for hydrolysis with fraction III is inactivated by heating between 45 and 50°C under the conditions described with Table IV. In addition, the activity has been observed to be sensitive to sulf- hydryl reactive reagents under a number of conditions. Data are presented in Table IV that depict inactivation by N-ethylmaleimide. Dithiothreitol has been found to protect the activity during further fractionation of fraction III from G-200

TABLE IV INACTIVATION OF FRACTION 1110

Treatment Enzymatic hydrolysis pmoles

40°C 45°C 50°C

N-ethylmaleimide N-ethylmaleimide + dithio-

threitol

1.82 1.52 0.06 0.12 1.63

a Undiluted fraction III, 0.82 mg protein per milliliter of solution B, was heated at the indicated temperature for 5.0 min or made 10 mu in N-ethylmaleimide by the addition of 0.2 M solution. After 15 min at 0°C samples containing N-ethylmaleimide were brought to 10 mu in dithiothreitol by the addition of 0.25 M solution. In one sample of fraction III, the dithiothreitol was added before the N-ethylmaleimide. Aliquots of 20 ~1 were removed from each sample of fraction III and assayed for Met-tRNAr”et hydrolase activity in the standard assay procedure.

I I I

I I I I I 0.5 1.0 1.5 2 .o

A260 UNITS, 60 S

FIG. 6. The effect of 66s ribosomal subunits on Met-tRNAtMat hydrolysis by fraction III. The indicated amount of 600s ribosomal subunits were added before incubation as the final component to each 1.0 ml of the preliminary incubation mixture, then hydrolysis was measured as described for the standard assay procedure.

Sephadex. These observations, with the chromatographic profile of Fig. 5, are consistent with relatively heat labile, sulf- hydryl sensitive single enzyme of a molecular weight in the order of 100,000.


Inhibition of Enzymatic Hydrolysis by 608 Subunits

The relationship between fraction III dependent hydrolysis and 6OS ribosomal subunits added to the standard assay system is shown in Fig. 6. The 60s subunits in the amounts indicated were the last component added to the first incubation mixture in which binding of Met-tRNAtMet to the 40s subunits is normally carried out. Hy- drolysis was measured as usual after a second incubation with fraction III. Hy- drolysis is reduced by about 80% with 1.5 A260 units of 60s subunits added to the reaction mixture. This level of 60s subunits corresponds to a molar subunit ratio of about one to one (17). Somewhat greater inhibition is observed with higher levels of 60s subunits. There are very few free 405 subunits present in the preliminary incubation mixture under the conditions used when 1.5 A260 units or more of 60s subunits are added to the system.

DISCUSSION

The data presented indicate an enzyme that is able to release methionine from Met-tRNAtMet bound to 405 ribosomal subunits. We propose the temporary name of Met-tRNA hydrolase for this enzyme until its properties and function are understood to a degree to justify formal nomenclature. This hydrolase activity may be related to therelease of methionine from Met-tRNArMet observed by Caskey and his co-workers in the absence of termination codons (18, Table I). However, the termination assay performed by these workers was carried out with both ribosomal subunits.

As outlined previously in the introduction, the original objective of this study was to detect and characterize an enzyme that wouldreleasemethioninefrom Met-tRNArMet bound to the small ribosomal subunit. Such an enzyme might account for deacylated tRNA$fet observed on ribosomes after inhibition of protein synthesis by NaF. The properties of Met-tRNA hydrolase are those anticipated. However, at least two necessary major points supporting this hypothesis have not been established in our studies to this time. The studies

described here use synthetic mRNA’s, generally ApUpG, with high Mg2+ to nonenzymatically bind Met-tRNArMet to the small ribosomal subunits. Specific hydrolysis with a Met-tRNAfMet ’40s subunit. mRNA formed enzymatically at low Mg2+ has not been conclusively demonstrated, due to the difficulty in forming and handling this initiation complex in the mammalian system. Also, it has not been practical to demonstrate conclusively that deacylated tRNAf”et formed by the action of the hydrolase remains attached to the initiation complex as appears to be the case during NaF inhibition. Despite these limitations in reaching a firm conclusion, it appears likely that enzyme may function during NaF inhibition of peptide initiation in intact reticulocytes.

It should be emphasized that the data presented provide little insight into the physiological role the enzyme might play in vivo. The apparent specificity of the enzyme for Met-tRNArMet bound to the 405 subunit suggests a function in peptide initiation or regulation of protein synthesis. The very low hydrolysis of N-acetylMet- tRNA observed in either the presence or absence of 40s subunits indicates that this factor is distinguishable from the acyl- aminoacyl-tRNA hydrolase studied in sev- eral laboratories (19, 20).

It is of particular interest that fraction III will hydrolyze efficiently both Met- tRNAfMet and Met-tRNAMMst in the absence of 405 subunits but appears to hydrolyze only Met-tRNAfMet under conditions of the standard assay system. Our very tentative interpretation of these data is that Met- tRNA hydrolase and Met-tRNArMet are bound to 40s subunits in a special configuration in which hydrolysis can occur. A corollary of this hypothesis is that Met- tRNAMMet is not bound in this configuration. Some indication may be seen in the binding data of Table II that Met-tRNAMMet and Met-tRNAfMet are not bound to 40s subunits in the same way, even though the corresponding tRNA species from E. coli have the same anticodons (21). Numerous studies (22) with systems derived from many species including rabbits (23) have shown


differences in binding of these two tRNA species.

ACKNOWLEDGMENTS

The authors are grateful to Merle Batty, David Konecki and Mildred Hardesty for their excellent technical assistance and to Margaret Cooper for her help in preparing the typescript. This work was supported in part by Grant HD 03803 from the National Institutes of Health, U.S. Public Health Service and Grant GB 30902 from the U.S. National Science Foundation.

REFERENCES

1. HOERZ, W., AND MCCARTY, K. (1971) Biochim. Biophys. Acta 228, 526.

2. CULP, W., MORRIBEY, J., AND HARDESTY, B. (1970) Biochem. Biophys. Res. Commun. 40, 777.

3. PAWELEK, J., GODCHAUX, W., GROSSO, J., SKOULTCHI, A., EISENSTADT, J., AND LEN- GYEL, P. (1971) Biochim. Biophys. Acta 232,289.

4. LIN, S., MOSTELLER, R., AND HARDESTY, B. (1966) J. MoZ. Biol. 21, 51.

5. OBRIG, T., IRVIN, J., CULP, W., AND HAR- DESTY, B. (1971) Eur. J. Biochem. 21, 31.

6. HARDESTY, B., MCKEEHAN, W., AND CULP, W. (1971) in Methods in Enzymology (Grossman, L., and Moldave, K., eds.), Vol. 12, Part C, p. 316, Academic Press, New York.

7. HARDESTY, B., AND MCKEEHAN, W. (1971) in Methods in Enzymology (Grossman, L., and Moldave, K., eds.), Vol. 12, Part C, p. 330, Academic Press, New York.

8. FALVEY, A. K., AND STAEHELIN, T. (1970) J. Mol. Biol. 63, 1.

9. KERWAR, S., SPEARS, C., AND WEISSBACH, H. (1970) Biochem. Biophys. Res. Commun. 41,78.

10. HAENNI, A., AND CHAPEVILLE, F. (1966) Biochim. Biophys. Acta 114, 135.

11. LEDER, P. (1968) in Methods in Enzymology (Grossman, L., and Moldave, K., eds.), Vol. 12, Part B., p. 837, Academic Press, New York.

12. GOLDSTEIN, J., BEAUDET, A., AND CASKEY, T. (1970) Proc. Nat. Acad. Sci. 87, 99.

13. NIRENBERG, M., AND LEDF,R, P. (1964) Science 146, 404.

14. RICHARDSON, C., AND KORNBERG, A. (1964) J. Biol. Chem. 239, 242.

15. WARBURG, O., AND CHRISTIAN, W. (1942) Biochem. Z. 310, 384.

16. HOUSMAN, D., JACOBS-LORENA, M., RAJ- BHANDARY, U. L., AND LODISH, H. (1970) Nature (London) 227, 913.

17. Ts’O, P., AND VINOGRAD, J. (1961) Biochim. Biophys. Acta 49, 113.

18. CASKEY, T., SCOLNICK, E., TOMPKINS, R., GOLDSTEIN, J., AND MILMAN, G. (1969) Cold Spring Harbor Symp. Quant. Biol. 34, 479.

19. DEGROOT, N., PANET, A., AND LAPIDOT, Y. (1968) Biochem. Biophys. Res. Commun. 31,37.

20. CHAPEVILLE, F., YOT, P., AND PAULIN, D. (1969) Cold Spring Harbor Symp. Quant. Biol. 34, 493.

21. CORY, S., AND MARCKER, K. (1970) Eur. J. Biochem. 12, 177.

22. LUCAS-LENARD, J., AND LIPMANN, F. (1971) Ann. Rev. Biochem. 40, 409.

23. GUPTA, N., CHATTERJEE, N., WOODLEY, C., AND BOSE, K. (1971) J. Biol. Chem. 246, 7460.

Met-tRNA hydrolase from reticulocytes specific for on 40S ribosomal subunits

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