Action of spleen phosphodiesterase upon soluble ribonucleic acid

ARCHIVES

Action

OF

BIOCHEMISTRY

.iNI)

of Spleen

125,

BIOPHYSICS

480.-487

Phosphodiesterase

JAY K. GUNTHER,2 fiiochemistry Received

Division, November

(1963)

upon

A. Y. CHANG,3 University

of Illinois,

20, 1967; accepted

Soluble AND

Ribonucleic

J. M. CLARK,

Urbana,

Illinois,

January

15, 1968

JR.

61811

The attack of spleen phosphodiesterase upon aminoacyl-sRNA’s results in a rapid loss in ability of the aminoacyl-sRNA to transfer its amino acid into polypeptide material. There is a direct correlation, within the degraded aminoacyl-sRNA’s, between this loss in amino acid transfer ability and a loss in aminoacyl-sRNA, mRNA, ribosome complex forming ability. This suggests that the loss in amino acid transfer ability within aminoacyl-sRNA upon treatment with spleen phosphodiesterase is due to inability of the degraded aminoacyl-sRNA to complex with mRNA r~?on ribosomes. The structural and ftmctional implications of this observation ne:essitate a more definitive study of the action of spleen phosphodiesterase lIpon sRNA and aminoacyl-sRNA. Evidence is presented supporting the concept that spleen phosphodiesterase, or a contaminant in same, catalyzes an initial endonttclenlytic cleavage within sRNA’s after which nucleoside monophosphat.cs are released sequentially from the site of endonucleolytic cleavage.

The important role that sR!SA’s play in protein biosynthesis has prompted great interest, in sRNA structure and function. The recent determination of the nucleotide sequence of several specific sRNA’s (l-4) has revealed that each amino acid specific sRNA comains a unique trinucleotide sequence that is both complementary to a mRNA codon of the amino acid carried by the sRn’A and potentially functional in that it’ is apparently not involved in intrachain hydrogen bonding. Final characterization of the exact role that these trinucleotides play in sRNA’s requires further evidence relating sRSA structure with sRNA function. A variety of structural modifications of sRn’A’s have yielded information about

structural features essential for sRNA function (5-7). The treatment of sRNA’s, and aminoacyl-sRNA’s, with spleen phosphodiesterase is of particular interest among these st,ructural modifications of sRNA’s. Such treatment inhibits the ability of sRNA’s to accept activated ammo acids (7, 8) and t,he ability of aminoacyl-sRNA’s to transfer their ammo acids into polypeptide material (7). These observations raise questions as to the sit,e of attack by spleen phosphodiesterase upon sRNA’s and the reason(s) for loss of function within sRNA’s as a result’ of attack by this enzyme. This paper presents data designed to answer these questions.

To Richard 8. Schweet, pioneer in the field of pept,ide synthesis--in memoriam. 1 This work was supported in part by IJnited States l’rlblic Health Service grants CM-O%47 and
Preparation of L-phenylalan?Jl-14C-sIZ:\‘n. LPhen~lalarlyl-‘4C-sI~NA was prepared by the method of Clark and Crmther (9) by using L-phenylalanine-“C and h’scherichia coli sRNA. The L-pherl!rlalallvl-14C-sl~NA obtained was fltrther pllrified by gel filtration throllgh Sephades (t-75 eclrlilibrated with 0.01 M ammonium formate titrated to pR 5 with concentrated formic acid. The elllted L-pherlylalanyl-‘4C-sl?NA was COW centrat.ed by lyophilization, precipitated with 3

EXPERIMENTAL

480

PlKK%~~GI~ES

ACTION

OF

SPLEEN

PHOSPHODIESTERAHE

volumes of cold ethanol, washed with cold absolute ethanol, vacuum dried, and then stored at -20” until rise. Hydrolysis of sRNA and L-phenylalan,yl-~4CsKX;A by the action of spleen phosphodiesterase. Various sRNA’s were hydrolyzed at 37” by incubating one unit of spleen phosphodiesterase activity (Worthington Biochemical Corp., Freehold, ?ITew Jersey) per millilit,er of 1.0% sRNA in 0.1 M pot,assium succinate buffer, pH 6.7. Reactions were t,erminated by heating the reaction mixt,ures for 5 minutes in a boiling-wat.er bath. The percentage of hydrolysis of the sRNA was determined from the acid-soluble nucleotides formed in O.l-ml aliquots of the reaction mixtllres as previo\&y described (8). rj-Phenylalanvl-~4C-sRNA was hydrolyzed at 30” by incubation of one unit of spleen phosphodiest)erase per milliliter of 0.5% L-phenylalanyllGsRNA in 0.1 M potassium succinate buffer, pII 5.5. Reactions were stopped by the addition of an eqllal volume of cold, water-satllrated phenol. The degraded L-phenylalanyl-‘“C-sRNA was then isolated by phenol extraction (9) before storage at -20”. Synthesis ojpoly-L-phenylalanine-WY. The transfer of L-phenylalanine-14C from L-phenylalanyl14C-sCNA to poly-L-phenylalanine-1°C was performed with the cell-free E. coli system of Xirenberg et al. (10) with the modification t.hat reactions were incubated for 10 minutes at 37” with 50 rg of 1’01~ U in the presence of the high salt, conditions of Conway (11) in place of added phosphoenolpyrnvate and pyruvic kinase. Under these conditions, the extent of poly-r,-phenylalanine-l% synthesis, measured as filterable, hot acid-insoluble material (9) is proportional to the inpni, of L-phenylalat~yl-14C--sl~NA. Formation and assag 01 aminoncyl-sRXA , mR;\rA, ribosome complexes derived from E. coli. L-l’henylalanyl-~4C-sI~NA, Poly U, and E. co/i ribosome complexes were formed under aminoacylsRNA limiting conditions in which complex formation was proportional to pherryla~lanyl-~4C-sRNA concent.ration. Format’ion of sllch complexes involved a 5-minute incubation (37”) of 0.25.ml reaction mixtures containing 25 pmoles XH,Cl, 2.5 rmoles Tris-Cl, pH 7.6, 0.6 rJmole 2.mercaptoethanol, A rmtrles KCl, 2.5 umoles magnesium acetate, 0.06 my of r,-pherlylalarl?rl-‘4C~sl~~A (colltainillg 1500 cpm of L-phenylalanine-14C after varying degrees of hydrolysis catalyzed by spleen ptlosphodiesterase), 1OOrg of Poly U, and sufficielLt E. coli ribosomes (10) to provide 0.5 mg of ribosomal protein as determined by 1 he method of Lowry et al. (12). ,5rlcrose densit.v gradient analyses of L-phenylalx11yl-‘~C-sl~N;.4, 1’01~ IT, E. coli ribosome com-

ON

sRNA

481

plex formation were performed by a modification of the method of Spyrides (13). Reaction mixtures of 0.25 ml were centrifuged into a cold 4.6-ml, 5-20% linear sucrose gradient in 0.01 M Tris-Cl, pH 7.5, 0.01 M magnesium acetate, 0.024 M 2-mercaptoethanol, and 0.1 M NH&l for 1 hour (O-2”) at 35,000 rpm in a Spinco SW-33 rot.or. After centrifllgation, consecutive IO-drop fractions were collected through a No. 20 needle. Aliquots (0.1 ml) of a sol\ltion of 5 mg yeast sRNA per milliliter were added to alternate fractions as carrier, after which macromolecules were precipitat,ed with 2.5 ml of 5% trichloroacetic acid. The samples were then washed onto filters, dried, and counted as previously described (9). The remaining alternate fract.ions were examined direct,ly for A260 mat.erial. Membrane filter adsorption assa)-s of L-phenylalanyl-1~C-sl~N.4, Poly U, E. coli ribosome complex formation employed a modification of the method of Nirenberg and Leder (14) in which 0.25-ml reaction mixtures were dilitted with 3 ml of cold 0.1 M Tris-acetate, pII 7.2, 0.04 n$ KCl, and 0.03 M magnesium acetate, and quickly poured onto membrane filters previously moistened wit,h the dillrting solution. The adsorbed materials were washed three times with cold 3.ml washes of the diluting solution, dried on the filters, and then count,ed as previously described (9). Formation and assag of aminoacgl-s&V&l , mlj;l, ribosome complexes derived ,from rabbit reficzclocules. The “binding enzyme (TFI),” dependent, formation of L-phenylalanyl-14C-sRNA, Poly IT, rabbit reticulocyte rihosome complexes was performed by the method of Arlinghaus el al. (15) using 0.25 mg of L-phenylalar~,vl-‘lCsRN,4 of E. co/i. 1Jnder these conditions, complex formation, as assayed by the alkali treatment method of Arlinghaus et al. (15) or the membrane filter adsorption assay (lfi), is proportional to aminoacyl-sKNA concentration. Flirther, this system was free of “peptide synthetase” (TFZ) act,ivity (15) for no poly-r,-phenylalanine-14C s,vnthesis coldd be detected during the reaction. =Issa!y of phosphonlonoesters present upon slZ~V:t’s. Commercial E. coli slbNh or partially hydrolyzed E’. coli sRNS (see spleen phosphodiesterase-catalyzed hydrolysis and subsequent phenol isolation procedllre above) was passed through a Sephadex (i-50 column (2 X 45 cm) equilibrated with 0.1 M ammonium formate to separate the large molecular components from residual small molecules. The resllltant large molecule portion (>95yo of total ;1?60 absorbing material) was further purified by lyophilizat,ion to dryness, solubilization in 2 ml of 29; potassilun acet,atc, pH 5, and final precipitation with alcohol followed by drying as described previollsly (9) Crystallille alkaline phosphatase of E. co2i was

482

GUNTHER,

CHANG,

freed of residual nuclease activity by passage (4”) through a Sephadex G-75 column (3 X 35 cm) equilibrated with 0.1 M KCl, 0.01 M Tris-Cl, pH 7.5, 0.001 M iLl&$ . The resultant concentrated enzyme (2 mg,/ml), as obtained by dialysis against 0.01 M Tris-Cl, pH 7.5, 0.001 M MgClz followed by partial lyophilization, did not release acid soluble A2~ absorbing material when incubated in the phosphomonoester assay described below. Twenty-five mg of intact or partially hydrolyzed E. coli sRNA was incubated (60’) with 0.5 mg of purified E. coli alkaline phosphatase in a final volume of 2 ml of 0.05 M Tris-Cl, pH 8.0. At l-hour intervals, 0.25-ml aliquots were removed and added to 0.5 ml of cold 0.6 M HClOa . The resultant precipitate was removed by centrifugation (O-2”)) after which the inorganic phosphate released was determined by t,he method of Fiske and SubbaR.ow (17). Source of materials. Guanosine 2’(3’) ,5’-diphosphate was prepared by dicyclohexylcarbodiimidedependent phosphorylation of the pyridinium salt of guanosine 2’,3’-cyclic phosphate (Sigma Chemical Co.) with 2-cyanoethyl phosphate followed by alkaline hydrolysis to open the 2’,3’-cyclic phosphate and remove the 5’(2-cyanoethyl) group. The product, guanosine 2’(3’) ,5’-diphosphate, as purified by anion-exchange chromatography, was characterized by its absorption spectra and paper chromatographic RF (two solvent systems). L-Phenylalanine-1% (sp. act. 110 mC/mmole) was obtained from the New England Nuclear Corp., Boston, Massachusetts. Membrane filters (type B-6) were obtained from Schleicher and Schuell, Keane, New Hampshire. Soluble RNA’s were obtained from General Biochemical Industries, Chagrin Falls, Ohio. Poly U was obtained from Miles Laboratories, Elkhart, Indiana. Spleen phosphodiest.erase and E. coli alkaline phosphatase were as provided by Worthington Biochemical Corp., Freehold, New Jersey. Other reagents and chemicals were as obtained from Sigma Chemical Corp., St. Louis, Missouri.

AND CLARK

+ 8

I

IO 20 % HYDROLYSIS FIG. 1. Effect of spleen phosphodiesterasecatalyzed hydrolysis of L-phenylalanyl-“C-sRNA upon the abilit,y of L-phenylalanyl-I%-sRNA to t.ransfer L-phenylalanine-14C into poly-n-phenylalanine-1%. These results are similar to those previously reported by Harkness and Hilmoe (7).

n-phenylalanineJ4C into polypeptide material in the cell-free, protein-synthesizing system of Nirenberg et al. (10). This loss in amino acid “transfer reaction” capacity is not due to the hydrolysis of the n-phenylalanine-14C from the aminoacyl-sRNA, for spleen phosphodiesterase does not remove amino acids from aminoacyl-sRNA (8). This loss in transfer reaction capacity must therefore reflect either an inability of the partially hydrolyzed aminoacyl-sRNA to bind into initial aminoacyl-sRNA, mRNA, ribosome complexes (“initial complexes”), or an inability of partially hydrolyzed, ribosome bound, aminoacyl-sRNA to function in the subsequent steps of the transfer reaction. Employing a cell-free system from E. co& one can determine which of these two RESULTS possibilities causes this loss in transfer Ability of partially hydrolyzed sRNA’s to reaction capacity. With such a system, it is function in protein synthesis. The work of possible to assay the ability of limiting Harkness and Hilmoe (7) and Clark et al. quantities of intact or partially hydro(8) has shown that hydrolysis of sRNA, as lyzed aminoacyl-sRNA’s to form aminocatalyzed by spleen phosphodiesterase, re- acyl-sRNA, mRNA, E. coli ribosome comsults in a rapid lossin the ability of sRNA to plexes under conditions where no peptide accept activated amino acids. Figure 1 bond formation occurs (13, 18). Such assays, demonstrates that spleen phosphodiesterase employing either a sucrose density gradient assay (Fig. 2) or a membrane filter adsorpcatalyzed hydrolysis of n-phenylalanyl-14G sRNA also results in a loss in the ability of tion assay (Fig. 3) for initial complex forthe L-phenylalanyl-14C-sRNA to transfer its mation, shows that partial hydrolysis of

ACTION

OF

SPIXES

PIIOSPHODIESTERASE

ap

ON

483

sRNA

I 20

IO %

HYDROLYSIS

FIG. 3. Membrane filter adsorption assay of the ability of intact and degraded L-phenylalanylI%-sRNA’s to form L-phenylalanyl-‘4C-sRN.4, Poly U, h’. coli ribosome complexes. FRACTION

NUMBER

FIG. 2. Sucrose density gradient assay of the ability of intact, and degraded L-phenylalanyl14C-sRNA’s to form L-phenylalanyl-1%sRNA, Poly U, E’. coli ribosome complexes. Sedimentation is from left to right. The percent values refer to the percentage of hydrolysis catalyzed by spleen phosphodiesterase on each sample. The solid line, which illustrates the migration of ribosomes and ~J-phenylalanyl-14C-slCNA, Poly U, ribosome complexes away from the smaller sRNA’s, represents t.he A260 assays. The dashed line, which illustrat,es the location of free and ribosome bound L-phen~lalarlyl-‘4C-sl~NA, represents acid-precipitable ‘C.

L-phenylalanylJ4C-sRNA of E. coli, as catalyzed by spleen phosphodiesterase, greatly reduces the ability of the L-phenylalanyl-W-sRNA to form L-phenylalanyl14GsRNA, Poly U, E. coli ribosome complexes. A comparison (Fig. 4) of the ability of the partially degraded L-phenylalanyl14C-sRNA t,o form initial complexes (Figs. 2 and 3) and the ability of these same materials

to

transfer

their

14C into polypept’ide react,ion correlation reaction

as

L-phenylalanine-

material (the transfer Fig. 1) reveals a direct

seen in between the loss capacity and the loss

in

transfer

in L-phenylPoly U, E. coZi ribosome ability. This relationship

alanyl-14C-sRNA, complex-forming strongly suggests that spleen phosphodiesterase

the

inability

treated

of

amino-

a9

IO %

20 HYDROLYSIS

FIG. 4. Effect of spleen phosphodiesterasecatalyzed hydrolysis upon the ability of L-phenylalanyl-W-sRNA to function in the transfer react.ion and to function in initial complex formation. This figure represents a sllmmation in which data on transfer reaction capacity (0, Fig. l!, sucrose densit,y gradient assays of init.ial complex forming ability (Cl, as in Fig. 2), and membrane filter adsorption assays of init,ial complex-forming ability (u, Fig. 3) are plotted jointly. The data from the sucrnse density gradient, assays represents points derived by summat,ion of the L-phellylalanyl-W-sRNA migrating with ribosomes into t,he gradient. These points are derived from Fig. 2 plus data from similar analyses.

acyl-sRKA’s to function in the transfer reaction represents an inability of these degraded molecules to partake in the initial step of the t.ransfer reaction, i.e., the formation of aminoacyl-sRNA, mRNA, ribosome complexes.

GUNTHER,

484

J

25 4’

k z i

\ IO %

CHANG,

20 HYDROLYSIS

FIG. 5. Effect of spleen phosphodiesterasecatalyzed hydrolysis of L-phenylalanyl-IGsRNA upon t,he ability of the n-phenylalanyl-~4CsRNA to form initial complexes. Initial complex formation is assayed by the alkali treatment method (0) of Arlinghaus et al. (12) or the membrane filter adsorption technique (U) (16).

The universality of the relationship between initial complex-forming ability and degree of hydrolysis of aminoacyl-sRNA’s can be tested using the well-resolved protein-synthesizing system from rabbit reticulocytes (15). This system requires GTP and a soluble “binding enzyme” (TF,) for the formation of aminoacyl-sRNA, mRNA, ribosome complexes. Thus, if one uses a purified binding enzyme, one can limit protein synthesis to the initial “binding reaction,” i.e., to aminoacyl-sRNA, mRNA, ribosome complex formation (15). A glutathione-requiring second enzyme, “peptide synthetase” (TFz), is subsequently required for the formation of the first peptide bond. The presence of both binding enzyme and peptide synthetase leads to the synthesis of multiple peptide bonds (th.e transfer reaction) and polypeptide materials. Binding enzyme dependent assays of L-phenylalanyl-14C-sRNA, Poly U, rabbit reticulocyte ribosome complex formation, employing either the membrane filter adsorption technique or the alkaline hydrolysis technique of Arlinghaus et al. (15) (Fig. 5), all reveal that partial hydrolysis of L-phenylalanyl-r4C-sRNA, as catalyzed by spleen phosphodiesterase, greatly reduces the ability of the L-phenylalanyl-14C-sRNA to partmakein initial complex formation. The

AND CLARK

results of these assays employing the rabbit reticulocyte system are quite similar to the data obtained for initial complex formation with degraded aminoacyl-sRNA’s in the E. coli system (Figs. 2-4). Thus, this effect of spleen phosphodiesterase-catalyzed hydrolysis upon the ability of aminoacyl-sRNA’s to form initial complexes appears universal. Mechanism of hydrolysis of sRNA’s catalyzed by spleen phosphodiesterctse.Interpretation of the above results requires a clearer picture of the mechanism of attack of spleen phosphodiesterase upon sRNA’s and aminoacyl-sRNA’s. Spleen phosphodiesterase acts as a 5’-exonuclease upon oligoribonucleotides, hydrolyzing these molecules into nucleoside 3’-phosphates derived from the 5’ end (19, 20). Preliminary evidence suggests that spleen phosphodiesterase acts analogously upon sRNA’s (8). The exact mechanism of attack of spleen phosphodiesterase on large molecule substrates needs further chemical characterization. The 5’ ends of sRNA’s usually exist as 5’-monophosphate esters (l-4, 21, 22). Therefore, if spleen phosphodiest,eraseacts as a Y-exonuclease upon sRXA, the most

1.0 ,8 t

0.5 ..

/I

200

400

600

MILLILITERS FIG. 6. Elution profile of small molecules derived from yeast sRN-4 upon treatment with spleen phosphodiesterase. The solid line illustrates the profile of the small molecules obtained when 25 mg of yeast sRN.4 is hydrolyzed to an extent of 9.2% by the action of spleen phosphodiesterase. The dashed line illustrates the elution profile of 0.5 mg of guanosine 2’(3’) ,5’-diphosphate. Both lines reflect the use of l-liter gradients of 0.140.28 M NaCl in 7.0 M urea, 0.02 M Tris-Cl, pB 7.0, after the method of Rnshizsky et al. (23).

ACTION

OF

SPLISI~S

1’HOSI’TIOI)IESTERBSP:

prevalent init,isl small molecule product removed from the sRNA’s should be a nucleoside 3’) 5’.diphosphate. DICAE-Sephadex column chromatography will easily distinguish such a nucleosidc 3’) %diphosphnte from t.he nucleoside Z-phosphates expect,ed as secondary products after initial removal of a nucleoside 3’) %diphosphate. If yeast sRNA is partially degraded (9.2 c% hydrolysis) by the a&on of spleen phosphodiesterase and the resultant small molecule products are then eluted from DE4ESephadex by the met’hod of Rushizky et al. (23), one obtains only a single pcnk of LdZ5oabsorbing material (Fig. 6). This material elutes at t.he position of a nucleoside monophosphat8estandard. Thus, the initial, and following, small molecule product,s removed from sRNA upon treat,ment \vit.h spleen phosphodiesterase are nnclcoside monophosphates. Further, no n s6oabsorbing mat#erialelutes at the position of a nucleoside 2’(3’) ,5’-diphosphate standard. It, follows t!hnt#the attack of commercial spleen phosphodiesterasc upon sRNA’s dots not initiate with a 5-cxonucleolytic cleavage to releasea nucleoside 3’ :5’.diphosphatc. The results of Fig. B force one to consider alternate mechanisms for the action of spleen phosphodiesterase upon sRKA’s. Spleen phosphodiest’erasedoes not act as a Zesonuclease t)o release nucleoside ;i’-phosphates from sRSL4’s (8) or oligonucleotides (19, 20). Our l;no\vledge of the mechanism of :&on of the enzyme upon oligonucleotides (19, 20) precludes it’s action as a 5’exonuclease that releasesnucleoside .j’-phosphates. These considerat’ions force one to t.hc conclusion that. commercial spleen phosphodiesteruse contains a small amount of endonucleolytic activity associated with, or as a contaminant in, the enzyme. If this \\-erc so, hydrolysis of sItKA could commence wit.h endonucleolyt,ic cleavage within the sRK:A so as t!o expose a new 5’-hydroxyl terminus. Cleavage between a phosphorus atom and a ?-carbon is required since spleen phosphodiestcrase does not alter the populat,ion of nucleosides released from sRS;\‘s by alkaline hydrolysis (8). The nc\vly exposed 5’.hydroxyl terminus could

OK

loo0

385

sRN.4

2000

3000

MILLILITERS

FIG. 7. Elation profile of intact and degraded E. coli sf{NA’s. The solid line illustrates the profile obtained whet) 15 my of E. coli sltN.4 is elated from I)II:.4I-Sephades, after the method of I
then serve as a site for successive release of nucleoside 3’-phosphates by the action of spleen phosphodiesterase. Several lines of evidence support t,his theory of act’ion of commercial spleen phosphodiesterase upon sRIYA’s. First, the exposed loops of sRSA’s that are apparently not involved in interchain hydrogen bonding (1-4) would be expected to serve as sites for the proposed init,ial endonucleolytic attack on sRNA’s. Significantly, these exposed loops contain eit.her an anticodon sit’e for attachment to mRKA’s upon ribosomes (1-4) or a ubiquitous nucleot,ide sequence (24) most probably involved in binding aminoacyl-sRKA’s to ribosomcs. Initial disrupt.ion of these structures nould be expected to Jield the result#sof I’igs. 2, 3, and 5, that) is, disruption of aminoacylsRr\‘A, mRKA, ribosome complex formntion. Second, spleen phosphodiesterusehas difficulty in hydrolyzing substrates tcrminat,ing Lvith a 5’-phosphat,e (19, 20). This point favors cndonucleolytic generation of a Zhydroxgl group before spleen phosphodiest’erase-catalyzed hydrolysis can occur. Furt,her, removal of the Y-terminal phosphate from sRSh’s does not, affect the rate

456

GUNTHER,

CHANG,

of subsequent hydrolysis of dephosphosRNA’s catalyzed by spleen phosphodiesterase (8). This supports the previous observation (Fig. 6) that initial attack does not occur at the 5’-end of sRNA. Third, endonucleolytic cleavage at one of the exposed loops of sRNA should drastically alter the size of the remaining large molecule portion of the sRNA. This change in size would be hard to detect as long as intrachain hydrogen bonds remain intact. Such hydrogen bonding probably accounts for the minimal alterations in Svedberg value and gel-filtration properties observed in heat-treated (5 minutes 100”) samples of sRNA that had previously been partially hydrolyzed by the action of spleen phosphodiesterase (8). The urea environment of the DEAE-Sephadex chromatography procedure of Rushizky et al. (23) precludes such hydrogen bonding. Further, if the elution gradient of this system is extended to higher salt concentrations, one can elute large polynucleotides. The experiment of Fig. 7 uses this system to compare the elution profile of intact sRNA of E. coli with the profile of E. coli sRNA that has been hydrolyzed to the extent of 7% by the action of spleen phosphodiesterase. The degraded sRNA clearly demonstrates heterogeneity as a result of cleavage(s). The exact nature of this cleavage is hard to interpret in light of the poor resolving power of the short column employed in this technique (23), yet the results are similar to those reported for a specific endonucleolytic cleavage of sRNA (25). The above theory of action of spleen phosphodiesterase upon sRNA’s requires that the residual large molecular weight portion(s) of the sRNA (with or without intraconnecting hydrogen bonds) contain its original 5’-phosphomonoester group and a new 3’-phosphomonoester generated by the endonucleolytic cleavage. Phosphomonoesterase attack upon such molecules should release, at equilibrium, two inorganic phosphates per original sRNA. Figure 8 reveals that the large molecular weight fragments of E. coli sRNA, present after appreciable (7.6%) hydrolysis catalyzed by spleen phosphodiesterase, contain the same num-

AND

CLARK

I

2

3

4

HOURS FIG. 8. Escherichia coli phosphomonoesterasedependent release of inorganic phosphate from intact and partial degraded E. coli sRNA. The E. coli sRNA was hydrolyzed to an extent of 7.6% by the action of spleen phosphodiesterase. Intact sRNA (0) and this 7.695 hydrolyzed sRNA (0 ) were then purified, treated with phosphomonoesterase, and subsequently analyzed for inorganic phosphate as described in EXPERIMENT.\L PROCEDURES.

ber of phosphomonoesters as the original RNA. This observation necessitates that the endonucleolytic cleavage proposed as the initial attack by commercial spleen phosphodiesterase upon sRKA result in a cyclic 2’,3’-phosphate, for it is unlikely that a 3’-phosphate would be inaccessible to E. coli alkaline phosphatase under the 60” conditions of the assay. DISCUSSION

Preliminary experiments have suggested that commercial spleen phosphodiesterase acts as a 5’-exonuclease upon sRNA’s (8). New and more definitive experiments presented here force a new interpretation of the action of commercial spleen phosphodiesterase upon sRNA’s. Specifically, such preparations cannot act as 5’-exonucleases for no nucleoside 3’) 5’-diphosphate is formed upon attack of sRNA’s (Fig. 6). This fact, combined with other data (8, 19, 20), leave endonucleolytic attack of sRNA’s as the only possible site for initial cleavage by commercial spleen phosphodiesterase. Preliminary evidence of the existence of endonucleolytic activity (Fig. 7) supports this conclusion. The exact site of such internal cleavage of sRNA’s by commercial spleen phosphodiesterase, or a contaminant

ACTION

OF

SPLEEN

PHOSPHODIESTERASE

in such preparations, is not known. Yet this site of initial attack is of obvious functional significance in that such cleavage has a drastic effect upon the ability of sRNA’s to accept activated amino acids (7, 8) and upon the ability of aminoacyl-sRNA’s to partake in aminoacyl-sRNA, mRNA, ribosome complexes (Figs. 4 and 5).

9. 10.

11. 12.

ACKNOWLEDGMENTS The authors are indebted t,o Dr. Richard S. Schweet for personally instructing us in the preparation and assay of the binding enzyme (TR) of rabbit reticuloryt,es (15). REFERENCES 1. HOLLEY, R. W., APGAR, J., EVERETT, G. A., M.~DISON, J. T., MAHQUISEE, M., MERRILL, S. H., PENSWICK, J. It., .IND Z.~MIR, A., Science 147, 1462 (1965). 2. MADISON, J. T., EVERE’I’T, G. A., .IND KUNG, H. K., Cold Spring Harbor Symp. Quant. Biol. 31, 409 (1966). 3. ZACH.\U, H. G., DUTTING, D., AND FELDM.~N, II., Angew. Chem. 78, 392 (1966). 4. EZJBHANDART, U. I,., CHBNG, S. II., STU~~RT, A., F.4ULKNER, R. D., HOSKINSON, R. M., AND KHOR.INA, H. S., Proc. X&l. Acad. Sci. U. S. 57, 751 (1967). 5. PRIESS, J., BERG, P., OFENGAND, E. J., BERGMAN, F. H., .\NU DIECKMAN, M., Proc. Natl. Acad. Sci. U.S. 46, 313 (1959). 6. ZUBaY, G., ANU TAK.~NAMI, M., Biochem. Biophys. Kes. Commun. 16, 207 (1964). 7. H,IRENE~S, D. R., .~ND HILMOE, R. J., Biochem. Biophys. Res. Commun. 9, 393 (1962). 8. CLSRK, J. M., JK., EYZ.\GUIIZ~LE, J. P., .IND

13. 14. 15.

16. 17. 18. 19. 20. 21. 22.

23. 24. 25.

ON

sRNA

487

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