Incorporation of phenyllactic acid in terminal position during polyphenylalanine biosynthesis

J. Mol. Biol. (1965) 13, 757-766

Incorporation of Phenyllactic Acid in Terminal Position during Polyphenylalanine Biosynthesis G.

HERvE .AND

F.

C1Lu'EVILLE

Service de B iochimie du D epartemeni de Biologie, CEN -Saclay Commissariat l'Energie Atomique, Gif-sur-Yvette, B .P. nO 2 S ein e et Oise, France

a

(Received 5 F ebruary 1965, and in revised form 30 May 1965) Phenyllactyl-sRNA was prepared from phenylalanyl.sRNA by nitrous acid treatment. This removal of the amino group increases by about ten times the resistance, to alkaline hydrolysis, of the ester linkage with the sR NA . When phenyllactyl-sRNA is incubated with an Es cherichia coli ribosomal system with p olyuridylic a cid, phen yllactic a cid is in corporat ed in the NH 2 " t erminal position of synt hesized polyphenylalanine, t hat is, t he extremity fr om wh ich polypep tide bio synthesis begins. This method permit s t ho spec ific labelling of the t erminal po sition of the synthesized cha ins ; a nd tho kinetics of this incorpor ation corres po nds to the kinetics of the init iation of p olyp eptide chains.

1. Introduction By converting an amino acid bound to sR NA into an other amino acid (Chapeville, 1962; Chapeville et al., 1962), it was possible to verify the adaptor hypothesis experimentally (Chapeville et al., 1962). Thus the transformation of cysteine either into cystei c acid or into alanine, and of tyrosine into DOPA to showed that these conversion products were incorporated into artificial or natural proteins synt hesized in vitro (Chapeville et al., 1962; Chapeville, Cartouzou & Lissitzky, 1963; von Ehrenstein, Weisblum & Benzer, 1963). Since cysteic acid and DOPA are incorporated, it follows that the transfer factors possess no specificity towards the amino acid. By using the same hybrids it was also demonstrated that, in the rev erse reaction for amino acylsRNA synthesis, the activating enzymes are specific for both the amino acid and the sRNA, and do not recognize a hybrid (Herve & Chapeville, 1963). Among the possible modifications, that of deaminating the sRNA-linked amino acid by nitrous acid is of particular interest, since it concerns the amino group involved in peptide bond formation. In the present work the transformation of phenyl. alanine into phenyllactic acid has been examined (Fig. 1). The experiment al results show that, after incubation in presence of an E scherichia coli ribosomal syste m, phenyllactic acid is found in the ~"'H2-terminal position of polyphenylalanine, which

t Abbreviations us ed: DOPA, dihydroxyphenylalanine; poly U , p olyuridylic acid; T CA, trichl oroacetic acid; SD S, sod ium d odecyl sulpha te ; PEP, phosphoenolpyruvate; PEP-kinase, phosphoenolpyruvate-kinase; Phe·s R NA, sR NA charged with phen yl alanine; Phelac- sRNA, sRNA ch a rged with phen yll ac tic acid; [14C]Phe-[3ZP]sRNA, 3zP-Iabelled sRNA charged with HC·labelle d phenylalanine; [l'CJP helac -[32P JsR NA. 3zP _Iabelled sR NA charged wit h HC-Ia belled phenyllactio acid. 757

758

G. HERVE AND F. CHAPEVILLE

is the position from which polypeptide biosynthesis begins. ;rhis result shows that: (a) the amino group of the amino acid is not involved in the formation of the tertiary ribosome-messenger-aminoacyl-sRNA complex; (b) the amino group is not a prerequisite for the initiation of polypeptide chains.

FIG. 1. Transformation of Phe-sRNA into Phelac-sRNA.

2. Materials and Methods Phe-sRNA. E. coli sRNA was charged with phenylalanine under the usual conditions (Chapeville et al., 1962). It was stored at - 20°C in 10- 4 M-potassium acetate buffer at pH 5. Phelac-sRNA. To 1 ml. of Phe-sRNA (5 to 10 mg/rnl.), 1 ml. of acetic acid and 1 ml. of sodium nitrite solution saturated at O°C were added. The mixture was maintained for 2 hr at 20°C, 2 vol. of ethanol were then added and after centrifugation at 4°C the sRNA was dissolved in 10- 4 M-acetate buffer (pH 5), reprecipitated with ethanol and redissolved in the same buffer. The material thus obtained was analysed by paper chromatography and paper electrophoresis (Plates I and II). Plate I shows that, prior to hydrolysis of the ester bond to sRNA, all the radioactivity is found at the level of sRNA, whereas after hydrolysis, it is at a spot corresponding to phenyllactic acid. From the results of paper chromatography, it appears that the oxidation of phenylalanine also leads to the formation of traces of an unidentified compound, possibly a nitroso derivative of phenyllactic acid (Plate II). [32 P]sRNA was prepared according to the method described by Gilbert (1963) and diluted with [31P]sRNA to a specific activity of 0·3 p.c/mg sRNA. It was then charged with [14C]phenylalanine, and a part was oxidized to [14C]Phelac_[32P]sRNA. E. coli ribosomes were prepared from frozen bacteria (Nathans & Lipmann, 1961). E. coli 105,000 g crude supernatantjraction was used as the source of activating enzymes and transfer factors; for certain experiments, purified transfer factor, prepared according to Nathans & Lipmann (1961), was used. To obtain [12C]phenyllactyl-phenylalanine, phenylalanyl-phenylalanine was oxidized by nitrous acid, absorbed on charcoal, and eluted with ethanol-water-ammonia (50; 47 : 3). Synthetic polynucleotides were prepared according to the method previously described (Chapeville et al., 1962). Pronase was purchased from the Enzyme Development Corp., [14C]phenylalanine from the New England Nuclear Corp., or from the Commissariat a l'Energie Atomique (360 or 200 mc/m-mole), [32P]phosphate from the Commissariat a l'Energie Atomique, [12C]phenyllactic acid from the Mann Research Lab., and pancreatic RNase from Boehringer.

3. Results (a) Properties of phenyllactyl-sRNA

The stability of Phelac-sRNA in alkaline medium was examined and shown to be far greater than that of Phe-sRNA; thus at pH 8·5 and 37°C their half-life times are 206 and 16 minutes, respectively. When Phelac-sRNA was incubated in the usual

POLYPHENYLALANINE BIOSYNTHESIS

759

conditions, in the presence of activating enzymes, AMP and pyrophosphate (Herve & Chapeville, 1963), no enzymic discharge occurred, under conditions where PhesRNA is rapidly discharged. When 500 times more phenyllactic acid than phenylalanine is added to the incubation mixture, it does not inhibit the charging of sRNA by phenylalanine. After stripping Phelac-sRNA at pH 10, the recovered sRNA could be recharged to the extent of 20 to 30% of the level reached with sRNA not previously treated by nitrous acid. (b) Incorporation of phenyllactyl-sRNA by E. coli ribosomal system Phelac-sRNA was incubated with E. coli ribosomes, 105,000 g crude supernatant fraction, all the necessary ingredients for transfer, and in the presence or absence of polyU. (i) Kinetics of incorporation

Figure 2 shows the kinetics of incorporation of phenyllactic acid from PhelacsRNA, with and without poly U. In the absence of poly U no significant radioactivity is recovered in the TCA precipitate, whereas in its presence 10% incorporation is observed. No incorporation occurred with poly A. If after incubation with poly U the Phelac-sRNA not used for incorporation was degraded by the addition of RNase, and the synthesized material precipitated by cold 5% TCA, the result was the same as with hot (15 minutes at 90°C) TCA precipitation.

c:

E <,

120

With poly U

"''""'

a

~

>- 80 .....

';;

'.oJ u 0

.2

-0 0

c::

40 Without poly U

0

30

60

90

120

Time (min)

FIG. 2. Incorporation of phenyllactic acid by E. coli ribosomal system. Each sample contained in a total volume of 0·3 m!.: 1 mg of ribosomes, 105,000 g crude supernatant (0·1 mg protein), 20 jLg of Phelac-sRNA (1300 ctsjrnin), 13 jLmoles of tris-HCl (pH 7·4), 8 jLmoles of KCl, 3 jLmoles of MgCI 2 • 4 jLmoles of GSH, 3 jLmoles of GTP, 8 jLmoles of PEP-kinase, 27 jLmoles of PEP and 5 jLgof poly U where used. After 45 min incubation at 37°C, 20 jLg of RNase were added and the mixture was maintained for 30 min at 37°C, 1 mg of serum albumin was added and the reaction was stopped in each tube by addition of 2·5 m!. of 5% TCA. The precipitate was washed twice with 5% TCA and once with 1: 1 ether-alcohol. It was then dissolved in formic acid, plated and counted in a Nuclear-Chicago gas-flow counter.

(ii) Essential factors

In order to compare the nature of phenyllactic acid incorporation with that of phenylalanine incorporation, the factors normally essential for protein biosynthesis were successively omitted. Table 1 shows how the omission of these factors affects

760

G. HERVE AND F. CRAPE VILLE

similarly the incorporation of both phenylalanine and phenyllactic acid. The results indicate that all the ingredients essential for phenylalanine incorporation are also essential for phenyllactic acid incorporation.

TABLE

1

Effect of omitting various factors on phenylalanine and phenyllactic acid incorporation Phe

Phelac

Complete system

3030

331

Without Without Without Without Without

1560 27 17 14 450

101 56 11 10 17

105,000 g supernatant energy source (GTP, PEP) ribosomes Mg 2 + poly U

The incubation and analysis were made as described in Fig. 2. Incubation time: 45 min.

(iii) Inhibitors

The effect on the incorporation of phenyllactic acid of various substances known to inhibit protein biosynthesis was examined; the results show (Table 2) that puromycin, aureomycin and chloramphenicol, which inhibit the incorporation of amino acids into proteins, also inhibit the incorporation of phenyllactic acid into hot TeA-insoluble material. TABLE

2

Inhibition of phenyllactic acid and phenylalanine incorporation

Incubations

Puromycin 3xlO- 4

0 Phe-sRNA 1850 cts/min

+ 0 0 0 0

0 Phelac-sRNA 3400 cts/min

+ 0 0 0 0

M

Aureomycin 10- 3 M

Chloramphenicol

0 0 0

0 0 0 0 0

2xlO- 3 M

+ 0 0 0 0 0

+ 0 0

The incubations were made as described in Fig. 2 for 45 min.

+ 0 0 0 0 0

+

Radioactivity incorporated (ctsjmin)

1250 695 1390 300 1300 885 370 44 306 84 350 222

Pheloc acid

+

Phe

I I

Phe - . RNA treated with N0 2 H

Before pH 11

After pH 11

PLATE 1. Analysis of nitrous acid-treated Phe-sRNA. The material prepared as described in Materials and Methods was fractionated by electrophoresis on Arches 302 paper for 90 min at 20 v lctt: in 0·5 M-formic acid. The eleetrophoregram was exposed to a Kodirex X-ray film.

[facing p. 760

..

---- - - - -

- -- - - - , I

IT

ill

v

N

Phe

Phelac ac id

PLATE II. Analysis by paper chromatography of the material synthesized from l14C]PhelacsRNA. Solvent: n-butanol-acetic acid-water (78:.5: 17). I. II. III. IV. V.

[14C]Phe-sRNA treated with nitrous acid and stripped at pH U. Materials synthesized by ribosomal system in the presence of [14C]Phelac-sRNA. Same material hydrolysed 15 hr at 105°C with 6 N-HCl. [14C]Phenylalanine. [14C]Phenylalanine treated with nitrous acid.

The chromatogram was exposed to a Kodirex X-ray film, and the ['2C]phenyllactic acid and phenylalanine used as carriers, were detected on the paper by the luminescence resulting from the ultraviolet absorption (2537 A) in liquid nitrogen. Phenylalanine was also detected by ninhydrin.

Elec tro phore sis pH 8,5

x Phe lac o cid

A2 EIec l ro ph or e sis pH 2

Phel cc x

P he

I I

I'

-I

81

82

83

I

84

PLATE III. Enzymic degradation by pronase of the material synthesized from Phelac-sRNA. Incubations of [14C]Phelac-sRNA were made using 105,000 g crude supernatant fraction, and in 'the presence (incubation mixture I) or in the absence (incubation mixture II) of free [14C]phenylalanine. The mixtures were then centrifuged for 60 min at 101i,000 g in 4 X 10- 2 M-tri3-HCI (pH 7), 10 - 2 M-MgC12 • The ribosomes carrying the synthesized material were resuspended in 330 Ill. of 0.·1 M-tris-HCl (pH 8,5) and .~O 1-'1. of pronase solution (5 mgjml.) and incubated at 37°C for 14 hr. The mixtures were then lyophilized, the residues dissolved in 1001-'1. of distilled water and chromatographed in n-butanol-acetic acid-water. Beside the undigested material which did not migrate, and the spot of [14C]phenylalanine in the case where it was added in the synthetic medium, one radioactive spot was found. This spot was eluted, rc-chromatographed in the same conditions, re-eluted and analysed by electrophoresis in 0·05 M-ammonium carbonate buffer (pH 8,5) and in 0·5 M-formic acid (pH 2). At pH 8,5, in both cases, it gave two spots. One of them was free phenyllactic acid (AI: coming from incubation mixture II; A2: coming from incubation mixture I). The other (spot X) was eluted and re-run at pH 2 before and after hydrolysis at 10lioC with 6 N-HCI for 15 hr (B I and B2 from AI, B3 and B4 from A2). After hydrolysis it gave [14C]phenyllactic acid only (B2), when coming from incubation mixture without ["4C]phenylalanine, and [14C]phenyllactic acid plus ["4C]phenylalanine (B4), when coming from incubation mixture containing ["4C]phenylalanine.

-- -@

Phe

Phelac ac id

PLATE lY. Localization of the phenyllactic acid residue incorporated. The incubations were made in the conditions described in Fig. 2 using 750 I-'gof the following materials, and in the presence of 2 rnumolcs of [14C]phenylalanine (80 me/mole). 1. [12C]Phelac-sRNA. 2. [14C]Phelac-sRNA. 3. [14C]Phe-sRNA. 4. [14C]Phelac-sRNA.

After 45 min of incubation the samples were treated with 5 % TCA at 90°C for 15 min. The precipitates were washed twice with 5% TCA and Once with ethanol-ether (1: 1). To samples I, 2 and 3, 1001-'1. of formic acid, 5001-'1. of 10% acetic acid, and 5001-'1. of saturated sodium nitrite were added. After 2 hr at 20°C the material was precipitated and washed twice with TCA, and once with ethanol-ether. The four samples were then hydrolysed for 15 hr at 105°C with 10 N-HCI. The hydrolysates were chromatographed in n-butanol-acetic acid-water, and the chromatogram exposed to a Kodirex X-ray film.

POLYPHENYLALANINE BIOSYNTHESIS

761

(c) Properties of the material synthesized from Phelac-sRNA by the ribosomal system

In the ease of Phelac-sRNA, as in the case of Phe-sRNA, the synthesized material is recovered with the ribosomes after centrifugation of the incubation mixture for 60 minutes at 105,000 g. With Phelac-sRNA the radioactive material recovered as 14C in the pellets was the same when incubations were at O°C and at 37°C, and there was no difference whether GTP was present or not. However, the material associated with ribosomes after incubation at 37°C in the presence of GTP was RNase-resistant, whereas it was RNase-sensitive in the absence of GTP (Table 3). The material synthesized from Phe-sRNA showed the same behaviour towards RNase. This might indicate that in the presence of GTP and at 37°C a chain was formed, whereas in the absence of GTP, Phelac-sRNA was only bound to the ribosomes; such a binding of sRNA to ribosomes in the presence or absence of messenger has been studied by Cannon, Krug & Gilbert (1963), by Kaji & Kaji (1963), and by Nirenberg & Leder (1964). It is also known that polypeptidyl-sRNA attached to the ribosomes is RNaseresistant (Cannon et al., 1963; Risebrough, 'I'issieres & Watson, 1962). TABLE

3

Properties of material bound to ribosomes with and without GTP Incubation conditions

Analysis conditions

CtsJmin

Without energy source

(1) centrifugation at 105,000 g (2) RNase and centrifugation (3) RNase and TCA

400 65 35

With energy source

(4) centrifugation at 105,000 g (5) RNase and centrifugation (6) RNase and TCA

470 420 422

[14C]Phelac-sRNA was incubated as described in Fig. 2 for 45 min at 37°C, in the presence or absence of energy source (GTP, PEP, PEP-kinase). Samples 2, 3, 5 and 6 were treated with RNase as described in Fig. 2. To samples 1, 2, 4 and 5,10- 2 M-tris-HCl (pH 7·4) containing 10- 2 M-MgC1 2 was added and the tubes centrifuged 1 hr at 105,000 g. The radioactivity of the ribosomal pellets was determined as described in Fig. 2.

Using the technique described by Slapikoff, Fessenden & Moldave (1963) to separate the nucleic acid fraction from the protein fraction of ribosomes, it was also possible to show that after incubation in the absence of GTP the material obtained with PhelacsRNA is recovered with the nucleic acid fraction, whereas in its presence it is found in the protein fraction. In all cases the material can be separated from the ribosomes by resuspension in a 1 % solution of sodium dodecyl sulphate, as shown in Table 4. Figure 3 shows that the behaviour of the material synthesized from Phelac-sRNA and subsequently incubated with puromycin is the same as that of the material synthesized from Phe-sRNA, when examined by sucrose gradient centrifugation in SDS solutions. In both cases puromycin treatment results in the formation of a peak with lower sedimentation coefficient (Gilbert, 1963; Traut & Monro, 1964). This shows that the material containing phenyllactic acid was attached to sRNA at the end of the synthetic reaction. 51

G . HER VE AND F. CH A P E V IL L E

762

TABLE

4

Separation of incorporated phenyllactic acid fr om ribosomes by sodium dodecyl sulp hate

Material u sed

R adioactivi t y of r ib osomes b efore SDS

R adioa cti vi t y of p ellet after SDS

Phelac·sRN A 135,000 cts/min

14,0 00

420

Phe·sRNA 26,00 0 cts /m in

10,000

685

The incubations wcr e made as d escr ib ed in Fig. 2 for 45 m in . Afte r treatment by RNase, 10- 2 Mtris-HCI (pH 7,4) containing 10- 2 M.MgCl. was added and the mi xt u res cen t rifuged 60 min at 105,000 g. The ribosomes of the co n t ro ls wer e then dissolved in formic ac id and their radioactivity determined. The ribosomes of t ho ot he r sam ples were treated 30 m in with 0 ·5 ml. sodium dodecyl sulphat e at 1 %, and centrifuged 60 min at 105,000 g in tris-HCI MgCI2 b uffer . The radioactivity of the pellets was then d eter mined as d escribed in Fig. 2.

c

~

160

160

..,'"

A

B

~

o-, 100 ..,

.;;

'';:; u

o

.2

-0

o

a:: 20

2

6

10

14

2

6

10

14

Fraction no. Fig . 3. Cen t rifugation in a sucrose gradient of material sy nthesized fro m, Phelac-sRNA. The inc ubations were carried out as indicated in F ig. 2 for 45 min an d the r ibosomes -isolat ed b y ce nt rifuga t ion at 105,000 g for 1 hr. Sa mples A and B wer e re-i ncu b ated 30 min at 20°C under co n di t ion s similar to those for p ro te in synthesis, except tha t B co ntaine d 10- 3 M-puromycin. They were then centrifuged 12 hr at 39,000 rev./min in a linear suc rose gradient (12'5 to 5 %) containing 0'5% SDS, 0·1 M-LiCI, and t ris- H CI (pH 7) in an SW3P ro to r at 15 to 18°C: drops wer e co llec ted from the bottom of t he tub e and counted in a Nuclear - Ch ica go liquid-scintillation counter (Traut & Monro, 1964).

(d) Nature of the material synthesized fr om Ph elac-sRN A by the ribosomal system (i) Hydrolysis by 6 lV-HCl

Th e material synthesized on r ibosomes from Phe-sR NA or from Phelac-sRNA does not migrate during pap er chroma t ography . Whcn t he material obtained fr om Phelac-sRNA is treated for 15 hours at 105°C wit h 6 N-lICl, an important fraction of the radioactive material is liberated and corresponds to free ph enylla ctic acid (Plate II).

POLYPHENYLALANINE BIOSYNTHESIS

763

(ii) Enzymic hydrolysis by pronase The material synthesized on ribosomes from [14C]Phelac-sRNA was treated with pronase for 14 hours at 37°C. The hydrolysate was purified by two successive paper chromatographic separations in n-butanol-acetic acid-water, and by paper electrophoresis at pH 8·5 (Plate IlIA). The products found were free phenyllactic acid and phenyllactyl-phenylalanine. This peptide was identified in the following manner. (1) By incorporating [14C]phenyllactic acid from Phelac-sRNA in the presence of free [14C]phenylalanine, followed by pronase digestion, the peptide was obtained, which yielded [14C]phenyllactic acid and [14C]phenylalanine upon acid hydrolysis (Plate IIIB). (2) By chromatography and electrophoresis it co-migrated with a sample of [12C]phenyllactyl-phenylalanine prepared by nitrous acid treatment of phenylalanyl-phenylalanine. (iii) Incorporation of phenyllactic acid in the presence of purified transfer factor Phelac-sRNA was incubated in the complete system required for polypeptide biosynthesis, using purified transfer factor prepared according to Nathans & Lipmann (1961), and in the presence or absence of free [12C]phenylalanine. The results given in Table 5 show that free phenylalanine is indispensable for the incorporation of phenyllactic acid from Phelac-sRNA. It was ascertained that such is not the ease for Phe-sRNA. TABLE

5

Role of free phenylalanine on the incorporation of phenyllactic acid Incubation conditions

Crude enzyme without poly U

Cts/min Phelac incorporated

Cts/min Phe incorporated

36

134

Crude enzyme poly U

710

1380

Purified enzyme polyU

141

1235

Purified enzyme + polyU 10 fig [12C]Phe

419

1330

Purified enzyme 200 fig sRNA 100 fig ATP 10 fig [12C]Phe without poly U

16

80

Purified enzyme 200 fig sRNA + 100 fig ATP 10 fig [12C]Phe with poly U

505

1450

+

+ +

+ + +

+ +

The incubations were made as indicated in Fig. 2, for 45 min, in the presence of purified transfer factor (55 fig of proteins), or of 105,000 g crude supernatant (270 fig of proteins).

764

G. HERVE AND F. OH A P E V I L L E

(iv) M odifi cations of the ratio 14Cj32p of [14C]Phelac-[32P ]sRN A and of [J4C]Phe[32P] sRN A during inco rporation by ribosomal system [14C]Phe_[32P]sRNA and [14C]Ph elac-[32P]sRNA were incubated wit h a ribo somal syst em in the presence or absence of an energy source (GTP, PEP) . By comparing the ratio l4Cj32P of the molecules bound to the ribo somes in the absence of a source of energy and this ratio for the cha ins formed in the same conditions plus GTP, the average number of radioactive residues incorporated per chain can be calculated. This is based on the findin gs that an aminoacyl-sRNA binds specifically to the messenger- ribosome complex, and non-specifically to the ribo somes (Cannon et al.,1963; K aji & Kaji, 1963; Nir enb erg & Leder, 1964), and that in t he pr esence of GTP only the aminoacyl-sRNA specifically bound to the complex participate s in polypeptide cha in formation, the chains remaining attached t o the ribo somes by the sRNA of the la st residue incorporated. During the growth.of the polypeptide chain, the ratio l4Cj32P will increase if several l4C-residues are incorporated per chain, but it will stay the same if only one residu e is incorporated per chain. It is thus possible to calculate the average number of radi oactive residues incorporated into a chain. Table 6 shows that after incorporation in t he presence of 105,000 g crude supern at ant fracti on , the r atio 14Cj32P in creases 9·5 times in the case of phenylalanine; but that it remains the same in the case of phenyllactic acid, indi cating that there is only one such residue per chain synt hesized . The number of [l4C]phenylalanine molecules incorporated p er chain was also determined by treating the synt hesized polyphenylalanine with nitrous acid. Thi s transforms the NH2- terminal residues of the chains into phenyllactic acid. In the presence of 105,000 g crude supernatant fr action, the average number of [14C]phenylalanines per chain was 10, whereas it was about 20 in the case of 105,000 g supernatant fra ction passed through Sephadex G25. The difference observed is most lik ely due to t he traces of [12C]phenylalanine found in the crude supernat ant solution, which is incorporated into the chains .

TABLE

6

Determination of the average number of residues per chain

14Cj32p ratio in the ribosomal-bound m atori al without GTP

140j32P ratio in the ribosomal-bound material with GTP

Average number of residues per chain

Pho

0·360

3·30

9·5

Phelao

0·425

0·415

1

The incubations were m ade as d escri bed in T abl e 2 with 0· 1 mg of [14C]P he -[32P]sR NA or with 0' 1 mg of [14C]P he lac -[32P ]sRNA. in t he p resence or ab sence of energy sou rces . After in cubati on , the samples were centr ifuged 1 hr at 105,000 g an d t he r adioa cti vi t y (14Ca nd 32P) of t he m ater ial s bo und to the r ibosomes was determined in a Nuclear-Chicago gas-flow counter, bya di ffer en t ial absorpti on method, using a scree n abso rbing all 140 and 62% of 32p Jl.parti cles.

POLYPHENYLALANINE BIOSYNTHESIS

765

(v) Localization of the phenyllactic acid residue

The results of the following experiments, described in Plate IV and Table 7, indicate that the phenyllactic acid residue is incorporated into the terminal position of the chains. When [12C]phenyllactic acid is incorporated from [12C]Phelac-sRNA in the presence of free [14C]phenylalanine, only traces of [14C]phenyllactic acid are formed after nitrous acid treatment of the synthesized material. When [l4C]phenyllactic acid is incorporated from [14C]Phelac-sRNA in the presence of free [14C]phenylalanine, nitrous acid treatment yields but little additional [14C]phenyllactic acid. It was found previously that treatment of normal polyphenylalanine with nitrous acid yielded 5 or 10% of phenyllactic acid, depending on whether crude supernatant fraction or supernatant fraction free of amino acids was used. In Table 7 are reported the radioactivities of the phenylalanine and the phenyllactic acid eluted from the chromatogram presented in Plate IV, and the ratio of phenylalanine to phenyllactic acid for each sample. The results show that about five out of six chains begin with phenyllactic acid. They also show that the chains synthesized in the presence of Phelac-sRNA are shorter than those synthesized in the presence of Phe-sRNA alone. TABLE

7

Radioactivity of the eluates of the chromatogram of Plate I V Sample

Phe

Phelac

1 2

ll05 790 4180 1920

30 137 214 212

3

4

PhejPhelac ratio

37 6

20 9

The spots corresponding to phenylalanine and phenyllactic acid were eluted by 1 ml. of ethanol and 1 ml, of water. The eluates were plated and radioactivity counted.

4. Discussion The above results indicate that phenyllactic acid, obtained by deamination of phenylalanine on the sRNA, is incorporated into the terminal position of the polyphenylalanine chains synthesized in the presence of poly U. This implies that: (a) the amino group is not a prerequisite for the formation of the tertiary messenger-ribosome-aminoacyl-sRNA complex; (b) the presence ofthe amino group of the NH 2-terminal amino acid is not necessary for the formation of the first peptide bond between the carboxyl group of that amino acid and the amino group of the following residue. Since phenyllactic acid is incorporated only in the NH 2-terminal position, the kinetics of its incorporation corresponds to the kinetics of the initiation of polyphenylalanine chains. The results show that in the conditions used, chains were continuously initiated, at a decreasing rate over a period of 30 minutes. Using other methods, Gilbert

766

G. HER VE AND F . CHA P E V I L L E

(1963) showed that , in t he system synthesizing polyphenyl alanine, initiation occurred throughout the incubation. Fi nally, it has been demonstrated that the este r bond between phenyllactic acid and sR NA is mu ch more stable t ha n that between ph enylalanine and sRNA. Other exp erim ents not reported here have given similar results for deaminated leucine, tyrosine and alanine derivati ves of sR NA. This in creased stability of the ester bond may be advantageous in certain st udies wit h conditions.which promote rapid hydrolysis of the ester bond. We thank Dr R ob ert Traut for t he suc rose gradient centr ifugation analysis rep ort ed in F ig . 3. REFERENCE S Cannon , M., Krug, R & Gilbert, W. (1963). J. Mol. Biol. 7,360. Ch apeville, F. (1962). Fed. P roc. 21, 414 d. Chapeville, F., Cartouzou, G . & Li ssitsky, S. (1963). Biochim. biophys. Acta, 496. Ch apeville , F., Lipmann, F., v on Ehrenstein, G., Weisblum, B., Ray, W. & Benzer, S. (1962). Proc. Nat. Ac ad . Wash. 48, 1086. vo n E hre n stein , G., W eisblum, B . & Benzer, S. (1963). Proc, N at. A cad. Sci., Wash. 49, 669. Gilbe rt, W . (1963). J. M ol. B iol. 6, 389. H er ve, G . & Ch ape ville , F. (1963) . Biochim. biophys. A cta, 76 , 493. K aji, A. & Kaji, H . (1963). B iochem . B iophy s. R es. Comm. 13, 186. Nat hans, D . & Lipmann, F. (1961). P roc, N at. Acad. Sci., W ash. 47,497. Nirenberg , M. & Led er , P. (1964). Scie nce, 145, 1399. N irenberg , M. & Ma t thaei , J . (1961). Proc. N at. Acad. s«; Wash. 47. 1588. R isebrou gh, R, 'I'iss ieres, A . & Watson, J. (1962). P roc. N at. A cad . Wash. 48, 43 0. Slapiko ff, S. , Fessenden, J. & Moldav e, K. (1963). J. B iol. Chem , 238 , 3670. Traut, R & Monro, R (1964) . J. M ol. B iol. 10, 63.

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