Effect of starvation on tRNA synthesis, amino acid pool, tRNA charging levels and aminoacyl-tRNA synthetase activities in the posterior silk gland of Bombyx mori L

BIOCHIMIE, 1979, 61, 2~29-24+3.

Effect of starvation on tRNA synthesis, amino acid pool, tRNA charging levels and aminoacyl-tRNA synthetase activities in the posterior silk gland of Bombyx mori L.

(*)

G6rard CHAVANCY and Alain FOURNIER.

D~partement de Biologie G~ndrale et A p p l i q ~ e , Laboratoire Associ~ au C.N.R.S. n ° 92, 43, Bouleoard du I1 Nooembre 1918, 69621 Villeurbanne, France.

R~sumd.

dans la arise en place de l'adtrptation des tRNA s i c e ph6nombne r6sulte bien, comme nous le pensons, d'un contr61e de la transcription de ces mol6cules.

Les variations des principaux composants de l'appareil de traduction ont 6t6 mesur6es dams la pattie post6rieure de la glande s6ricig~ne, lots du jefine et de la r6alimentation des larves de Bombyx mori. Pendant le je~me, les synth6ses des tRNA et des RNA ribosomiques sont nulles. Les quantit6s des diff6rents RNA diminuent para]l~lement. Ainsi, la demi-vie des divers tRNA est sensiblement identique et l'adaptation de ces tRNA & la traduction du rnRNA de la fibroine persiste pendant le je~ne. De la m~me faqon, le rapport tRNA/rRNA reste constant (12 mo16cules de tRNA par ribosome) et semblable ~ celui des t6moins pendant le je~ne at apr6s r6alimentation. L'activit6 des mninoacyl-tRNA synth6tases et le taux de charge des tRNA diminuent 6galement a u cours du jefine. Le taux maximal d ~ c y lation, qui correspond chez les t6moins & la phase de synth~se prot6ique maxirnale, est recouvr6 apr~s 24 h de r~limentation. Les chmagements observ6s doms les quantit6s d'acides amin6s libres different selon Yacide mnin6 et les niveaux atteints a n cours du jefme ne sont pas tr~s difi6rents de ceux trouv6s chez les t6moins. Nos r6sultats suggbrent que la production des divers 616ments de l'appareil de traduction est coordonn6e et aiust6e & l'activit6 prot6osynth6tique. Les m6c~nL~mes qui permettent ces ~ustemenis et les interactions entre ces mol6cules sont discut6s dans le cadre de la ,, r6guhrtion m6tubolique +, d6crite chez les procaryotes et la levure. Le taux de charge des tRNA semble jouer un r61e central et pourrait ~tre impliqu6

Summary. Changes in the translational machinery components of the Bombyx mori posterior silk gland were a n a l y s e d during starvation and refeeding and compared to the regularly fed larvae. During starvation, tRNA and ribosomal RNA synthesis are stopped. The m o u n t s of difJerent RNA classes and ot the different tRNA species slow down at the same rate. Thus various tRNA show similar half-lifes and the preexisting tRNA adaptation to fibroin mRNA translation persists durinq starvation. Similarly, the tRNA/tRNA ratio is constant during starvation and tefeeding (12 tRNA molecules for one ribosome) as in silk glands of control animals. Aminoacyl-tRNA synthetases and tRNA charqing levels are decreased during starvation. The maximal tRNA charging level obtained during maximal protein synthesis in control animals is regained after 24 h refeeding of starved larvae.

(*) This work is part X1V of a series of Funetion+al Adaptalion of t R N A s to Protein Idiosyntnesis ; for part XIII, Chevallier, A. and Garel, J.-P. Studies on tRNA adaptation, l~iochimie 61, this issue 16

23'0

G. Chaoancy and A. Fournier.

Changes observed in the free amino acid pool are not similar from one amino acid to another and levels reached after starvation do not differ strongly from the controls. Our results suggest that the production of translation apparatus components is coordinated and adjusted to the protein synthesis activity. Whether this coordination occurs in the silk gland is discussed on the basis of the ,, metabolic regulation ,,, primarily described in prokaryotes and Yeast. Transfer RNK charging

T r a n s f e r RNA and a m i n o a c y l - t R N A s y n t h e t a s e s p l a y a ~ e y role in the m e c h a n i s m of p r o t e i n biosynthesis. T h e s e molecules, as welt as a m i n o a c y l tRNA, a r e also i n v o l v e d in o t h e r processes. F o r example, s y n t h e t a s e s are c l e a r l y i m p l i c a t e d in r e p r e s s i o n of th.e b i o s y n t h e t i c p a t h w a y of t h e i r cognate a m i n o a c i d [1, 2, 3], and a m i n o a c y l - t R N A in f u r n i s h i n g a m i n o a c i d s for n o n - r i b o s o m e dep e n d a n t p r o t e i n s y n t h e s i s [for r e v i e w see 4]. I n the p o s t e r i o r si'l~ g l a n d t h e r e exists a s t r o n g c o r r e l a t i o n b e t w e e n mRNA a n d tRNA s p e c i e s of c o n s i d e r a b l e p r e c i s i o n [5, 6, 7, 8] and a ¢ quantit a t i v e , r e l a t i o n s h i p b e t w e e n tRNA and a m i n o acyl-tRNA s y n t h e t a s e s [9]. All c o m p o n e n t s of the protein synthesis apparatus, including ribosome and m e m b r a n e s p r o t e i n s are also c o o r d i n a t e d to the fibroin mRNA t r a n s l a t i o n [7, 10]. This c o o r d i n a t i o n poses the p r o b l e m of r e g u l a t i o n of the synthesis of these molecules. T~his question has been e x t e n s i v e l y s t u d i e d in b a c t e r i a . The c o n t r o l of ¢ s t r i n g e n t r e s p o n s e >) in b a c t e r i a is r e l a t e d to the level of a m i n o a c y l - t R N A , w h i c h , u n d e r a m i n o a c i d d e p r i v a t i o n , leads to a r e p r e s sion of rRNA s y n t h e s i s [11, 12]. The s y n t h e s i s of tRNA and o t h e r factors of the t r a n s l a t i o n a l a p p a ratus (elongation factors, r i b o s o m a l p r o t e i n s , etc.) and some aminoacyl-tR~N,A s y n t h e t a s e s are u n d e r s t r i n g e n t c o n t r o l [13]. R e c e n t w o r k w i t h E. coli, s h o w e d c l e a r l y that tR'NA s y n t h e t a s e p r o d u c t i o n is c o r r e l a t e d w i t h g r o w t h r a t e [14, 15, 16]. T h i s p h e n o m e n o n , called <> of a m i n o a e y l - t R N A synthetases, is also f o u n d in l o w e r e u k a r y o t e s : w h e n g r o w t h rate is s l o w e d b y a m i n o a c i d l i m i t a t i o n or b y t r a n s f e r i n g ceils into a less r i c h m e d i u m , the s y n t h e t a s e s a n d a m i n o a c y l - t R N A c o n c e n t r a t i o n d e c r e a s e ; h o w e v e r , w h e n the g r o w t h rate is shifted up, the s y n t h e t a s e level i n c r e a s e s [17, 18]. T r a n s f e r RNA a n d a m i n o a e y l a t e d tRNA concent r a t i o n s are also i n c r e a s e d in f a s t - g r o w i n g cells

BIOCHIMIE, 19'79, 61, n ° 2~.

levels seem to play a key role in the process of regulation and could be implicated in the mechanism of tRNA adaptation if this phenomenon results as expected from a t r a n s c r i p t i o n a f control.

Key works: Amino acid, tRNA, acylation, tRNA ligase, silk gland, Bombyx mort, starvation.

[18]. This r e g u l a t i o n is lir~k.ed w i t h r i b o s o m a l RNA and p r o t e i n s y n t h e s i s [17]. W h e n ba.cteria are s t a r v e d for a given a m i n o acid, p a r a d o x i c a l results are o b t a i n e d : the conc e n t r a t i o n of the cognate a m i n o a c y l - t R N A synthetase increases. This d e r e p r e s s i o n p h e n o m e n o n is a f u n c t i o n of aminoacyl-tRN~A level w h i c h can act as a r e p r e s s o r or a c o r e p r e s s o r of the s y n t h e tase gene. On the w h o l e , <> and ~ r e p r e s s i o n - d e r e p r e s s i o n >> c o n c e p t s i n d i c a t e that, in b a c t e r i a , tRNA and aminoacyl-tR.NA synthetase p r o d u c t i o n s are u n d e r c o o r d i n a t e d control, w h i c h acts b y r e c i p r o c a l p o s i t i v e or negative f e e d b a c k b e t w e e n these t w o m o l e c u l a r components. I.n this view, a m i n o a c y l - t R N A level a n d a m i n o a c i d s s u p p l y p l a y the role of p r i m a r y signal [for r e v i e w see 19]. Our k n o w l e d g e of these p h e n o m e n a is p o o r in e u k a r y o t e s s p e c i a l l y in a n i m a l systems. I n r a t liver, s t a r v a t i o n r e d u c e s p r o t e i n s y n t h e s i s a n d i n d u c e s p o l y s o m a l b r e a k d o w n [20], but n o decrease in a m i n o a c i d c o n c e n t r a t i o n or tRNA charging level w a s f o u n d E21]. The authors c o n c l u d e d that c o n t r o l of h e p a t i c p r o t e i n s y n t h e s i s is m e d i a ted at the i n i t i a t i o n step, w i t h o u t r e q u i r i n g d e a c y l a t e d tR~A. Conversely, in c u l t u r e d h u m a n cells u n c h a r g e d tRNA seems r e s p o n s i b l e for p o l y s o m e b r e a k d o w n t h r o u g h d e c r e a s i n g the rate of transl a t i o n a l i n i t i a t i o n [22]. I.n the Bombyx mort si/dk gland, s t a r v a t i o n is k n o w n to i n d u c e p o l y s o m e b r e a k d o w n [7] ,and a d r a s t i c d e c r e a s e in p r o t e i n s y n t h e s i s [23], F u r t h e r m o r e , n a t u r a l s t a r v a t i o n p e r i o d s o c c u r in larval life at the e n d of each i n s t a r a n d d u r i n g moulting. On this basis, the p o s t e r i o r silk gland of Bombyx mort seems a p p r o p r i a t e for s t u d y i n g the r e g u l a t o r y m e c h a n i s m s on the t r a n s l a t i o n a p p a r a tus. In o r d e r to o b t a i n a p r e l i m i n a r y answer, w e f o l l o w e d the s y n t h e s i s of tRNA, the a c t i v i t y of

tRNA ligase activity in silk gland. aminoacyl-tBNA synthetases, the charged/uncharget tRNA l~ev,el and the amino acid pool during starvation and refeeding of silkworm larvae.

231

o e e u r e d a t differcn~ t i m e s d u r i n g t h e l a s t l,arval i n s t a r . T h e s e t~mes, were. stredu.le~d a s i n d i c a t e d in t h e figure 1. R a d i o a c t i v e l a b e l l i n g of R N A a n d n u c l e o t i d e s w a s o b t a i n e d b y i n j e c t i o n o f 100~ IxCi of [32p] p h o s p h a i e i n 10 ~tl of w a t e r . L a b e l l i n g p e r i o d s a n d d u r a t i o n s a r e i n d i c a t e d o n t h e figure 1 a n d in (( R e s u l t s , .

Materials and Methods.

Silkworm

tRNA.

PREPARATION AND ANALYSIS OF

ANIMALS.

l a r v a e ,are h y b r i d s

of E u r o p e a n

R N A w a s e x t r a c t e d f r o m t h e p o s t e r i o r silk g l a n d s of [24]. All s t e p s of the iso'Iation p r o c e d u r e w e r e c a r r i e d o u t a t 0°C. T r a n s f e r R~N'A w a s i s o l a t e d b y c h r o m a t o g r a p h y o n a DEAE-eell'ulose c o l u m n e q u i l i b r a t e d w i t h s o d i u m a c e -

strains

2.(~0 a n d 31~0. C o n t r o l an,im~a'lv vce,re ~ed ad l i b i t u m w~t]z

B. m o r i l a r v a e a s d e s c r i b e d b e f o r e

m u l b e r r y 1,e,aves ( f o u r m,ea4s pe,r &ay) a t a t e m p e , r a tuane of 21 + l°C. Cocoo,n spinr~ing l~egins o n t h e n ~ n t h clay o f ~l~e f i f t h l a~rvat i n s t ' a r . S,tavvl~tion a n d re f~edin.g

TIME (HOURS) AFTER THE FIRST MEALOF THE FIFTH LARVAL INSTAR EXPERIMENTS

12

24

36

48

60

72

84

96

I

I

I

I

I

1

I

I

RNA synthesis

I

I

I

ale

It

~-¢

-"

Xl

X~

V

X$

V

•

V

-"

"+

=

~

V

4-

÷

of tRNA

~7 ~

4-

4-

Free amino acids

V

4-

4-

Aminoacyl-tRNA synthetases activity

4-

=

I

A

I

t

Charging level

--

!

.L

4-

4-

t

4

4-

Schemalic experimental schedule for starvation and refeeding experiments.

C o n t r o l a n i m a l s (eon t i n a o u s l y fed). B,eginning of ,~tarvation. B e g i n n i n g of refved.ing. I n j e c t i o n of r a d i o a c t i v e p h o s p h a t e [32p]. Sacrifice of l a r v a e .

B I O C H I M I E , 1979, 61, n ° 2.

I

"-

"1"

• V b * +

I

V--X-I-

of tRNA

Fro. 1.

I

V

Nucleotides

Half-life

108 120 132 144 156 168 180 I

~;

232

G. Chaoancy and A. Fottrnier.

tare buffer (0.05 M, pH 5.1)) eon[aining NaCl (0.1 M) and MgCl, (6.{)ffl M). After washing the column, tRNA was eluted by acetate buffer containing 1.2 M NaC1 and precipitated by 2.5 volumes of 95 per cent e t h a n o l at - - 2 0 ° C . In s,band~a,rd conditio.ns, the pellet w~as dissolved in Tris-ttG1 huffer (1.8 M, pH 8) and incubated 1 h o u r at 37°C to deacylate the tRNA : a f t e r ethanol precipitation, the tRNA p r e p a r a t i o n was dried u n d e r vacum and stored at - - 2 0 ° C . TotM RN~A was analysed by c h r o m a t o g r a p h y on a Sephadex G-100 column swollen in sodium acetate buffer (1.0-2 M, pH ~ 5.5~),. Radioactivity of each fraction [32p] was d e t e r m i n e d b y Ceren,kov counting or a f t e r trichloracet~ic acid~ (10 per cent) precipitation on a W h a t m a n 3 MM p a p e r disc w i t h a n a p p r o p r i a t e scintill,ation cocktail. tRNA preparatio~s v~ere eleetrophoresed as previously described [25, 26]. Separation was done only for ,the first d i m e n s i o n . RNA b a n d s were located by staining and gaI s c a n n i n g (with a Joyce Ghromosean), t h e n cut ; [3~p] ~adi~activity was m e a s u r e d by Cerenkov counCting. PEnlODATE OXIDATION OF tRNA.

After e t h a n o l p~eeipitation and drying of tRNA eluted f r o m DEAE-cellulos~ column, the pellet was dissolved directly (200, ~tg/ml) w i t h o u t Tris-buffcr t r e a t m e n t , in sodium acetate buffer (0.1 M, pH 4.6). One h a l f was used as. centre,1, tire o t h e r h a l f wa~s treated w i t h sodium period'ate (N'aIO]) to a final concent r a t i o n of 0.2 M. I n c u b a t i o n was at room t e m p e r a t u r e in the dark, for 30 minutes. The reaction was stopped by adding glucose (0.2 M final concentration). After 15 m i n u t e s in l h e dark, 0.1 volume of p o t a s s i u m acetate buffer (20 per cent, ,lair 5.2') was added, t h e n the tRNA precipitated b y 2~.5 volumes of 95 per cent ethanol at --2.0.°C. Control tRNA and periodate treated tRNA were stripped of a m i n o acids, by incubation in Tris-buffer (see above), and recovered by ethanol precipitation. Am,ino acid accepter activity was measured as indicated below. The ratio of treated to non-perledate treated samples gave the in r i v e tRNA <>. Using a k n o w n a m o u n t of [14CIaminoaeyl-tRNA (prepared as below) mixed w i t h crude homogenate, we m e a s u r e d deacylation duriag the extraction process before periodate oxid,a~tion,. Less t h a n 8 per cent deacylation was found for all aminoacyl-tRNA tested. PREPARATION OF AMINOACYL-tRNA SYNTHETASES. Crude syntheta~se (F~C 6.1~.L) was prepared as described earlier [24] from the post-ribosomal superrm~tant of t/re posSe river part of the si'lk gl,and. The post-ribosomal s u p e r n a t a n t was stripped of amino acids and tRNA by filtration on Sephadex G-25 and by DEAE-cellulose c o l u m n s c h r o m a t o g r a p h y . The enzyme p r e p a r a t i o n was stored at - - 20°C in Tris-buffer (50 raM, pH 7.4) con~taining KC1 (25 raM), MgCl~ (10 raM), ~ m e r o a p t o e t h a n o l (10 mM) and glycerol (50 per .cent, w / w ) . ASSAY FOR in v i t r o AMINOACYLAT1ONOF tRNA. Standard condiHons for m e a s u r i n g a,mino acid acoeptor activity of tRNA have been previously desB I O C H I M I E , 1979, 61, n ° 2.

cribed [24]. Each assay contained 12.5 ~tmotes of Tris, 5 ~males of ATP, 8 ~tmoles of MgC]~ 10, to 40 ~tg of tRNA, 0.2 to 0.5 mg of p r o t e i n s (cru4e enzyme), 0.1 i~Ci of [14G] a m i n o acid, 20 per cent (w/w) glycerol, in a final volume of 200 ~1 (pH 7.4). After incubation at 37°C during 25 minutes, reaction was stopped by TCA (Triehloracetic acid): 10 per cent and lif0 ~tl of the sample were filtered on ,a circle (2.5 cm diameter) of W h a t m a n (3 MM) filter paper. F i l t e r s were washed by cold TCA 10 per cent during 30 minutes, TEA 5 per cent during 15 minutes, t h e n b y ,ethanol 95 per cent during 5 minutes. They were dried by e t h e r and radioactivity was m e a s u r e d in a Packard Spectrometer w i t h a classical scintillation liquid (4~ g of P.P.O. a n d 400 mg of POPOP in 1 liter of toluene). PREPARATION AND AI~ALYSIS OF NUCLEOTIDES AND FREE AMINO ACIDS,

To an~iyse t~e appe~arence of ¢ Magic spots >> (ppGpp and p p p ~ p p ) during starvation, extraction of nueleotides was done as described by Cashel [27]. Dissected silk glands were homogenized i n formic acid (1 M, pH 3 . 0 ) a t 00{3. After c e n t r i f u g a t i o n at 6500 r p m in a SS 34 $orwall rotor, ~he superrmta~nt ~as chromatographical o n a PEI-eellulose t h i n l a y e r plate (Merck). This ascending c h r o m a t o g r a p h y was p e r f o r m e d w i t h p h o s p h a t e buffer (1.5 M ; pH 3.4) at room temperature. Results were a n a l y z e d by r a d i o a u t o g r a p h y w i t h Kodak X Omat X R 1 film. Free a m i n o acids of the posterior silk gland weTe extracted by homoge~i~atio,n in 5 pry cent sulfosallcyt~c acid (1 ml for t w o silk ~ands)~ Th~ acidosoluble fraction was t h e n ¢h~'om'atLo~raphie'd on a ~ech,n~eon autoarmlyser ~s previ'ot~sly described [28]. CHEMICALS.

All radioactive chemicals were purchassed from C.E.A. ( S a c l a y - France). Specific activity of [32p] sodium phosph~tte was 50Ci/mg ; specific activity of [14C]amino acids were between 100 and 20,0 mCi/ mMoles. Adenosine trip~os]~hate was provided by Boehringer-Mannheim, DEA4E-cellulose (DE~_o) and filter paper 3 MM b y W h a t m a n , Sephadex (G-25 and G-100) b y Pharmacia~ all other chemicals by Merck.

Results and Discussion. SYNTHESIS OF TRNA AND nRN,A. L a b e l l i n g of R N A w a s d o n e d u r i n g g r o w t h a n d s t a r v a t i o n as i n d i c a t e d i n M e t h o d s f o r t w o d i f f e r e n t p e r i o d s . In the first case, the s t a r v a t i o n start e d 48 h a f t e r t h e b e g i n n i n g of t h e f i f t h i n s t a r (fig. 1). I n t h e s e c o n d c a s e , t h e s t a r v a t i o n s t a r t e d 120 h o u r s a f t e r t h e f i r s t m e a l of t h e f i f t h i n s t a r , w h i c h w a s t a k e n as t h e o r i g i n of t i m e (fig. 1). T h e s e t w o p e r i o d s c o r r e s p o n d to d i f f e r e n t d i e t a r y s t a g e s : s t a r v e d l a r v a e d o n o t e n t e r final n y i n p h o -

tRNA ligase actioity in silk gland. rpfn for pair of D o s t e r i o r

233

sdk~llands

290~0

100000 f

J

...fl

,q

200QO

:'-j

.k

C',

10 O00

~

1

rRNA

5S RNA

%

i

/

t RNA

FIG. 2. - - In vivo pulse-labelling of posterior silk gland RNA from two days old

larvae of the fiJth larval instar.

Groups of five s i l k w o r m s f r o m ~ last larval insta,r were starved during 48 h, t h e n injected w i t h radioactive phosphate. Posberior silk glands were dissected out 4 hours tater. O~Jler groups, a f t e r the starvation; were refed during 8 hours, t h e n inje,eted .and ~ e posterior silk glands remov,ed and homogenized after 4 hours labelling. Con,trol anim:als, fed continuously, were 1.ikewrise injected a,nd dissected (see fig. I). All pos,t.erior silk ~lands were treated a.s described in <> to obtain I~NA and to separate differenrt R~NA elasses b y Sephadex G-I~0 column c h r o m a tography.

[]

BIOCHIMIE, 1979.

C~n~rol

,,,

C o n t r o l of r e f e d l a r v a e

48 h starved

~.%

12 h o u r s ,refeed~ng

61. n ° 2,.

234

G. Chavancg a n d A. Fournier.

sis, d u r i n g the first three d a y s w h e r e a s after the t h i r d d a y they s p i n a cocoon and u n d e r g o metam o r p h o s i s E23]. R a d i o a c t i v e labelling [a2p] of r i b o s o m a l RNA, 5 S RNA and tRNA is s h o w n in figures 2 and 3.

Cpm for pair of p o s t e r i o r

fifth i n s t a r [7, 29]. On the o t h e r hand, c o m p l e t e stop of RNA s y n t h e s i s is p r o b a b l y r e s p o n s i b l e for the d e c r e a s e in RNA content d u r i n g fasting. Conversely, since s y n t h e s i s is g r e a t e r in r e f e d animals than in c o n t r o l ones, the tR~N~Alevel i n c r e a s e d , ten-

silkglands

7500

,/

5000

2500"

--

i:il

I I I I I rRNA 5S R N A t RNA Fro. 3. - - In vivo pulse-labelling of posterior silk gland RNA from five days old larvae of the fifth larval instar. For legends, see figure 2. In s t a r v e d animals, the s y n t h e s i s of all RNA classes is almost null. H o w e v e r , the RNA l a b e l l i n g is m u c h h i g h e r i n sil~ glands of r e f e d animals than in the c o n t r o l ones. R,esutts w e r e s i m i l a r in the first p a r t (fig. 2) and s e c o n d p a r t (fig. 39 of the fifth instar. Nevertheless, w i t h these r e f e d a n d c o n t r o l animals, l a b e l l i n g levels w e r e v e r y different b e t w e e n the above two periods, the incorp o r a t i o n rate beeing about 30 fold h i g h e r d u r i n g the first p a r t t h a n d u r i n g the s e c o n d half of the l a r v a l instar. These results e x p l a i n p r e v i o u s o b s e r v a t i o n s w h i c h s h o w e d that rRNA and tRNA a c c u m u l a t i o n rates w e r e m u c h h i g h e r at the b e g i n n i n g of the

BIOCHIMIE, 1979, 61, ~o 2,.

ding to r e g a i n that o b s e r v e d in E29]. The a g r e e m e n t b e t w e e n our s u r e m e n t s of R,NA content s h o w ling of RNA is r e p r e s e n t a t i v e of synthesis.

c o n t r o l animals results and meathat pulse labelthe actual RNA

The effect of s t a r v a t i o n w a s v e r y s i m i l a r on rRNA and tRNA suggesting t h a t a c o m m o n signal i n h i b i t e d t h e i r t r a n s c r i p t i o n . It is w e l l k n o w n that in p r o k a r y o t e s , g u a n o s i n e p o l y p h o s p h a t e s are resp o n s i b l e for the (~ s t r i n g e n t r e s p o n s e >) in RNA s y n t h e s i s i n h i b i t i o n E27, 30]. These p o l y p h o s p h a t e s (ppGpp a n d p p p G p p ) are a c c u m u l a t e d w h e n cells are r e s t r i c t e d in a m i n o acids or starved. To see if such a signal exists • in the p o s t e r i o r silk gland,

tRNA

235

l i g a s e a c t i o i t g in s i l k g l a n d .

we have studied r a d i o a c t i v e phosphate i n c o r p o r a tion i n nucleotides d u r i n g starvation. No labelled spots c o r r e s p o n d i n g to guanosine polyphosphates exist o n the r a d i o a u t o g r a p h y of c h r o m a t o g r a m m (fig. 4). These results were not s u r p r i s i n g , since RNA synthesis control b y ppGpp or pppGpp has been only described i n b a c t e r i a or in chloroplasts [30, 31].

before starvation (fig. I) : the first time 12 h o u r s after the first meal of the fifth instar, the second time 2,4 hours l a t e r . Twelve hours after the last injection, larvae were starved, then lots of 5 animals w e r e dissected .each 12 hours. T r a n s f e r RNA extracted from the silk glands was subjected to first-dimensional p o l y a c r y l a m i d e gel electrophoresis. After staining, the gel was s c a n n e d , and b a n d s c o r r e s p o n d i n g to major tRNA species in the silk gl,and (fig. 5) w e r e cut to measure the [32p] radioactivity.

O.D.

arbitrary scale

2

7

3

10

1

::::,:::if::¸¸ : :: i • i

A Fro. 4 . -

Radioautography of pulse-labelled nueleotides from posterior silk gland. Starx~e~l an.im,als reeve divided in two groups : the first got va&ioactiv.e [32p] phospla~te a,fter 6 hours starvation, the other after 2~4hours (fig. 1;). Silk gl~ands were dissected 3 hours after ,inject~ion and treated as 5eseribed in ¢ M~ethods,, to analyse la'helled nueleotides. A : Control animals ; t~ : 6. h starved animals• ; C : 24 h starved animals. Xj and X,~ indicate ~he pl,aee of .expected ¢ Magic SIm.ts >> (ppGpp and pppGpp).

HALF-LIFE OF TR'~A. Since tR,NA synthesis was stopped by starvation, decay of tRNA r e p r e s e n t s only d e g r a d a t i n g activities. To estimate the half-life of tR,IVA, s i ~ w o r m larvae were injected twice w i t h [32p] phosphate, BIOCHIMIE, 1979, 61, n ° 2,.

e Fro. 5. - - Eleelrophoregram of transfer RNA from the posterior silk gland. • Ala Band 1 contains tR~NA2a , band 2 tRtN,A~bla , band -r Ser 3 tRNA~ Lv , band 6 tRNA~l'v , band 10 tttNA',, and Ser band 1"5 tR~N'A1 the major eomponents of the tRNA poo.1 [26J.

F i g u r e 6 shows that t h r o u g h o u t the fasting period, the relative labelling for each b a n d remained constant. This suggests that the half-lives of the different tRNA species are very similar. These results agree well with those o b t a i n e d by acylation m e a s u r e m e n t [29] c o n f i r m e d b y a d d i t i o n a l e x p e r i m e n t s on changes in each separate iso-tRNA species ~26!. This shows that the cellular tRNA p a t t e r n is not c h a n g e d by starvation and then that the q u a n t i t a t i v e a d a p t a t i o n of tRNA p o p u l a t i o n to mRNA codon f r e q u e n c y r e m a i n s d u r i n g starvation.

G. Chauancy and A. Fournier.

236 CHARGING

LEVELS

TRNA.

OF

T h e assay for d e t e r m i n i n g in vioo levels of c h a r ged tRNA ~vas b a s e d on the a b i l i t y of p e r i o d a t e to i n a c t i v a t e the u n a c y l a t e d tRNA [ ~ ] . These levels w e r e d e t e r m i n e d for tRN0~ specific to a m i n o acids w h i c h m a k e up the b u l k of siLk fibroin (glycine a n d alanine) e x c e p t for s erine, Mnce it w a s r e p o r t e d that p e r i o d a t e t r e a t m e n t resulted in d i s c h a r ging of the a m i n o a e y l - t R N ~ [3~] ; w e also determ i n e d tRNA specific for a m i n o acids p r e s e n t at low level or absent in fibroin.

m o z m

w i t h those of D.elaney a n d S i d d i q u i [8], w h i c h s h o w e d that the i n c r e a s e of a c y l a t e d tR~lAOly and tRNA A~a a m o u n t is m o r e i m p o r t a n t t h a n the i n c r e a s e of o t h e r c h a r g e d tRNA content. Thus, d u r i n g the siLk synth,esis p e r i o d , the q u a n t i t a t i v e a d a p t a t i o n of the tR~NA pool to the c o d o n distrib u t i o n of fibroin m R N A co~responds to the quantitative a d a p t a t i o n of the aminoacyl-tR~5?A p o p u lation. The i n c r e a s e of acyl~ation level d u r i n g the g r o w t h p h a s e (from the first meal V1, to the fourth

I"I

30.

U) _4 UJ

I

_~£

=

•

-3

-o-

o

o 1 2

" ~o

'13 ÷2

15.

I-- 0 &

o~ ¢3 n-

0

P-

"6-

•

JI.

vs

lo

AO

&

++

+,4

;~' Hours

of

• ,h

starvation

F~m. 6. - - Changes of prelabelled tRNA during starvation. After in vivo ,labelling of tR~A' by [32p], animals were starved from the fift~ d,ay of the fifth ,instax (s~e fig. 1). l~a+eh 12, J~ours, tR,NA from ~he posterior silk gla.nds were separa:ted by polyaerylamide gel el.ectrophores,is (as in fig. 4) and radioa,ebivity of gel bands was measured. l~vla¢ive rad~o~eti~z~ty of ba'nds 1, 2, 3. 6, 10, 13, is pl.o~¢ed du.ring ~ 7'2,~ou,rs s~a,r w ~ t i ~ period. T a b l e I s h o w s results o b t a i n e d w i t h contin u o u s l y fed larvae. B,efore the first meal (V 0 stage) the level of a c y l a t e d tRNA in the p o s t e r i o r silk gland is v e r y low (20 to 40 p e r cent of total tR'NA). The c h a r g e d f r a c t i o n of e a c h tRNA i n c r e a s e s r a p i d l y at the b e g i n n i n g of the fifth instar, r e a c h i n g a m a x i m u m after 72 h, then it r e m a i n s c o n s t a n t until the 8th day. No n o t i c e a b l e differences appear e d betveeen tRNA c o r r e s p o n d i n g to the m a j o r a m i n o .acids of fihroin and specific for m i n o r amino a c i d s : all tRN, A species w e r e 75-100 p e r cent charged. Consequently, since th, e total a m o u n t of tRNAOly and tRNAA1a i n c r e a s e s d r a m a t i c a l l y comp a r e d to o t h e r tR,NA E8, 24] d u r i n g the g r o w t h p e r i o d of the p o s t e r i o r silk gland ('fixst four days), the level of available glycyl- and alanyl-tRNA i n c r e a s e s in the same ratio. These results agree

BIOCHIMIE, 1979, 61, n ° 2.

d a y ) c o u l d be r e l a t e d to the step up in p r o t e i n s y n t h e s i s d u r i n g this p e r i o d [7]. F r o m this p o i n t of view, it is s u r p r i s i n g that tRNA are not m a x i m a l l y a c y l a t e d d u r i n g the w h o l e r a p i d g r o w t h p e r i o d . P e r h a p s tRNA a.re s y n t h e t i z e d faster t h a n t h e y can be acylated. The a m i n o a c y l - t R N A synthetase activities go u p v e r y significantly d u r i n g this p e r i o d [9] and must a c c o u n t for the i n c r e a s e in c h a r g e d tRN~A f r a c t i o n (table I,). After this g r o w t h phase, the p o s t e r i o r silk gland r e a c h e s a steady-state since tR~NA, rRNA and synthetases a m o u n t do not i n c r e a s e f u r t h e r [7, 9]. This s t e a d y state level c o r r e s p o n d s to m a x i m u m fibroin synthesis a n d c o n s e q u e n t l y to the h i g h e r rate of p r o tein s y n t h e s i s ,in the silk gland. If w e try to i n t e r p r e t these results w i t h <> m o d e l s p r o p o s e d for p r o -

tRNA ligase activity in silk gland. k a r y o t e s , the g r o w t h p e r i o d could c.orrespond to a d e r e p r e s s i o n of s y n t h e t a s e a n d tR'NA s y n t h e s i s b y the i n c r e a s i n g a m o u n t of u n c h a r g e d tRN.A resulting from h i g h tRNA t u r n o v e r on the ribosomes. W h e n the steady-state is r e a c h e d , synthetase a n d tRNA levels a r e both a d j u s t e d to the rate of p r o t e i n synthesis and the tRNA c h a r g i n g levels are c o n t i n u o u s l y m a i n t a i n e d . This c o n c e p t i o n c o u l d e x p l a i n <~m e t a b o l i c r e g u l a t i o n • in p r o k a r y o t e s a n d y e a s t : in f,ast-gro~ving cells h i g h p r o tein s y n t h e s i s c o u l d i n d u c e tRNA and synth.etase i n c r e a s e b y u n c h a r g e d tRNA m e d i a t e d d e r e p r e s sion up to the steady-state level. On the c o n t r a r y , w h e n cultures w e r e shifted down, the d e a c y l a t i o n rate of tRNA w a s l o w e r , s y n t h e t a s e s ,and tR,N'A p r o d u c t i o n b e e i n g c o n s e q u e n t l y r e p r e s s e d or less d e r e p r e s s e d . I n steady-state after s h i f t - d o ~ n , conc e n t r a t i o n s of tRtNA a n d s y n t h e t a s e s are louver.

237

and c h a r g i n g levels (fig. 7) g r o w e d up l e a d i n g to the r e c o v e r y ,of a h i g h a m i n ~ a c y l - t ~'R~A c o n t e n t available for p r o t e i n b i o s y n t h e s i s . These d a t a .can be c o m p a r e d w i t h results obtained at the V o stage (table I) w h e n a n i m a l s a r e not y e t fed. In b o t h cases, p r o t e i n s y n t h e s i s a n d RNA t r a n s c r i p t i o n w e r e s t o p p e d a n d tR~NA c h a r g i n g level w a s low. Such a c o o r d i n a t i o n b e t w e e n tR~NA s y n t h e s i s a n d level of c h a r g e d tRNA has been obser~red in m a m m a l i a n systems [36]. The a u t h o r s s h o w e d that an i n h i b i t i o n of 4he tRNA a m i n o a c y lation i n d u c e d a d e c r e a s e of the rate of tRNA synthesis a n d that drugs s t i m u l a t i n g the a m i n o a c y l a tion of ~RN.A cause a r i s e of the rate of tR'N,A synthesis. F r o m these data, t h e y c o n c l u d e d t h a t the tR~NA s y n t h e s i s d e p e n d s on the level of c h a r g e d tRNA. Then, tR,NA s y n t h e s i s c o u l d be m o d u l a t e d

TABLE I.

In vivo tRNA charging levels in posterior silk gland during the fifth instar. Hours of the fifth in,~tar tRNA

Alanine Aspartate Glutamate Glyeine Isoleucine Leucine Methionine Phenylalanine Valine

Vo

V~4

V4s

V~2

V~

Vl~n

34 27 20 29 40 34 25 22 20

46 44 33 40 76 52 55 28 25

76 70 61 75 80 69 75 68 78

84 76 70 88 90 90 100 76 82

86 80 75 85 100 87 95 75 82

87 78 73 85 100 83 100 78 80

Results indicate the ratio of elmrged on total tRNA (pe~- cent) estimated by aeeeptor activity (average of 2 assays): CAmrged tKNA correspond to unoxidized tRNA after perioda~e treatment (see <
I n fact, this e x p l a n a t i o n does not agree w i t h results of the l i t e r a t u r e w h i c h s u p p o r t the <> c o n c e p t [16, 18] a n d is also c o n t r a d i c t e d b y o u r results : w h e n a n i m a l s w e r e s t a r v e d after t h r e e d a y s of r e g u l a r feeding, the c h a r g i n g level of all tRNA b e c a m e l o w e r (fig. 7). T h e c h a r g e d v~rsus tot,al tRNA ratio d i d not d e c r e a s e continously, but r e a c h e d m i n i m u m values b e t w e e n 3~ a n d 4 , 8 h starvation. These lowest values c o r r e s p o n d to about the half value of the control. The d e c r e a s e of the a m o u n t of IRNA r e c o r d e d d u r i n g fasting p e r i o d s [29] and r e s u l t i n g from a n i n h i b i t i o n of tRNA s y n t h e s i s (see above) was then a c c o m p a n i e d b y a d e c r e a s e of c h a r g i n g levels, the amoun~ of a c y l a t e d tRNA b e c o m i n g l o w e r than e x p e c t e d . D u r i n g refeeding, both IRN~A

BIOCHIMIE, 1979, 61, n ° 2.

b y c h a r g e d tR~NrA a c t i n g as a c t i v a t o r s o r u n c h a r ged tR~NA acting as r e p r e s s o r s . All o u r data, e v e n t h e ones concern.tug t h e g r o w t h p h a s e can be e x p l a i n e d w i t h this model. Nevertheless, it r e m a i n s to k n o w h o w the tRNA c h a r g i n g level is l o w e r e d in s t a r v e d a n i m a l s a n d i n c r e a s e d after s u b s e q u e n t refeed,ing. The m a i n two w a y s b y w h i c h c h a r g i n g level can be m o d i f i e d are the a m i n o a c i d a v a i l a b i l i t y a n d the a m i n o a c y l tl~NA s y n t h e t a s e activity. F R E E AMINO ACID P O O L S .

The free amino a c i d pools of the p o s t e r i o r silk gland ~v.ere m e a s u r e d u n d e r fasting con~lltions, s t a r t i n g from d a y 4 of the fifth insta'r. In b o t h the

G. C h a v a n c y and A. F o u r n i e r .

238

D u r i n g starvation, the Level of amino acids was .either lo-wered (Alanin.e, Valine, Leuc.ine, Histidin,e, Lysine) or slightly increased (glutamic acid), or almost u n c h a n g e d . The observed changes occured essentially d u r i n g the first 12 hours. No significant v a r i a t i o n s were found after this period

control and starved animals, the different pools for each a m i n o acid were f o u n d very u n b a l a n c e d (table II)~. These results, confirmed by previous exhaustive d,ata [28], showe.ed that these pools did not reflect the an~ino acid c o n t e n t of the synthesized proteins. If w,e ,expr.ess the amino acids con-

t

,_.

50-

g III e-

t

~e

Z,,,¢

•

• o glycine l

• ~ alanine

C m_

'

''

,D

"

50.

.Cr

....

©

[3"~'"£3

si;,

;:S/

U')

glutamate

• z~ leucine

,.'"

/

o

,,,... i,.,

l

• o aspartate

• o glutamine l

• A phenylalanine

A zs valine •

• cl ~soleucine

0 2'4

4'8

7'2 Hour2'6_

of

2'4

starvation

4'8

7'2

tJ

methionine

9'6

FIG. 7. Changes of tRNA charging level during starvation. Results. are ,expressed as in table I (average of 2 experimenets). Closed circles, squares or ~triangles correspond to s,tarved anim~als. O.pe~ed signs correspond to refed animals. Arrows indicate the time when re feeding started. Th~ zero lim.e corresponds to the 4th day of the fifth instar (see fig. 1). -

-

tent as the n u m b e r of mi'nutes of fibroin synthesis that each a m i n o acid can s u p p o r t (table III), the effective c o n c e n t r a t i o n s vary by at least 700 fold, the most a b u n d a n t beeing histidin,e (2200 minutes) a n d the less a b u n d a n t glycine and a l a n i n e (2 to 3 minutes).

BIOCHIMIE, 1979, 61, n ° 2.

except for p h e n y l a l a n i n e a n d tyrosine. Moreover, after 48 hours, the a m o u n t of free a m i n o acids in the silk gland of starved animals were not very different from the control except for p h e n y l a l a n i n e and tyrosine (1/10 and 1/4 of the control values),

t R N A ligase activity in silk gland. Some possibility existed that o b s e r v e d changes of tRNA c h a r g i n g level d u r i n g s t a r v a t i o n (fig. 7) could be affected by the v a r i a t i o n s of i n t r a c e l l u l a r a m i n o a c i d .concentration s h o w n on table IL F r o m this p o i n t of view, tRNA l)h~ c h a r g i n g level c o u l d be t h e most affected and tRNAC~y not at all. If tRNA e~e w a s really less acylat,ed than the others, tRNAOly c h a r g i n g level ~was also d e c r e a s e d (fig. 7). It seems that c h a n g e s in a m i n o acid c o n c e n t r a tions w e r e too slight to i n d u c e significant variations of the a m o u n t of c h a r g e d tRNA. F u r t h e r m o r e , as w e s h o w e d that the anmunt of tRNA l o w e r e d i'n s t a r v e d glands, the tR~b~A a c y l a t i o n could be

239

after 48 h s t a r v a t i o n (table II). These results i n d i cate that u n d e r fasting, both u p t a k e and i n t r a c e l lular synthesis of a m i n o acids w e r e l o w e r e d . AMINOACYL-TRNA SYNTHETAS]~S.

Measurements of s e v e r a l a m i n o a c y l - t R N A synthetase activities w.ere d o n e in p o s t e r i o r silk gland c r u d e extracts i'n excess of tRNA (see Methods). S t a r v a t i o n started after t w o days of regular f e e d i n g in the fifth instar. F i g u r e 8 s h o w s that after 24 h starvation, some synthetase activities r e m a i n e d the same as the c o n t r o l ones (gly-

TABLE II.

Free amino acids pool of lhe hemolgmph and posterior silk gland during starvation. Hemolymph - ~

Centre| 0

Ser Glu Gly Ala Vat Met Pro Leu Tyr Phe Lys His Arg

Posterior silk gland

12

8.26 11.51 2.13 2.99 7.26 10.54 3.05 2.73 3.83 2.39 3.24 0.93 1.35 1,49 1.96 1.43 1.81 0.90 1.39 5.73 7,83 17.59 10,75 2.28 7,52

Starved

26

36

11.65 2.38 10.05

2.03 0.46 1.07 3.27 1.63 2.65

2.72 3.31 0.92 1.38 1.11 1.61 0.76 1 50 0.85 5.15 5.38 18.06 18.88 3.06 1.81

~8 6.64 1.24 5.10 8.49 1.79 3.40 0.72 0,98 0.58 0.77 6.12 17.77 1.98

t2

26

8.78 1.65 7.41 7.66

10.05 1.02 7.56 6.33

2.54 0.19 0.30 0.66 0.45 2.89 18.39 0.95

2.00

2.88 24.22 0.87

Control 48

0

0.587 7.75 0.185 0.054 0.51 0.128 3.04 0.031 0.22 0.059 0.37 0.130 0.37 0.056 0.38 0.569 3.71 0.057 17.45 0.448 1.58 0.049

I~2

0.869 0.429 0.137 0.190 0,098 0.091 0.222 0.051 0.860 0.080 0.579 0.124

24

Starved 36

12

36

48

0.107 0.142 0.415 0.614 0.420 0.519 0.693 0.148 0.206 0.144 0.200 0.162 0.041 0.079 0.016 0.023 0.067 0 051 0.025 0.027 0.140 0.024 0.026 0.022 0.020 0.028 0.039 0.020 0.020 0.014 0.124 0.060 0.048 0.028 0.025 0.028 0.017 0.007 0.008 0.565 0.553 0.220 0.060 0.068 0.047 0.028 0.022 0.032 0.389 0.438 0.343 0.237 0.406 0.054 0.069 0.023 0.057 0.033

In hemolymph, results aa,e expressed in ~tM/ml, in silk gland in p~M/gland. Starvation began the 4th day of the fifth ir~star fsee fig. 1.).

p e r f o r m e d w i t h a l o w e r a m i n o aoids availability. I,n fact, p r e v i o u s results [28] s h o w e d that i n t r a c e l lular free amino acid pool is c o m p a r t i m e n t a l i z e d in p o s t e r i o r silk gland and that the i m m e d i a t e p r e c u r s o r s of p r o t e i n synthesis come f r o m the e x t r a c e l l u l a r pool. Finally, it appears that in e u k a r y o t e s the levels of acylaCed tRNA cannot be c o o r d i n a t e d w i t h the c o n c e n t r a t i o n of free a m i n o acids [2'1, 3~, 35]. An o t h e r i n t e r e s t i n g feature ,is the b e h a v i o u r of silk gland s y n t h e s i s e d am_Lino acids (such as al~anine) w h i c h did :not differ strongly f r o m that of a m i n o :acids p u m p e d in the h e f n o l y m p h (phen y l a l a n i n e and glutamate) although great a m o u n t s r e m a i n e d available outside the silk gland, even

BIOCHIMIE, 1979, 61, n ° 2.

cine, isoleucine, and serine) w h e r e a s the others were already lowered. W h e n animals w e r e r e f e d after 48 h starvation, some synthetase activities g r e w up (glycine, ala~ine, leucine) the others r e m a i n e d almost constant (fig. 8). In all cases, e v e n after 48 h of continous r e f e e d i n g , activities w e r e l o w e r t h a n in controls, l~henylalanine-, arginyl- and valyl-tRN~A synthetases d e c r e a s e d after refeeding, the level r e a c h e d after 48 h b e e i n g not significantly differ e n t f r o m that of s t a r v e d animals.

In vitro ,assays to m e a s u r e s y n t h e t a s e activities in c r u d e extracts set always the p r o b l e m of relationslfip w i t h actual in viva activities. P r e v i o u s results [9]. o b t a i n e d in the same c o n d i t i o n s , w i t h

240

G. Chaoancy and A. Fournier. 20

GLYCYL-IRNA

~

J

15

[ !

ALANYL" tRNA 5YNTHETAS[

r

!

? o10 Ij

E 5

SERYL- tRNA SYNTHETASE

ISOLEUCYL'IRNA SYNTHETASE

? 10

LEUCYL -IRNA SYNTH£TA~...,..

/

PHENYL-ALANYL tRNA SYNTHETAS[

? -~ 20 x

E u

0 50

ARGINY£ - IRNA SYNTHETASE

m

x 25

U

,,Jr

E

10

Fl~. 8. - - Changes of aminoacyl-tRNA synthetases actioity during starDation and rcfeeding. • ASPARTYL " t RNA SYNTH|TAS|

r. GLUTAMYL

SYNTHETASE

t RNA

/

?

E Q.

..,-,4

R e s u l t s a r e e x p r e s s e d i n e p m of [14C] a m i n o a c i d c h e r g e d on t R N A f r o m p o s t e r i o r si]k g l a n d b y in oitro a m i n o acylaLion with crude extracts synt~eta~es corresponc~ing to o n e g l a n d . (For d e t a i l s , see M~tuhods >>). E a c h p~i~nt i s tthe m~an, v a l u e o f 3 e x p e -

y..

rime,n~sl.

e--@ I1--11 3

4

5

nAYS

OF THE

BIOCHIMIE, 1979, 61, n ° ~.

2

3

A--A 4

FIFTH LARVAL INSTAR

5

Control extracts. Syn~Jleta, ses of s t a r v e d a n i m a l s . S y n C h e t a s e s o f vefed a n i m a l s .

t R N A ligase activity in silk gland.

241

TABLE III.

Quantitative relationship belween several compot, ents of the translation apparatus in the posterior silk gland during the fifth larval in,tar. Synthetase activity

tRNA

Free amino acids

tRNA

rRNA

tRNA (3)

Free amino acids amino acids ineorporalcd into fibroin/minote (~)

11 ~- 2 3.5 1.8 0.9

150 18 9 80 260 44 900 200

3 3 16 300 25 2200 45

(t)

Control animals

Starved animals

Total Gly Ala Ser Leu Tyr His Asp Total Gly Ala

2 2 1.1 1.2 0.7

~- 0.2 --I- 0.3 -+- 0.5 ~___0.2

1.9 0.7

Set

1.5

Leu Tyr

0.3

His

Asp

(~)

12 +~_ 2 3.6

180 32

3

2 0 1,2

13 120

1 4

50 2 1620 350

60 3 2100 25

(1) Arbitrary units.. For re gula,rly fed animals, the aetivi,ty of s.ynC~eta~s wa.s obtairmd from previo.us results [9] and quanUity of tRNA from several published d,ata [5, 8, 24, 25]. The ratio indi, eates the mean value of 8 m,easuremen.ts done a,t the different days of the fifth instar. This rat4o remains the same, whatever the synVhetase and eRNA, ~and although changes in tRN'A pool and synOletase occur during all the last larval tinstar in this control andmals [5, 9]. For 48 h starved animals quantity of tRNA were o'btoined by ,aeceptor activity [29] and syntheease activitie.s from our experiment. (2) Total tRNA/rRNA ra.tio are expressed in n u m b e r of ~tI~N~AmolJeeules by rRNA molecules. Calculation was done from our exp,e~imer~ts and af.ter separation of silk gland RNA by S~ph,adex G-100 column chromatography. Fo.r cofitrol animals 9 independent m~asurem, ents were pooled eorrespont$ing to 9 different days of the fifth larval insbar. For 48 h starved an,ima.ls result is the mean value of 10~independan't e.xperiments done at 4iffere~t stages of the f~i~¢h insear. Individual t l ~ A vcere e~leulated from first-dimensional polyacrylamide gel eleotropho~esis (fig. 6) in a single experiment. (3) Number of fr~c amino acids molecules for one tRNA molecu,le. Calculation was done with five 4ays old an,imals (eo~trols) and 48 h starved animals (previously fed during five days). (4) Th~is ratio gives the t,ime (minutes) of fibroin synthesis ,that ea.c~h intTaeellular free amino acids pool can support calculated f r o m fiwe days old larvae and 48 h s¢arved animals after five days of regular feeding.

c r u d e e x t r a c t s of n o r m a l l y f e d a n i m a l s s e e m e d r e p r e s e n t w e l l t h e v a r i a t i o n s of in viva levels, s i n c e the a d a p t a t i o n of s y n t h e t a s e s to f i b r o i n c o m p o s i t i o n f o u n d in t h e s e e x p e r i m e n t s c a n n o t be obtained by chance. Nevertheless, the aminoacylati~)n w i t h e x t r a c t s of r e f e d silk w o r m s w e r e s u r p r i s i n g for v a l i n e a n d argir~ine (fig. 8). W e k n o w that, d u r i n g s u c h a p e r i o d , p r o t e i n a n d R N A s y n t h e s i s s t a r t fastly. T h e i n c r e a s e of t R N A a m o u n t m u s t n e e d a i n c r e a s e in s y n t h e t a s e s to be c o n v e n i e n t l y ,acylated. It m i g h t be a r g u e d t h a t tR~N,AA~g et tRNAVal d i d not s i g n i f i c a n t l y g r o w up, s i n c e their concentration into fibroin is very low; u n f o r t u n a t e l y , t h e m e a s u r e m e n t of t h e s e i n d i v i d u a l t R N A has n o t b e e n d o n e in t h e s e e x p e r i mental conditions.

BIOCHIMIE, 1979, 61, n ° 2.

Ex.cept f o r v a l i n e a n d argini'ne, v a r i a t i o n s encountered during starvation and refeeding were in g o o d a g r e e m e n t w i t h t h e f a c t t h a t t R N A a m o u n t decreased under fasting and increased during r e f e e d i n g . N e v e r t h e l e s s , it is d i f f i c u l t Iv a s s u m e t h a t v a r i a t i o n s in t R N A c h a r g i n g l e v e l s d u r i n g s t a r v a t i o n (fig. 7)' d e p e n d u p o n d e c r e a s e of s y n t h e t a s e a c t i v i t i e s w h e r e a s the <> of s y n t h e t a s e s e e m e d s h o r t e r (24' to 50 h o u r s ) t h a n half-lif~e of t R N A [29]. M e a s u r e m e n t of r e a l q u a n t i t y of e n z y m e s a l o n e c a n give the a n s w e r . At least, v a r i a t i o n s of s y n t h e t a s e a c t i v i t i e s c a n n o t be e x p l a i n e d b y <> m e c h a n i s m s i n c e to r e d u c e d levels of a m i n o a c y l tRNA correspond low synthetase activity. Our

242

G. C h a v a n c [ i a n d A . F o u r n i e r .

observations rejoin those w i t h E. colt a n d yeast I14, 15, 16, 18~ whi~ch s u p p o r t the concept of <>.

Conclusions. Our w o r k h a n d shows that c o m p o n e n t s of the t r a n s l a t i o n a l apparatus in the posterior s~lk gland are c o o r d i n a t e d w i t h p r o t e o s y n t h e t i c activity u n d e r different physiological c o n d i t i o n s (feeding, starvation or refeed:ing). T r a n s f e r RNA a n d ribosomal RSI& synthesis are i n h i b i t e d u n d e r fasting ; s i m u l t a n e o u s l y , aminoacyl-tRN~A synthetase a n d aminoacyl-tRN~A levels decrease. The quanii'Ly of rRNA and tRNA u n d e r various c o n d i t i o n s remained i'n a .constant raldo (table III). The ratio of amino,acyl-tRNA synthetase activity per tRNA a m o u n t is also c o n s t a n t in control animals d u r i n g all the fifth larval i n s t a r (table HI). These results sugges~ that all c o m p o n e n t s of the t r a n s l a t i o n mac h i n e r y .are ~ n e l y t u n e d a n d adapted to the rate of p r o t e i n synthesis. E v i d e n c e s for such an adjust m e n t exist in louver eu~karyotes and E. colt [13, 17] w h e r e c o o r d i n a t e d regulation occurs d u r i n g steady~state growth betvceen r i b o s o m a l proteins, ribosomal RN:A, ,elongation factors EF-G and EF-Tu, tRNA and aminoacyl-tRNA synthetases. This <>>> .is not yet well explained. It w o u l d appear [17, 18] that the changes i~ synthetase level are due to c h a n g e s i n t r a n s c r i p t i o n a n d transl.at4on Tat,es of the corresp o n d i n g genes. This response seems to follow the p a t t e r n of r i b o s o m a l RNA a n d t r a n s f e r RNA transc r i p t i o n and suggests a c o o r d i n a t e d response of genes c o r r e s p o n d i n g to t r a n s l a t i o n apparatus components. This c o o r d i n a t i o n could be a s s u m e d b y a single sign,al such as guan.osine tetraphosphate. In the posterior silk gland, as well as in h i g h e r eukaryotes, nevertheless, guanosine tetraphosphate cannot be a c a n d i d a t e for this role. It is more p r o b a b l e that all c o m p o n e n t s i n t e r a c t in this regulation. F o r example, a m i n o a c y l - t R ~ A level is p r o b a b l y i~nplicated in the regulation of tRNA synthesis [367. The w a y by w h i c h aminoacyl-tRNA levels vary is unclear. Our results show that free a m i n o acids pool has n r o b a b l y a little effect if not n o n e on a m i n o a c y l a t i o n rate a n d that this pool is not correlated w i t h other c o m p o n e n t s (table III) ; conversely, the f.all down of sy,nihet~se level d u r i n g starvati'on, could explain the decregse of tRNA c h a r g i n g level, but results obtained with crude ,extracts must be taken carefully. One ~-ay BIOCHIMIE, 1979, 61, n ° 2.

or a n o t h e r the p r o b l e m of regulation of synthetase level r e m a i n s open, since <> pro oess c a n n o t be i n v o k e d to e x p l a i n our results n o r the <(metabolic regutation >> f o u n d i n other systems [15, 18]. Similarly, our results are i n good agreement w i t h the hypothesis of the control of the translational ~initiation b y u n c h a r g e d tRN~A [20, 22, 36] but the m e c h a n i s m s b y w h i c h aminoacyl-tRNA is l o w e r e d or i n c r e a s e d poses once again the problem of the aminoacylati,on control a n d then of synthetase level control. F i n a l l y , as we have already suggested [26, 29] a d a p t a t i o n of tR,NA populati,on to fibroin mRNA codon f r e q u e n c y i n posterior silk gland could result from a differential tRNA t r a n s c r i p t i o n . Our results confirm that in vivo d e g r a d a t i o n of tRI~A, at least d u r i n g starvat~ion, is s i m i l a r for all tRNA species ; o n the other h a n d , tRNA c h a r g i n g level is correlated to the rate of p r o t e i n synthesis and could control the tRNA synthesis [36]. W e can i m a g i n e than, w h e n fibroin mRN,A is actively translated, tRNA oly, tRNA Ala and tRNASer are more r a p i d l y acylated t h a n the other tRNA and consequently, the c o r r e s p o n d i n g genes t r a n s c r i p t i o n could be higher. This view argues i n favor of a <> of tRNA to c o d o n freq u e n c y of t r a n s l a t e d mRI~A a n d opposes to a constitutive tRI~A adaptation. Experi~nents are done i n our l a b o r a t o r y to test more d i r e c t l y these hypothesis.

Acknowledgements. We would like to thank Dr J. Bonnet, J.-P. Garel, R. Granlham and J.-H. Well for their critical advising and reading the final manuscript, and J.-M. Aulheman for his technical assistance.

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