Biochimie ( 1991 ) 73,599-605
599
© Soci6t6 franc aise de biochimie et biologie mol6culaire / Elsevier, Paris
How does ppGpp affect translational accuracy in the stringent response? AM Rojas*, M Ehrenberg** Department of Molecular Biology, Uppsala Universi~, Biomedical Center, Box 590, S-751 24 Uppsala, Sweden (Received 5 July 1990; accepted 4 April 1991 )
S u m m a r y ~ With an in vitro poly(Phe) synthesis system we have tested recent models concerning translational accuracy in the stringent response during aminoacid starvation. We have found that cognate, deacylated tRNA of very high concentrations is unable to block the A-site. No influence of EF-Tu.ppGpp on ribosomal proofreading has been found. Alternative mechanisms to keep translational errors low by the stringent response are discussed.
stringent response / translational accuracy / ppGpp / magic spot / aminoacid starvation
Introduction When bacteria are starved for an aminoacid, eg by a nutritional downshift, they produce high amounts of the guanine nucleotide analogue ppGpp in the so called stringent response [ 1-4]. One function of ppGpp is to prevent the translation machinery from being overwhelmed by errors at starved codons [1, 5-8]. Relaxed strains (relA-), unable to respond quickly to aminoacid deprivation, show significant protein heterogeneity in P-I-'I o~1~ I l l i n d l o n t i n o h i o h ~rrnr levels in translation. According to O'Farrel [ 1] ppGpp protects bacteria from high errors by inhibiting the overall rate of protein synthesis so that the charging level of the aminoacid starved tRNA isoacceptors is allowed to increase to more normal levels. O'Farrels model predicts that the charging level of a starved tRNA is much lower in relaxed (relA-) than in wildtype (relA+) strains. However, experiments show almost identical charging levels for starved tRNAs in relA- and relA+ strains [9-11]. Recently, a number of papers have dealt with new mechanisms that help bacteria to keep translation errors low during aminoacid starvation [12-14, 16-18]. Some of these models allow for unchanged charging levels of the starved tRNA isoacceptors by the stringent response [13, 16-18]. Gallant [12] and later Liljenstrrm [ 14] suggested that deacylated tRNA can bind to the ribosomal A-site at starved codons and inhibit non cognate peptide bond formation. Their . . . .
~
. . . . . .
j
~ v . ~
t
~
JI
a a a ~ a ~ a a ~ : : ~
aaz~:~aa
~a
Z ~at
*Present address: Catedra de Bioquimica, Facultad de Farmacia, Universidad Central de Venezuela, Venezuela **Correspondence and reprints
model [12, 14] is a special case of O'Farrels general hypothesis [ 1]. Ninio's idea [ 13] is, in short, that ribosomes have two functional states: a high and a low accuracy configuration. The transitions between these states have the property that codons read by ternary complexes at low concentrations are translated with higher precision than others. This mechanism accomodates the apparently contradicting experimental observations that the errors in relA+ strains are kept low, that the charging levels for starved tRNAs are the •
~
1111
e~ll.li~l
l k l l
I
1~1.l~11~1.1111~l
I.IIlIlUl
I.ilLJI./
lli~bl~
K.IL
other than the starved codons are unaffected by the stringent response [ 15]. Previously we suggested [16, 17] that ppGpp in complex with elongation factor Tu (EF-Tu) could influence the accuracy of translation by modulating ribosomal proofreading. However, when we tested this hypothesis no supporting evidence was found [M Ehrenberg, unpublished data]. It was therefore surprising to discover that Dix and Thompson thought our model to be correct from their finding that EF-Tu.ppGpp seems to modulate the proofreading of non cognate tRNAs in vitro [18]. The first excitement about this result faded rapidly when we realized that their experiments were uninterpretable as discussed below. In the present communication, we report in vitro tests of the models of Gallant [ 12] and Ninio [ 13] using a poly(U)-translation system optimized for accuracy [19] and rate [20]. Our experiments contradict these models, and we also demonstrate that EF-Tu in complex with ppGpp has no effect on ribosomal proofreading, in contrast to the claims by Dix and Thompson [18]. Alternative ways that accuracy may be achieved by the stringent response are discussed.
0(X)
AM Rojas, M Ehrenberg
Materials and M e t h o d s Chemicals Phenylalanine (Phe). leucine (Leu), phosphoenolpyruvate (PEP). GTP, GDP, ATP, putrescine, spermidine, myokinase (MK) (BC 2.7.4.3), ppGpp and ppApp were from Sigma, St Louis, MO, USA. Pyruvate kinase (PK) (EC 2.7.3.40) from Boehringer Mannheim, Germany. Poly(U) from Pharmacia, Sweden. 14C-Phe, 3H-Leu, 14C-Leu and 3H-GDP were from Amersham International, Bucks, England. 1,4-Dithioerythritol (DTE) from Lab Kemi AB, Sweden. Methanol, trifluoroacetic acid (TFA) were from Merck, Darmstadt, Germany, TLC plates were polygram CEL 300 PEI from Macheray-Nagel, Germany.
containing 7.5 g/l casamino acids. 3H and 14C backgrounds were obtained in the absence of poly(U). Filtrations and calculations were as in [20, 261.
Dipeptide assays
Ribosomes were prepared from frozen E coil cells (017 strain UDtG4, wild-type) [21] and stored a t - 8 0 ° C in polymix. EF-G was purified as in [22], EF-Tu as in [20, 23], EF-Ts as in [24], Phe- and Leu-tRNA synthetases as in [20]. Preparation of tRNAPh% IR_NA~U~ t_R_N_A~ Lea a_n.d N-acety!-14C-Phe-tRNAPhe, or N-acetyl-3H-Phe-tRNA phe as by Ruusala et al [25].
Two mixtures were prepared on ice (I and II). Mixture I was as for the elongation assay above with 3H-labelled N-acetyl-PhetRNA ohe and in addition ppGpp 0.5 mM and EF-Tu.GDP as specified. Mixture II contained in 50 lal ATP (2 mM), PEP (20 mM), PK (5 lag), MK (0.3 lag), 3H-GDP (80 pmol, 3000 cpm). For Phe-dipeptides and fc-analysis it contained 14C-Phe (5 mmol, sa 30 cpm/pmol), Phe (80 units), tRNA Phe (120 pmol), EF-Tu (100 pmol). For Leu-dipeptides and fw-analysis it contained laC-Leu (5 mmol, sa 30 cpm/pmol), LeuS (3 units), tRNA T M or tRNA Leu (700 pmol), EF-Tu (500 pmol). The Phe- and Leu-mixes were divided: to one part (A) was added H20 and to the other (B) unlabelled GDP (about three times the concentration of EF-Tu). The mixes I, IIA and liB were incubated separately for 10 min at 37°C. Dipeptide formation was started by addition of 50 lal of mixture I into mixtures IIA and liB. The assay time was 10 s. Backgrounds were measured in samples where poly(U) had been omitted. For peptide analysis, the assay was stopped by adding 1.5 ml 20% TCA containing 7.5 g/1 casamino acids. For GTP-hydrolysis 15 !11 aliquots were quenched in 5 lal conc HCOOH. The TCA stopped samples were spun in an Eppendorf centrifuge and the pellets were resuspended in 200 lal 0.5 M KOH. Then 15 lal conc HCOOH was added and the samples were centrifuged. The supematant was collected and run through an RP-18 LiChroCart column from Merck, Darmstadt, Germany, with an HPLC (Waters). Constant fractions of buffers A and B were used. 50% of A and B for N-acetyl-3H Phe-laC-Phe dipeptide and 48% of A for N-acetyl-3H-Phe-14CLeu dipeptides. Isotopes were counted in A Raytest-Ramona LS and evaluated in an IBM PC. Samples quenched with HCOOH were centrifuged and applied to thin layer plates with buffer C. GDP and GTP spots were cut out and counted.fc- and f.,-values were calculated as described in [26].
Elongation assays
Ternary complex waiting times
Two mixtures (I and II) were prepared on ice in polymix buffer with DTE (1 mM). Mixture I contained in 50 lal ribosomes (43 pmoles total, 30% active), lac or 3H labelled N-acetyl-Phet R N A ~ (60 pmoles) with a specific activity (sa) of 1 cpm/ pmoi and 150 cprn/pmol respectively, poly(U) 20 lag and zacPhe (30 mmol, sa 5 cpm/picomole) in the case ef the starved assays. Mixture II normally contained in 50 lal ATP (2 mM), PEP (20 mM), PK (5 lag), MK (0.3 lag), EF-G (20 pmol), EF-Ts (60 pmoi), 3H-Leu (2 mmol, sa 1500 cpm/pmol), leucyl-tRNA synthetase (LeuS) (3 units), tRNA_Iseu or tRNA~u (500 pmol), GTP (2 mM) or when either ppGpp or ppApp (1.6 mM) were present GTP (0.4 mM). For the step time experiments mix II contained, in. addition, pheny!a!anyl-tRNA synthetase (PheS) (80 units), 14C-Phe (3 pmol), tRNA Phe (120 pmol) and EF-Tu as specified. For the kc~,t/Km (Rrfactor) detgrminations either tRNA l'he, PheS and Phe or tRNA2t'eu or tRNA~ eu, LeuS and Leu were present. In the deacylated tRNA experiments there were three units of PheS, 520 pmoles of EF-Tu and tRNA Phe as specified. Mixtures I and II were preincubated 10 min at 37°C. The reaction was started by addition of 50 lal of mixture I into mixture II. The reaction was stopped by adding 5 ml 20% TCA
The waiting times (T) in figure 2 were calculated from kcat/KM for EF-Tu in poly(Phe)-synthesis, which according to table I is 1.4 x 107 M -I s - l , and the concentration of EF-Tu according to:
Buffer for the in vitro syswm Polymix buffer [191 contained 95 mM KCI, 5 mM NH4CI, 5 mM MgAc2, 0.5 mM CaC1,., 5 mM putrescine, 1 mM spermidine, 5 mM potassium phosphate, pH 7.5. Working strength buffer was prepared by mixing 100 mM potassium phosphate (pH 7.5) and a ten-fold concentrate without phosphate (pH 7.5) according to I191.
Buffers for HPLC A: 10% methanol, 0.1% TFA passed through nitrocellulose filter. B: 90% methanol, 0.1% TFA passed through paper filter.
Buffer for thin layer chromatography C: 0.5 M HK2PO4, pH 3.5.
Purifications and preparations
1
T=
(kcat]gM) • [EF-Tu]
Results P h e n y l a l a n i n e s t a r v a t i o n w a s a r r a n g e d b y k e e p i n g the P h e S at a s m a l l rate l i m i t i n g c o n c e n t r a t i o n in the p r e s e n c e o f tRNAIr eu as d e s c r i b e d p r e v i o u s l y [22, 27]. T h e c o n c e n t r a t i o n o f tRNAPhe w a s s u b s e q u e n t l y titrate d f r o m z e r o to 3 x 10 -5 M. T h e p o l y ( P h e ) - s y n t h e s i s rate a n d the e r r o r level w e r e m e a s u r e d w i t h a n d w i t h o u t p p G p p (fig 1). T h e r e w a s a s m a l l i n c r e a s e in the p o l y ( P h e ) - s y n t h e s i s rate w i t h i n c r e a s i n g t R N A Phe c o n c e n t r a t i o n in t h e b e g i n n i n g o f t h e t i t r a t i o n c u r v e a n d t h e n t h e r a t e w a s c o n s t a n t (fig l a). A c c o r d i n g to
How does ppGpp affect translational accuracy'? Table I.
k~,,]KM (R-factor)
-ppGpp +ppGpp
tRNAehe
tRNA Le"
tRNAL,',
1.4 • 107 0.64.107
7 • 102 3.6.102
2.1 • 103 1.2.103
Error (measured)
-
Error -ppGpp (calculated)* +ppGpp
-
1.0 • 10-4 6.0. 10-4 0.5 • 10-4 0.6.10-4
1.5 • 10-4 2.0- 10-4
*With k~,,,/KM for Phe(RC) and k~.~,/KMfor Leu~ or Leu4 (R w) the errors a r e P E calc - 1 ! + RC/Rw
Gallant [12] and Liljenstrrm [14] one would expect the error level to decrease significantly as the concentration of t R N A Phe is increased and the A-site, by hypothesis, becomes more occupied by deacylated tRNA Phe. In contrast, the error level is almost constant at all concentrations of tRNAPhe, with as well as without p p G p p (fig 1b). This indicates that deacylated tRNAPhe cannot block the A-site even at high concentrations. The a priori possibility that this in vitro result is caused by the absence of a crucial component like the stringent factor that existes in vivo is made less likely by the correspondence between our in vitro observations and some in vivo results by Ulrich and Parker [28]. T h e y t'ound that overproduction of deacylated tRNAnis does not enhance the accuracy at starved His codons. We also note that if deacylated tRNAPh~ binds to the ribosomal E-site [29], this binding does not influence the efficiency of non cognate reading in our system. The error level is under our experimental conditions lower in the presence than in the absence of ppGpp (fig lb). This is because only the amount of Leu ternary complex is decreased by the presence of ppGpp. The concentration of Phe ternary complex is unaffected since the poly(Phe)-synthesis rate is synthetase limited as explained by Wagner and Kurland [22]. This type of inhibition of non cognate peptide bond formation at starved codons by ppGpp m a y a priori play a role in the stringent response [22, 27]. However, the very high concentrations of aminoacyl-tRNA that exist in vivo seem to make this type of inhibition insignificant [ 17, 30]. From N i n i o ' s model [13] it follows that the accuracy of poly(Phe) translation should increase as the time ribosomes spend waiting for Phe-ternary complexes becomes longer. Variation in waiting times was achieved experimentally by changing the concentration of EF-Tu at constant amounts of t R N A Phe and t R N A ~ u or t R N A ~ " . In figure 2 we have plotted the
601
tRNA Le" and t R N A Le" errors as functions of waiting time (Materials and Methods). The errors were approximately constant with a tendency to increase instead of decrease at very long waiting times (fig 2). This tendency is more pronounced for tRNAt4-eu than for tRNA2Leu and can be explained as due to an EF-Tu independent binding of the tRNALeu-isoacceptors to the ribosome. This 'back' flow reaction is rather significant for t R N A ~ " but is very small for t R N A ~ u [31 ]and in the latter case there is hardly any change in the error level over a wide range of waiting times with as well as without ppGpp (fig 2b). These results contradict Ninio's hypothesis. This negative conclusion was checked in yet another type of experiment.
•-= 200 E
1A
n
0 •
0
E 150
•
O
o (O
0
~e - 100 ffl i
e-
~- 50 0
I
f
I
10
20
30
1B
o
*~ 6 - 0
o
o
g
•
•
! lO
! 20
4
o
..d
2
-ID
o I
30
tRNAPhepM
Fig 1. A. Extent of poly(Phe)-synthesis during 3 min under PheS limitation as a function of the concentration of tRNA 0he. • • • 0.2 mM GTP, 0.8 mM ppGpp; o o o 0.2 mM GTP, 0.8 mM ppApp. B. Error level from tRNA4Leu in poly(Phe)-synthesis on poly(U) as a function of tRNA P"e concentrations. • • • 0.2 mM GTP, 0.8 mM ppGpp; o o o 0.2 mM GTP, 0.8 mM ppApp.
602
AM Rojas, M Ehrenberg
2
2B
uJ 0 _.l
.'0
0
0
O
"e
Q
u
@o Q
I
I
10
20
2A
8-
I
==4•
.--I
O O
U
2
I0
20 seconds
waiting time
Fig 2. A. Error level f.mm ,. 4,_~u in poly(Phe)-synthesis .~. ,r,,., ,i,,,~r, on poly(U) as a function of the waiting time for Phe-temary complex (see Materials and Methods). • • • 0.2 mM GTP, 0.8 mM ppGpp; o o o 0.2 mM GTP, 0.8, mM ppApp. B. Error level from tRNA~ u in poly(Phe)-synthesis on poly(U) as a function of the waiting time for Phe-temary complex (see Materials and Methods). • • • 0.2 mM GTP, 0.8 mM ppGpp; o o o 0.2 mM GTP, 0.8 mM ppApp.
Here we took advantage of the prediction of Ninio's model according which, in the absence of cognate tRNA, ribosomes will be predominantly in their hyper accurate mode. When, on the other hand, there is only cognate t R N A present, ribosomes will be in their normal, "sloppy mode'. This feature of the model implies that k~JKM-values for right (kcaJgM) c and wrong (kc=/KM), tRNAs will differ much more when measured separately than would be expected from errors (PE) measured with both isoacceptors present simultaneously [32]. In other words, if Ninio's model is correct the expectation is that (kcaJKM)w/(kcaJKM) c
599
© Soci6t6 franc aise de biochimie et biologie mol6culaire / Elsevier, Paris
How does ppGpp affect translational accuracy in the stringent response? AM Rojas*, M Ehrenberg** Department of Molecular Biology, Uppsala Universi~, Biomedical Center, Box 590, S-751 24 Uppsala, Sweden (Received 5 July 1990; accepted 4 April 1991 )
S u m m a r y ~ With an in vitro poly(Phe) synthesis system we have tested recent models concerning translational accuracy in the stringent response during aminoacid starvation. We have found that cognate, deacylated tRNA of very high concentrations is unable to block the A-site. No influence of EF-Tu.ppGpp on ribosomal proofreading has been found. Alternative mechanisms to keep translational errors low by the stringent response are discussed.
stringent response / translational accuracy / ppGpp / magic spot / aminoacid starvation
Introduction When bacteria are starved for an aminoacid, eg by a nutritional downshift, they produce high amounts of the guanine nucleotide analogue ppGpp in the so called stringent response [ 1-4]. One function of ppGpp is to prevent the translation machinery from being overwhelmed by errors at starved codons [1, 5-8]. Relaxed strains (relA-), unable to respond quickly to aminoacid deprivation, show significant protein heterogeneity in P-I-'I o~1~ I l l i n d l o n t i n o h i o h ~rrnr levels in translation. According to O'Farrel [ 1] ppGpp protects bacteria from high errors by inhibiting the overall rate of protein synthesis so that the charging level of the aminoacid starved tRNA isoacceptors is allowed to increase to more normal levels. O'Farrels model predicts that the charging level of a starved tRNA is much lower in relaxed (relA-) than in wildtype (relA+) strains. However, experiments show almost identical charging levels for starved tRNAs in relA- and relA+ strains [9-11]. Recently, a number of papers have dealt with new mechanisms that help bacteria to keep translation errors low during aminoacid starvation [12-14, 16-18]. Some of these models allow for unchanged charging levels of the starved tRNA isoacceptors by the stringent response [13, 16-18]. Gallant [12] and later Liljenstrrm [ 14] suggested that deacylated tRNA can bind to the ribosomal A-site at starved codons and inhibit non cognate peptide bond formation. Their . . . .
~
. . . . . .
j
~ v . ~
t
~
JI
a a a ~ a ~ a a ~ : : ~
aaz~:~aa
~a
Z ~at
*Present address: Catedra de Bioquimica, Facultad de Farmacia, Universidad Central de Venezuela, Venezuela **Correspondence and reprints
model [12, 14] is a special case of O'Farrels general hypothesis [ 1]. Ninio's idea [ 13] is, in short, that ribosomes have two functional states: a high and a low accuracy configuration. The transitions between these states have the property that codons read by ternary complexes at low concentrations are translated with higher precision than others. This mechanism accomodates the apparently contradicting experimental observations that the errors in relA+ strains are kept low, that the charging levels for starved tRNAs are the •
~
1111
e~ll.li~l
l k l l
I
1~1.l~11~1.1111~l
I.IIlIlUl
I.ilLJI./
lli~bl~
K.IL
other than the starved codons are unaffected by the stringent response [ 15]. Previously we suggested [16, 17] that ppGpp in complex with elongation factor Tu (EF-Tu) could influence the accuracy of translation by modulating ribosomal proofreading. However, when we tested this hypothesis no supporting evidence was found [M Ehrenberg, unpublished data]. It was therefore surprising to discover that Dix and Thompson thought our model to be correct from their finding that EF-Tu.ppGpp seems to modulate the proofreading of non cognate tRNAs in vitro [18]. The first excitement about this result faded rapidly when we realized that their experiments were uninterpretable as discussed below. In the present communication, we report in vitro tests of the models of Gallant [ 12] and Ninio [ 13] using a poly(U)-translation system optimized for accuracy [19] and rate [20]. Our experiments contradict these models, and we also demonstrate that EF-Tu in complex with ppGpp has no effect on ribosomal proofreading, in contrast to the claims by Dix and Thompson [18]. Alternative ways that accuracy may be achieved by the stringent response are discussed.
0(X)
AM Rojas, M Ehrenberg
Materials and M e t h o d s Chemicals Phenylalanine (Phe). leucine (Leu), phosphoenolpyruvate (PEP). GTP, GDP, ATP, putrescine, spermidine, myokinase (MK) (BC 2.7.4.3), ppGpp and ppApp were from Sigma, St Louis, MO, USA. Pyruvate kinase (PK) (EC 2.7.3.40) from Boehringer Mannheim, Germany. Poly(U) from Pharmacia, Sweden. 14C-Phe, 3H-Leu, 14C-Leu and 3H-GDP were from Amersham International, Bucks, England. 1,4-Dithioerythritol (DTE) from Lab Kemi AB, Sweden. Methanol, trifluoroacetic acid (TFA) were from Merck, Darmstadt, Germany, TLC plates were polygram CEL 300 PEI from Macheray-Nagel, Germany.
containing 7.5 g/l casamino acids. 3H and 14C backgrounds were obtained in the absence of poly(U). Filtrations and calculations were as in [20, 261.
Dipeptide assays
Ribosomes were prepared from frozen E coil cells (017 strain UDtG4, wild-type) [21] and stored a t - 8 0 ° C in polymix. EF-G was purified as in [22], EF-Tu as in [20, 23], EF-Ts as in [24], Phe- and Leu-tRNA synthetases as in [20]. Preparation of tRNAPh% IR_NA~U~ t_R_N_A~ Lea a_n.d N-acety!-14C-Phe-tRNAPhe, or N-acetyl-3H-Phe-tRNA phe as by Ruusala et al [25].
Two mixtures were prepared on ice (I and II). Mixture I was as for the elongation assay above with 3H-labelled N-acetyl-PhetRNA ohe and in addition ppGpp 0.5 mM and EF-Tu.GDP as specified. Mixture II contained in 50 lal ATP (2 mM), PEP (20 mM), PK (5 lag), MK (0.3 lag), 3H-GDP (80 pmol, 3000 cpm). For Phe-dipeptides and fc-analysis it contained 14C-Phe (5 mmol, sa 30 cpm/pmol), Phe (80 units), tRNA Phe (120 pmol), EF-Tu (100 pmol). For Leu-dipeptides and fw-analysis it contained laC-Leu (5 mmol, sa 30 cpm/pmol), LeuS (3 units), tRNA T M or tRNA Leu (700 pmol), EF-Tu (500 pmol). The Phe- and Leu-mixes were divided: to one part (A) was added H20 and to the other (B) unlabelled GDP (about three times the concentration of EF-Tu). The mixes I, IIA and liB were incubated separately for 10 min at 37°C. Dipeptide formation was started by addition of 50 lal of mixture I into mixtures IIA and liB. The assay time was 10 s. Backgrounds were measured in samples where poly(U) had been omitted. For peptide analysis, the assay was stopped by adding 1.5 ml 20% TCA containing 7.5 g/1 casamino acids. For GTP-hydrolysis 15 !11 aliquots were quenched in 5 lal conc HCOOH. The TCA stopped samples were spun in an Eppendorf centrifuge and the pellets were resuspended in 200 lal 0.5 M KOH. Then 15 lal conc HCOOH was added and the samples were centrifuged. The supematant was collected and run through an RP-18 LiChroCart column from Merck, Darmstadt, Germany, with an HPLC (Waters). Constant fractions of buffers A and B were used. 50% of A and B for N-acetyl-3H Phe-laC-Phe dipeptide and 48% of A for N-acetyl-3H-Phe-14CLeu dipeptides. Isotopes were counted in A Raytest-Ramona LS and evaluated in an IBM PC. Samples quenched with HCOOH were centrifuged and applied to thin layer plates with buffer C. GDP and GTP spots were cut out and counted.fc- and f.,-values were calculated as described in [26].
Elongation assays
Ternary complex waiting times
Two mixtures (I and II) were prepared on ice in polymix buffer with DTE (1 mM). Mixture I contained in 50 lal ribosomes (43 pmoles total, 30% active), lac or 3H labelled N-acetyl-Phet R N A ~ (60 pmoles) with a specific activity (sa) of 1 cpm/ pmoi and 150 cprn/pmol respectively, poly(U) 20 lag and zacPhe (30 mmol, sa 5 cpm/picomole) in the case ef the starved assays. Mixture II normally contained in 50 lal ATP (2 mM), PEP (20 mM), PK (5 lag), MK (0.3 lag), EF-G (20 pmol), EF-Ts (60 pmoi), 3H-Leu (2 mmol, sa 1500 cpm/pmol), leucyl-tRNA synthetase (LeuS) (3 units), tRNA_Iseu or tRNA~u (500 pmol), GTP (2 mM) or when either ppGpp or ppApp (1.6 mM) were present GTP (0.4 mM). For the step time experiments mix II contained, in. addition, pheny!a!anyl-tRNA synthetase (PheS) (80 units), 14C-Phe (3 pmol), tRNA Phe (120 pmol) and EF-Tu as specified. For the kc~,t/Km (Rrfactor) detgrminations either tRNA l'he, PheS and Phe or tRNA2t'eu or tRNA~ eu, LeuS and Leu were present. In the deacylated tRNA experiments there were three units of PheS, 520 pmoles of EF-Tu and tRNA Phe as specified. Mixtures I and II were preincubated 10 min at 37°C. The reaction was started by addition of 50 lal of mixture I into mixture II. The reaction was stopped by adding 5 ml 20% TCA
The waiting times (T) in figure 2 were calculated from kcat/KM for EF-Tu in poly(Phe)-synthesis, which according to table I is 1.4 x 107 M -I s - l , and the concentration of EF-Tu according to:
Buffer for the in vitro syswm Polymix buffer [191 contained 95 mM KCI, 5 mM NH4CI, 5 mM MgAc2, 0.5 mM CaC1,., 5 mM putrescine, 1 mM spermidine, 5 mM potassium phosphate, pH 7.5. Working strength buffer was prepared by mixing 100 mM potassium phosphate (pH 7.5) and a ten-fold concentrate without phosphate (pH 7.5) according to I191.
Buffers for HPLC A: 10% methanol, 0.1% TFA passed through nitrocellulose filter. B: 90% methanol, 0.1% TFA passed through paper filter.
Buffer for thin layer chromatography C: 0.5 M HK2PO4, pH 3.5.
Purifications and preparations
1
T=
(kcat]gM) • [EF-Tu]
Results P h e n y l a l a n i n e s t a r v a t i o n w a s a r r a n g e d b y k e e p i n g the P h e S at a s m a l l rate l i m i t i n g c o n c e n t r a t i o n in the p r e s e n c e o f tRNAIr eu as d e s c r i b e d p r e v i o u s l y [22, 27]. T h e c o n c e n t r a t i o n o f tRNAPhe w a s s u b s e q u e n t l y titrate d f r o m z e r o to 3 x 10 -5 M. T h e p o l y ( P h e ) - s y n t h e s i s rate a n d the e r r o r level w e r e m e a s u r e d w i t h a n d w i t h o u t p p G p p (fig 1). T h e r e w a s a s m a l l i n c r e a s e in the p o l y ( P h e ) - s y n t h e s i s rate w i t h i n c r e a s i n g t R N A Phe c o n c e n t r a t i o n in t h e b e g i n n i n g o f t h e t i t r a t i o n c u r v e a n d t h e n t h e r a t e w a s c o n s t a n t (fig l a). A c c o r d i n g to
How does ppGpp affect translational accuracy'? Table I.
k~,,]KM (R-factor)
-ppGpp +ppGpp
tRNAehe
tRNA Le"
tRNAL,',
1.4 • 107 0.64.107
7 • 102 3.6.102
2.1 • 103 1.2.103
Error (measured)
-
Error -ppGpp (calculated)* +ppGpp
-
1.0 • 10-4 6.0. 10-4 0.5 • 10-4 0.6.10-4
1.5 • 10-4 2.0- 10-4
*With k~,,,/KM for Phe(RC) and k~.~,/KMfor Leu~ or Leu4 (R w) the errors a r e P E calc - 1 ! + RC/Rw
Gallant [12] and Liljenstrrm [14] one would expect the error level to decrease significantly as the concentration of t R N A Phe is increased and the A-site, by hypothesis, becomes more occupied by deacylated tRNA Phe. In contrast, the error level is almost constant at all concentrations of tRNAPhe, with as well as without p p G p p (fig 1b). This indicates that deacylated tRNAPhe cannot block the A-site even at high concentrations. The a priori possibility that this in vitro result is caused by the absence of a crucial component like the stringent factor that existes in vivo is made less likely by the correspondence between our in vitro observations and some in vivo results by Ulrich and Parker [28]. T h e y t'ound that overproduction of deacylated tRNAnis does not enhance the accuracy at starved His codons. We also note that if deacylated tRNAPh~ binds to the ribosomal E-site [29], this binding does not influence the efficiency of non cognate reading in our system. The error level is under our experimental conditions lower in the presence than in the absence of ppGpp (fig lb). This is because only the amount of Leu ternary complex is decreased by the presence of ppGpp. The concentration of Phe ternary complex is unaffected since the poly(Phe)-synthesis rate is synthetase limited as explained by Wagner and Kurland [22]. This type of inhibition of non cognate peptide bond formation at starved codons by ppGpp m a y a priori play a role in the stringent response [22, 27]. However, the very high concentrations of aminoacyl-tRNA that exist in vivo seem to make this type of inhibition insignificant [ 17, 30]. From N i n i o ' s model [13] it follows that the accuracy of poly(Phe) translation should increase as the time ribosomes spend waiting for Phe-ternary complexes becomes longer. Variation in waiting times was achieved experimentally by changing the concentration of EF-Tu at constant amounts of t R N A Phe and t R N A ~ u or t R N A ~ " . In figure 2 we have plotted the
601
tRNA Le" and t R N A Le" errors as functions of waiting time (Materials and Methods). The errors were approximately constant with a tendency to increase instead of decrease at very long waiting times (fig 2). This tendency is more pronounced for tRNAt4-eu than for tRNA2Leu and can be explained as due to an EF-Tu independent binding of the tRNALeu-isoacceptors to the ribosome. This 'back' flow reaction is rather significant for t R N A ~ " but is very small for t R N A ~ u [31 ]and in the latter case there is hardly any change in the error level over a wide range of waiting times with as well as without ppGpp (fig 2b). These results contradict Ninio's hypothesis. This negative conclusion was checked in yet another type of experiment.
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Fig 1. A. Extent of poly(Phe)-synthesis during 3 min under PheS limitation as a function of the concentration of tRNA 0he. • • • 0.2 mM GTP, 0.8 mM ppGpp; o o o 0.2 mM GTP, 0.8 mM ppApp. B. Error level from tRNA4Leu in poly(Phe)-synthesis on poly(U) as a function of tRNA P"e concentrations. • • • 0.2 mM GTP, 0.8 mM ppGpp; o o o 0.2 mM GTP, 0.8 mM ppApp.
602
AM Rojas, M Ehrenberg
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waiting time
Fig 2. A. Error level f.mm ,. 4,_~u in poly(Phe)-synthesis .~. ,r,,., ,i,,,~r, on poly(U) as a function of the waiting time for Phe-temary complex (see Materials and Methods). • • • 0.2 mM GTP, 0.8 mM ppGpp; o o o 0.2 mM GTP, 0.8, mM ppApp. B. Error level from tRNA~ u in poly(Phe)-synthesis on poly(U) as a function of the waiting time for Phe-temary complex (see Materials and Methods). • • • 0.2 mM GTP, 0.8 mM ppGpp; o o o 0.2 mM GTP, 0.8 mM ppApp.
Here we took advantage of the prediction of Ninio's model according which, in the absence of cognate tRNA, ribosomes will be predominantly in their hyper accurate mode. When, on the other hand, there is only cognate t R N A present, ribosomes will be in their normal, "sloppy mode'. This feature of the model implies that k~JKM-values for right (kcaJgM) c and wrong (kc=/KM), tRNAs will differ much more when measured separately than would be expected from errors (PE) measured with both isoacceptors present simultaneously [32]. In other words, if Ninio's model is correct the expectation is that (kcaJKM)w/(kcaJKM) c
