345

Biochimica et Biophysica Acla, 1088 (1991) 345-358 © 1991 Elsevier Science Publishers B.V. 0167-4781/91/$03.50 ADONIS 016747g ~9100098Y BBAEXP 92222

Potassium salts influence the fidelity of mRNA translation initiation in rabbit reticulocyte lysates: unique features of encephalomyocarditis virus RNA translation R i c h a r d J. J a c k s o n Department of Biochemistry, University of Cambridge, Cambridge (U. K.) (Received 12 July 1990) (Revised manuscript received 16 October 19901

Key words: Translation initiation; Encephalomyocarditis virus~ capped mRNA; (Rabbit reticulocyte lysate)

It is widely assumed that in vitro translation of mRNA is more efficient in the presence of potassium acetate rather than KCI, that the optimum concentration of potassium acetate is higher than for KCL and that uncapped RNAs exhibit a lower optimum salt concentration than capped mRNAs. When these assumptions were examined using several different mRNA species in four batches of rabbit reticulocyte lysate, some notable exceptions were found. The translation of encephalomyocarditis virus (EMCV) RNA exhibited a salt optimum unusually high for an uncapped mRNA, and w a s very much more efficient and accurate with KCI rather than potassium acetate. It was also unique in being strongly activated by low concentrations (5-10 raM) KSCN in the presence of 90 mM potassium acetate. For the translation of other uncapped RNAs (pollovirus RNA, cowpea mosaic virus (CPMV) M RNA and bacteriophage MS2 RNA) amino acid incorporation at the optimum potassium acetate level was significantly greater than could be achieved using KCl. However, KCI was found to be restrictive and potassium acetate permissive for the synthesis of abnormal products thought to arise from initiation at incorrect sites, with the reset that KCI gave a product pattern closer to that observed in vivo. In the particular case of the reticulocyte lysate system, accurate translation therefore requires the use of KC! rather than potassium acetate, but the choice of salt was found to be less critical in cell-free extracts from HeLa or L-cells.

Introduction

Protein synthesis shows a strong requirement for K +, and in cell-free translation experiments this requirement was traditionally met by the addition of 80-120 mM KCI [1]. Some years ago, however, Weber et al. [2] showed that if potassium acetate was used in place of KCI, the optimum K + concentration was significantly higher, whilst the overall incorporation was enhanced, slightly in the case of reticulocyte lysates, and more significantly in other cell-free systems. The difference between the two potassium salts was shown to be mainly

Abbreviations: CPMV, cowpea mosaic virus; EMCV, encephalomyocarditis virus; TMV, tobacco mosaic virus: Mops, 4-morphofinepropanesuiphonic acid. Enzymes: creatine phosphokinase (EC 2.7.3.2). Correspondence: R.J. Jackson, Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 IQW, U.K.

due to an in~bition of initiation, specifically of the binding of the small ribosomal subunit to mRNA, by CI- in excess of 50 mM [2]. It was argued that apart from the advantage of slightly higher incorporation, the use of potassium acetate rather than KCI was more physiologically representative, since it allowed cell-free translation assays to be conducted at a K + concentration closer to the intracellular level [2]. Notwithstanding the fact that acetate is an unphysiological anion, this argument has been widely accepted with the result that the majority of current users of the system choose potassium acetate. Whilst it has been recognised for some time that the K + optimum for translation may differ according to the species of mRNA tested [3,4], with uncapped mRNAs generally, but not invariably [3], exhibiting a lower optimum than capped RNA [5-7], it seems to have been a tacit assumption that the replacement of KCI by potassium acetate has no effect on the pattern of translation products or the specificity of initiation. However, on an occasion when other considerations required us to

346 use potassium acetate rather than KCI for some translation assays of EMCV RNA in the reticulocyte lysate system, we were surprised to find a product pattern radically different from previous results [8-11]. This observation prompted a wider comparison of the two salts on the translation of several RNAs in four different batches cf lysate. The results described here show that although potassium acetate promotes higher amino acid incorporation than KCI (except with EMCV RNA), the ratio of authentic products to incorrect products was significantly higher when KCI was used. EMCV RNA translation responds to different salts in a way that is atypical of other RNAs (whether capped or uncapped), which may possibly be related to the recently described novel mode of initiation on this RNA via an internal ribosome entry site [12]. Materials and Methods

Materials Tobacco mosaic virus (TMV) was a gift from Drs. P.G. Butler and D. Zimmern, MRC Laboratory of Molecular Biology, Cambridge. The virus was dissociated by heating to 65°C in the presence of 1% (w/v) sodium dodecyi sulphate, and viral RNA was isolated by phenol extraction. Cowpea mosaic virus M RNA was generously provided by Dr. R. Goldbach, Department of Molecular Biology, University of Wageningen, The Netherlands; poliovirus RNA (Type 1, Mahoney) by Drs. B.L. Semler and E. Wimmer, Department of Microbiology, State University of New York, Stony Brook, NY; EMCV RNA by Prof. R.A. Laskey, Department of Zoology, University of Cambridge; and bacteriophage MS2 RNA by Dr. C.W. Anderson, Department of Biology, Brookhaven National Laboratory, Upton, NY. All potassium salts were Analar grade, or were prepared from Analar grade acids and KOH. L-[35S]Methionine (1100-1300 Ci/mmol) was obtained from Amersham International. Catalogue No. SJ204 was used for the experiment shown in Fig. 10, and SJ1515 for all other experiments: the latter has the advantage that it does not cause background labelling of the reticulocyte lysate 42 kDa protein [13].

Cell-free translation systems Rabbit reticulocyte lysate was prepared and treated with micrococcal nuclease as described previously [1,13]. All preparations were supplemented with 60 # g / m l calf liver tRNA (Boehringer, Mannheim) after nuclease treatment [13]. Cell-free extracts from HeLa cells and L-cells were prepared as described previously [141, and were treated with micrococcal nuclease by incubation at 12°C for 60 rain with 0.15 mM CaCI 2 and 150 units/mi micrococcal nuclease (Boehringer, Mannheim). The digestion was stopped by the addition of 2 mM EGTA, as

for reticulocyte lysate digestion, and calf liver tRNA was also added to these systems.

Cell-free translation assays and analysis of products Nuclease-treated reticulocyte lysates were incubated under the basic conditions described previously [13]. The final assay mixture consists of 0.8 voi. nucleasetreated lysate and 0.2 vol. of a cocktail which contributes a final concentration in the assay mix of the following components: 0.5 mM MgCI 2, 10 mM creatine phosphate, 50 # g / m l creatine kinase, 5 mM dithiothreitol, 100 #M in each of 19 unlabelled amino acids (lacking methionine), [35Slmethionine at 0.2 m C i / m l unless otherwise stated, and potassium salts as specified in the figure legends. In common with usual practice, no buffer is added to such assays, since the high protein concentration is effective in buffering the pH at the optimum [15]. Unless otherwise stated, the following final concentrations of RNAs were used in these assays: 36 # g / m i TMV RNA, 36 # g / m l EMCV RNA, 16 # g / m l CPMV M RNA, 8 # g / m l poliovirus RNA and 40 # g / m l bacteriophage MS2 RNA. For the first three of these RNAs this represents a concentration of about 15 nM, and is just below the saturation level for most batches of reticulocyte lysate. Poliovirus RNA was tested at a lower concentration because higher levels lead to a more complex product pattern which is difficult to interpret and is the result of more frequent initiation at internal sites [10,16,17]. Bacteriophage MS2 RNA was tested at a significantly higher concentration because it is a very inefficient message and gives a poor signal [18]. For translation assays using HeLa or L-cell extracts, the final assay mix consisted of 0.66 vol. nuclease-treated extract, and 0.34 vol. of a cocktail contributing final concentrations of the following components: 90 mM pota:;sium acetate, 20 mM Mops-KOH (pH 7.2), 1.0 mM MgC! 2, 15 mM creatine phosphate, 50 p g / m l creatine kinase, 4 mM dithiothreitol, 0.5 mM ATP, 0.1 mM GTP, 1 mM EGTA (additional to that present in the nuclease-treated extract), 100 #M in each of 19 unlabelled amino acids, 3 mM 2-aminopurine, and [35S]methionine at 0.6 mCi/ml. The concentrations of RNA tested are given in the figure legends. Incubation was at 30°C for 60 rain, then the assay mix was supplemented with 50 # g / m l pancreatic ribonuclease and 5 mM EDTA, incubated at 30°C for a further 10 rain, and then diluted ten-fold in the gel sample buffer described previously [9]. The treatment with ribonuclease eliminates the background bands of labelled peptidyl-tRNA resulting from the translation of fragments of globin mRNA [13], and was usually omitted when TMV RNA or EMCV RNA were assayed since the translation of these efficient mRNAs suppresses the background labelling of peptidyl-tRNA to negligible levels [1,131.

347 An aliquot of 10 pl of of the diluted material (corresponding to 1 #! of the original assay mix) was taken for assay of incorporation into trichloroacetic acid precipitable protein as described previously [13]. Labelled products were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) using 15% (w/v) acrylamide gels under the conditions described previously [9], loading 10 ttl of diluted sample per track unless otherwise stated. The labelled proteins used as markers were: fl-galactosidase (116 kDa), phosphorylase (97 kDa), glutamate dehydrogenase (56 kDa), and creatine kinase (40 kDa). The gels were fluorographed using Amplify (Amersham International), and the fluorograms exposed to Fuji RX Medical X-ray film for 15-18 h unless otherwise stated. Fluorograms were scanned, where approprizte, using a Transidyne 2955 Scanning Densitometer. Results

Translation of capped RNAs: tobacco mosaic virvt~ RNA The experiments reported here were carried out using four different batches of lysate (I-IV), which span the range of activity we have observed in 10 years' experience of lysate preparation. With TMV RNA as template, lysates 1-11I were slightly above average in efficiency, whilst batch IV was significantly below a,'erage (Fig. 1). However, it should be appreciated that this hierarchy of efficiencies is dependent on the type of mRNA assayed: the relative efficiency in EMCV RNA translation was II > I >> IV > III, with batch I close to the average. The difference in efficiencies was not directly proportional to differences in ribosome content, which was highest in the case of batch I1, with batch I about 10% lower, and batches II1 and IV 20% lower in ribosome content. Except for those aspects of translation which showed strong batch-dependent variations, data relating only to batch I will be presented below. As expected from previous reports [2,7], the concentration of added potassium acetate (120-130 mM) required for maximum amino acid incorporation with TMV RNA, and for maximum yield of the two major products of 126 kDa and 183 kDa (which both originate from the same 5'-proximal initiation site [19]), was higher than the KCI optimum which was about 100 mM (Figs. 1 and 2). When the two salts were used at their respective optimum concentrations, potassium acetate gave greater incorporation than KCi (Fig. 1), but the difference was quite small, ranging from a 20% stimulation in the case of lysate 1I (data not shown) down to almost none in the case of lysate IV (Fig. 1). There are some smaller products which do not conform to this pattern, and although they are made in only trivial yield, they are of interest because their synthesis responds to potassium salts in a way that resembles the translation of the uncapped RNAs de-

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scribed belov,. The salient feature of these products is that their formation is favoured by low salt concentrations, irrespective of the nature of the potassium salt used, but their synthesis is inhibited more strongly by increasing KC1 than by increasing potassium acetate concentrations (Fig. 2, panels B and C). Therefore, at 100 mM salt, their yield is considerably higher if potassium acetate is used rather than KCl, and as Fig. 2 (panel D) shows, this category of products corresponds very closely to those products whose synthesis is resistant to, and even stimulated by addition of mTGTP cap analogue. (Note that the over-exposure necessary to reveal these minor products in Fig. 2 gives the impression that the synthesis of the 126 kDa and 183 kDa polypeptides is more resistant to inhibition by cap analogue than is in fact the case.) Thus, the synthesis of these chloride-inhibited products is cap-independent, and they will therefore include products translated from fragmented RNA templates, together with any products initiated at internal sites in intact genomes, if such internal initiation occurs. The products in this category larger than 30 kDa must arise from initiation at sites within the major open-reading frame coding for the 126/183 kDa proteins [19]. The smaller products in this

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I chloride I I acetate I I@ Itamate II acelate II chloride I Fig. 2. The effect of potassium salts and cap analogue on the products of TMV RNA translation. Standard assays were carried out using lysate i with the followingpotassium salts: potassium glutamate (panel A) at Ca) 40, (b) 60, (c) 80, (d) 100, (e) 120. and (O 140 raM; potassium acetate (panel B) at (a) 50, (b) 70, (c) 90, (d) 110, (e) 130 and (O 150 raM: KC! (panel C) at (a) 40, (b) 60, (c) 80, (d) 100, (e) 120 and (f) 140 mM. For the experiment shown in panel D, lysate l was translated in the presence of 100 mM added KCI (a-d) or 100 mM potassium acetate (e-h), and the following concentrations of mTGTP:Ca,h) 0.6 mM, (b,g) 0.3 mM and (c-f) none, Assays supplemented with m'~GTPalso received an equimolar amount of MgCI2. The major TMV RNA encoded products of 183 kDa, 126 kDa and 30 kDa [19-211 are shown, and the unlabelled arrows highlight the minor products whose synthesis is inhibited more by increasing KCI than by increasing potassium acetate (panels B and C), but is resistant to inhibition by mTGTP(panel D), as discussed in the text.

class are probably translated from a downstream openreading frame which has the potential to code for a 30 kDa protein, and which just overlaps the 3'-end of the reading frame coding for the 183 kDa product [19-21]. In infected plant cells, only one protein product from this reading frame has been detected, probably the 30 kDa polypeptide, but in cell-free translation assays several polypeptides are synthesised, which are coterminal at the C-terminus [20,21], and must therefore originate from initiation at different sites within this open reading frame. Their yield is known to be increased if the viral R N A is deliberately fragmented [22], or if the R N A is extracted from short virus rods [21], which confirms that translation of fragmented R N A makes certainly a major contribution to the synthesis of these products. Although the yield of these minor products showed some batch-dependent variation (data not shown), the consistent feature was that their synthesis in all lysates was more strongly suppressed by increasing KCI than by increasing potassium acetate concentrations (Fig. 2, panels B and C), although it is interesting that the yield of the 30 kDa protein, which is probably the only authentic product of this group, seemed to be less influenced by these parameters than was the yield of the smaller polypeptides (Fig. 2). In summary, although KCI is somewhat less effective than potassium acetate in promoting translation from the 5'-proximal cap-dependent initiation site of T M V

RNA, KCI selectively inhibits the synthesis of products resulting from initiation on uncapped fragments of R N A (or any initiation at internal sites of intact R N A as may occur), whereas potassium acetate is permissive for such initiation.

Translation of uncapped RNAs: cowpea mosaic virus M RNA and polioviru~ RNA As uncapped R N A s we would expect these to be translated at lower efficiency and with a lower optimum salt concentration than capped mRNAs. In fact, the concentration of added KCI giving maximum incorporation with both of these R N A s was found to be about 80 mM, (Figs. 3 and 5), some 20 m M lower than the concentration optimal for T M V R N A translation, whereas the optimum concentration of added potassium acetate was essentially the same (130 raM) for C P M V M R N A , poliovirus R N A and T M V R N A translation. At concentrations below about 80 mM, the choice of potassium salt has very little influence on amino acid incorporation, but as the concentration is increased above this level KCI is inhibitory in contrast to potassium acetate (Figs. 3 and 5). Analysis of the products of C P M V M R N A translation showed that in addition to the two authentic products of 116 kDa and 102 k D a [23] (which were formerly designated as 105 kDa and 95 kDa [24,25]), a number of lower molecular weight polypeptides were synthesised (Fig. 4). As these smaller products appeared very early

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Fig. 3. The optimum K + concentration for CPMV M RNA translation in lysate I. Incorporation per/xl assay mix is plotted in panel A against the final concentration of added KCI (Q Q), or potassium acetate (A . . . . . . ,L). Background incorporation in the absence of added RNA has not been subtracted. The sum of the yields of the two authentic CPMV products (o Q), and the sum of the yields of two representative incorrect products (A. . . . . . ,t,), determined by scanning densitometry of the fluorograph shown in Fig. 4, are plotted against the concentration of added KC! (lower portion of panel B), or added potassium acetate (lower portion of panel C). The upper portions of these panels show the ratio of these yields (total yield of both authentic products/total yield of the two incorrect products) plotted against KCI concentration (B), and potassium acetate concentration (C). The yield ratio at 140 mM KCI is not shown since the yield of incorrect products was too low to be determined accurately.

in time-course experiments, they are unlikely to arise through processing of the full-length product, and they most probably originate from initiation at sites internally located within the genome, though it is not known whether such translation is exclusively from fragmented R N A molecules rather than initiation at internal sites of intact RNA. The synthesis of these smaller products was more strongly inhibited by KC! concentrations above 80 m M than was the synthesis of the full-length products, so that the signal to noise ratio (the relative yield of correct versus incorrect products) increased quite markedly with increasing KCI (Fig. 3, panel B). On the other hand, increasing potassium acetate exerted almost no differential effect on the synthesis of abnormal as opposed to authentic products (Fig. 3, panel C). It has already been shown that the translation of poliovirus R N A in the rabbit reticulocyte lysate gives not only the capsid precursor (P1) and other authentic virus-coded polypeptides, but also a number of abnormal products arising from initiation at incorrect sites [16,17]. The synthesis of these aberrant products

can be suppressed by the addition of factors from H e L a cells which are either missing from reticulocyte lysates or present only in relatively low abundance [10,16,17]. These incorrect products include the P3-related polypeptides bracketed in Fig. 6 (though this group of polypeptides includes some authentic P3-related polypeptides originating from translation initiated at the correct site and subsequent processing of the polyprorein product). There are also some prominent incorrect products of lower molecular weight (Fig. 6), including a very characteristic and reproducible pair of p.~lypeptides previously designated Y and Z [10,16]. Like the larger P3-related abnormal products, Y and Z can be labelled when translation is carded out in the presence of N-formyl-[35S]methionybtRNAt [16], and as they are specifically precipitated by 'antibodies against 3CD (Jackson, R.J., Hanecak, R. and Semler, B.L., unpublished observations), they too must arise from internal initiation in the 3'-segment of the viral genome [10]. The synthesis of Y and Z and the other incorrect products occurred at all potassium acetate concentrations in the range 40-120 m M in lysates I and IV (Fig.

350 6, panels A and C). and was only slightly inhibited at 140 mM in lysate I. Their synthesis in lysate II was more susceptible to inhibition by increasing potassium acetate concentrations, but even at 140 mM was not completely suppressed (Fig. 6, panel B). In contrast, their synthesis was almost completely inhibited in all lysates by KCI at 105 mM, and even by 85 m M KCI in the case of lysate !I (Fig. 6, panels D-F), It was also noted that the pattern of P3-related products bracketed in Fig. 6 was less complex when high KCI (105-125 mM) was used than in assays supplemented with equivalent amounts of potassium acetate, a n d we interpret this as the result of at least partial inhibition nf internal initiation at these sites specifically by KCI. Clearly, inhibition of incorrect product synthesis occurs more readily in lysate 11 than in the other batches, but even with batch Ii the difference between the two potassium salts as effectors of this inhibition was maintained. It is possible that lysate II is atypical in having an unusually

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Translation of bacteriophage MS2 RNA

Fig. 4. The effect of potassium salts on the pattern of products translated from CPMV M RNA. CPMV M RNA was translated in lysate I with the following potassium salts: potassium acetate (panel A) at (a) 50 (b) 70, (c) 90, (d) 110, (e) 130 and (0 150 mM final concentration; KCI (panel B) at (a) 40, (b) 60, (c) 80, (d) 100, (e) 120 and (f) 140 raM. The extreme right track was loaded with radioactive marker proteins which are designated (in kDa) in the right margin. The two authentic viral products of 116 kDa and 102 kDa [23] are designated on the left, and the arrows labelled (O) show the two representative incorrect products whose yield was determined by scanning densitometry for plotting in Fig. 3 (panels B and C).

Bacteriophage MS2 R N A translation was e x a m i n e d because although this R N A directs the synthesis of specific products in eukaryotic cell-free systems [18], it is a very poor message a n d can be considered to have n o initiation sites specifically evolved for recognition in eukaryotic systems. It might therefore provide a model for the synthesis of incorrect products from R N A s such as poliovirus RNA. T h e results partly s u p p o r t this premise in that MS2 R N A translation, whether assayed as a m i n o acid incorporation (Fig. 5) or as the labelling of specific protein products ( d a t a not shown), was more efficient if potassium acetate was used rather t h a n KCI a n d the o p t i m u m potassium acetate c o n c e n t r a t i o n was clearly higher than the KCI o p t i m u m (Fig. 5). However, the o p t i m u m concentrations of b o t h salts was even

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Fig. 6. The effect of potassium salts on the pattern of products translated from poliovirns RNA. Assays were carried out using lysate I (panels A and D), lysate I1 ( panels B and E) and lysate Iil (panels C and F) using the following potassium salts: potassium acetate (panels A-C inclusive) at (a) 40, (b) 60, (c) 80, (d) 100, (e) 120 and (f) 140 raM; KCI (panels D-F inclusive) at (a) 25, (b) 45, (c) 65, (d) 85, (e) 105 and (f) 125 mM. The assays shown in panel G were carried out using (a-e inclusive) lysate IV, (~ lysate II1, (g) lysate I, and (h) lysate 11 with the following added salts: (a) 85 mM KCI, (b) 105 mM KCI, (c) 100 mM potassium acetate, (d) 120 mM potassium acetate, (e-h inclusive) l0 mM KSCN, 90 mM potassium acetate. The track labelled P shows 14C-labelledpoliovirns-coded proteins labelled in vivo in infected HeLa cells; these were mixed with unlabelled reticulocyte lysate before gel electrophoresis. Staining of the gel with Coomassie Blue R250 prior to fluorography showed that the migration of the reticulocyte proteins in this track was slightly slower than in other tracks. The authentic capsid precursor P1 (la) is designated in both margins, and other authentic poliovirus products are shown by 9 in the empty tracks between panels B and C, and between F and G. The arrows in the left margin and repeated between panels A and B, and again between D and E, show incorrect products including polypeptides Y and Z observed previously [10,16]. The brackets in the left margin show the P3-related products some of which arise from internal initiation [16], and some by translation initiated at the authentic site. lower than the respective o p t i m u m concentrations for incorrect product synthesis from C P M V M R N A (Figs. 3 a n d 4) a n d from poliovirus R N A (Fig. 6).

Translation of EMCV RNA E M C V R N A is atypical of u n c a p p e d R N A s in that, provided KCI is used, it is translated very efficiently in the reticulocyte lysate and, unlike poliovirus RNA, does not give detectable a m o u n t s of incorrect products [8-11 ]. It is also atypical in showing a high KCI optimum, a n d in p r o m o t i n g higher incorporation in the presence of KCI than with potassium acetate (Fig. 7). The optimum concentration of added KCi was 120 m M with all batches of lysate (Fig. 7), higher than the 100 m M optimal for T M V R N A translation. In contrast, incorporation in the presence of potassium acetate showed n o sharp o p t i m u m in the range 5 0 - 1 5 0 mM, and was significantly lower than when KCI was used (Fig. 7), particularly in the case of the less active lysates such as b a t c h IV. In order to evaluate the influence of potassium salts o n initiation at the authentic site, the c o m b i n e d yield of all the capsid proteins a n d their precursors (L-P1-2A, P1-2A, P1, 1ABC a n d VP1) was determined by scanning densitometry of the fluorograms (using shorter exposures t h a n that shown in Fig. 8). T h e results, plotted in the lower panels of Fig. 7, more than reinforce the conclusion that KCI is markedly superior to potassium

acetate for the correct translation of this RNA. Even in the case of the most active lysate (batch II) the yield of authentic products was much lower when potassium acetate was used rather t h a n KCI, whilst with the least active lysate IV scarcely any authentic E M C V R N A encoded products could b e detected when potassium acetate was used, although some n o r m a l products were synthesised at 100-120 m M KCI (Fig. 7 a n d Fig. 8, panel G). With all lysates, the KCI o p t i m u m for the synthesis of authentic capsid precursors was m u c h sharper than the o p t i m u m for overall a m i n o acid incorporation (Fig. 7). A t KC! concentrations below 80 m M in the case of lysates I and IV, or 40 m M for lysate II, very little authentic capsid precursor synthesis occurred (Fig. 7). Most of the a m i n o acid incorporation occurring at low concentrations of a d d e d KCI, a n d at all potassium acetate levels must therefore be into incorrect products, a n d c a n be regarded as 'noise'. Inspection of Fig. 8 reveals a large n u m b e r of products synthesised in low yield under these conditions. Three of these incorrect products, highlighted in Fig. 8, were considered representative indicators: a polypeptide migrating just a h e a d of the authentic p r o d u c t 1ABC, a n d a doublet situated just below the 40 k D a marker. These are synthesised in all lysates at all concentrations of potassium acetate that were tested, a n d only in the case lysate II is their synthesis suppressed slightly by high concentrations

352 e x a c t l y the s a m e w a y as d o e s t h e f o r m a t i o n o f t h e incorrect p r o d u c t s o f p o l i o v i r u s t r a n s l a t i o n d i s c u s s e d previously. Again, inhibition of the synthesis of these i n c o r r e c t p r o d u c t s w a s m o r e easily a c h i e v e d in l y s a t e II t h a n in the o t h e r b a t c h e s , but, as w i t h p o l i o v i r u s R N A

(Fig. 8, p a n e l s B - D ) . T h e y are also s y n t h e s i s e d in all lysates in the p r e s e n c e o f 4 0 - 6 0 m M KCI, b u t h i g h e r KCI c o n c e n t r a t i o n s in t h e r a n g e 1 0 0 - 1 2 0 m M c o m pletely inhibit their f o r m a t i o n (Fig. 8, p a n e l s E - G ) . T h e i r s y n t h e s i s t h u s r e s p o n d s to p o t a s s i u m salts in

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K concentration (mM) Fig. 7. Effect of K "~ concentration on EMCV RNA translation. Three different lysates were assayed under standard conditions: (A) batch L (B) batch !1 and (C) batch IV. The upper panels show the incorporation per ~al assay mix plotted against the final concentration of added KCI (O O), potassium acetate (a . . . . . . A). and potassium glutamate (ll- . . . . 41). Background incorporation in the absence of added RNA has not been subtracted. The lower panels show the effect of varying concentrations of potassium salts (symbols as in the upper panels) on the total yield of all authentic capsid proteins and capsid precursors determined by densitometry of different exposures of the fluorogram shown in Fig. 8, and adjustment for the different methionine content of these products [30].

353

A

B

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Fig. 8. The effect of potassium salts on the products of translation of EMCV RNA. Standard a~says were carried out using lysate 1 (panels A, B and E), lysate It (panels C and F) and lysate IV (panels D and G) with the following potassium salts: potassium glutamate (panel A) at (a) 40, (b) 60, (c) 80, (d) 100. (e) 120 and (f) 140 mM; l~otassiumacetate (panels B-D inclusive) at (a) 50, (b) 70, (c) 90, (d} 110. (e) 130 and (f) 150 mM; KCI (panels E-G inclusive) at (a) 40, (b) 60, (c) 80, (d) 100, (e) 120 and (f) 140 raM. The extreme left and right hand tracks were loaded with radioactive marker proteins, which are designated in the left margin (in kDa). Authentic EMCV RNA products Iwhich are most readily seen in panels C and F) are designated in the right margin. The arrows in the left margin labelled with (O), and repeated in track a of panel D. show the three representative incorrect products discussed in the text.

translation in this lysate, KCi was nevertheless more effective in p r o m o t i n g this inhibition than was potassium acetate. In time-course experiments these a b n o r m a l E M C V translation products appear very early (data not shown), which argues that they are not formed by proteolytic processing of larger precursors a n d favours the view that they arise from initiation at incorrect sites. This is supported by the observation that in the presence of potassium acetate, polypeptides L-P1 and P1, the first proteolytic processing products of the capsid precursor L-P1-2A [9], actually appear earlier in the time course than when KCI is used (data not shown). This early appearance of L-P1 a n d P1 has also been observed by other workers whose assays used potassium acetate [26,27], but has never been found in this laboratory using KC1 [8-10]. Since the conversion of L-P1-2A to L-PI a n d P1 is catalysed by a proteolytic enzyme encoded in the Y-region of the E M C V genome [28-30]. this observation impfies that potassium acetate promotes the p r e m a t u r e translation of the distal portion of the R N A , which in turn implies that it promotes initiation at internal (incorrect) sites.

Effect of other anions These results p r o m p t e d investigations into a wider range of salts. Potassium glutamate was tested because it is closer to KCi than to potassium acetate in its ability to support a m i n o acid incorporation in mouse

liver cell-free s"stems [31], and in HeLa or L-ceU extracts (Buhl, W.-J. a n d Jackson, R.J., u n p u b l i s h e d observations), with an o p t i m u m concentration of 100-120 mM. However, potassium glutamate at concentrations up to 140 m M was somewhat less effective than KC! in p r o m o t i n g overall a m i n o acid incorporation in the presence of T M V R N A (Fig. 1), a n d very inefficient with E M C V R N A (Fig. 7). Analysis of the translation products showed that it was more permissive to the translation of the m i n o r low-molecular-weiDlt T M V products than was potassium acetate (Fig. 2, panel A), whilst with E M C V R N A it failed to direct the synthesis of any authentic polypeptides a n d produced only 'noise' (Fig. 7 a n d Fig. 8, panel A). N o n e of the other potassium salts tested could support protein synthesis when used o n their own. Their influence could only be examined using 100 m M potassium acetate as the control, a n d assaying the effect of replacing some of this potassium acetate by other potassium salts up to the limit where strong inhibition of incorporation was observed. In the case of E M C V R N A translation, substitution of up to 40 m M potassium acetate by potassium p h o s p h a t e (Fig. 9, panel D), or potassium sulphate (data not shown), h a d very little influence on the pattern of translation products. Progressive substitution of potassium acetate by KCI resulted in a gradual increase in the yield of authentic products, already discernible at 40 m M KCI a n d peaking at 80 m M KCI, whilst the synthesis of incorrec:

354

A

B

C

(a b o d e l ~ { ' - ~ ' - ~ - - V a b c d e

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~-,-,,-KOAc/KCI

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.

.

.

KOAc/KSCN

.

.

.

.

.

.

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Fig. 9. The effect of other potassium sails on the pattern of products translated from E M C V R N A in lysate I. Assays of E M C V R N A translation

were carried out in the presence of the potassium salts specified below plus (A-D inclusive)sufficient potassium acetate, or (El sufficient KCI, to give a final concentration of 100 mM added K*: KCI (panel A) at (a) 0, (b) 20, (c) 40, (d) 60, (el 80 and if) 100 mM final concentration; KBr (panel B) at (a) 0, (b) 20. (c) 40. (d) 60, and (el 80 raM; KSCN ipanel C) at (a) 0, (b) 5, (c) 10, (d) 15. (el 20, (f) 25 and (g) 30 raM; K2HPO4 (panel D) at (a) 0. (b) 3, (c) 6, (d) 10. (el 13, if) 17 and (g) 20 mM; KSCN (panel E) at Ca) 0, (b) 5, (cl 10. (d) 15. (el 20, and If) 25 raM, The principal authentic EMCV products are labelled in the left margin 6;ompare Fig. 8); the three representative incorrect products are designated by arrows which are labelled(II) in the left margin and which are repeated in some of the empty tracks. products declined if the KCI was raised above 60 mM (Fig. 9, panel A). Significant enhancement of authentic product synthesis was therefore observed at lower KC! concentrations than were needed to effect detectable suppression of incorrect products. This argues that these changes in product pattern are not causally interrelated: enhancement of authentic initiation is probably not a simple consequence of suppression of incorrect product syr~thesis, or vice-versa. This is supported by the observation that replacement of potassium acetate by KBr resulted in no significant enhancement of authentic product synthesis, but did reduce the yield of incorrect products if the KBr was raised above 40 m M (Fig. 9, panel B). The most striking effect was achieved by KSCN, which caused a dramatic increase in authentic product synthesis at concentrations as low as 5 mM, without reducing incorrect product synthesis; higher K S C N concentrations gave no further enhancement of authentic product synthesis, but reduced the yield of incorrect products, until a general inhibition set in at levels above 20 m M (Fig. 9, panel C). Although KSCN and KCI are both capable of enhancing correct translation their influence is not additive: gradual replacement of KCI by KSCN resulted only in progressive inhibition of overall translation and correct product synthesis (Fig. 9, panel El. In similar assays with TMV RNA, the capacity of the various potassium salts to suppress the synthesis of the minor small products could be ranked in the order KSCN > KBr > KCI, with potassium phosphate and

sulphate ineffective (data not shown), and, again, the synthesis of the 30 kDa product was more resistant to this inhibition than the other minor products. This is similar to the ranking of the potassium salts for the suppression of the synthesis of incorrect E M C V products, but the specific enhancement of authentic E M C V product synthesis does not conform to this pattern and is a particular property of K S C N and KCI.

Other cell-free systems Whilst KCI has been used in this laboratory as the standard for reticuloeyte lysates, we have used the acetate salt as standard for the study of endogenous activity of other cell-free systems such as mouse liver [31], HcLa or L-cell extracts [14], since incorporation in the presence of potassium acetate was quite significantly higher than when KCI was used. It was therefore interesting to test whether potassium acetate was also permissive for the translation of incorrect products in these systems. The result was striking in that L-cell and HeLa cell-free extracts synthesised only authentic products from E M C V R N A (Fig. 10, panels C and D) and poliovirus R N A (Fig. 10, panels (3 and H) in the presence of potassium acetate, conditions under which the reticulocyte lysate control gave barely any correct products from either R N A (Fig. 10, panels A and E). Whereas the reticulocyte lysate system translates EMCV R N A with much greater efficiency than poliovirus R N A (certainly if KCI is used, but with most lysates also in the presence of potassium acetate), the two R N A s are translated with similar efficiency in the

355 11

B

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C

P

ml

....

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~

E

F

t-H

P

I

j

K L l~ N

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Fig. 10. Comparison of translation of EMCV RNA. poliovirus RNA and MS2 RNA in cell-free extracts from rabbit reticulocytes, HeLa cells and L-cells. Time-courses of translation of ( A - D inclusive) EMCV RNA at 32 # g / m l final concentration, and ( E - H inclusise) poliovirus RNA at l0 # g / m l were carried out in the following cell-free systems: (A. E) reticuiocyte lysat¢ incubated under conditions normally used for HeLa and L-cell extracts, (B) reticulocyte lysate under standard conditions with 100 mM added KCI. (F) reticulocyte lysate under standard conditions except that the final concentration of added KCI was 60 mM, (C, G) nuclease-treated L-cell extract and (D, H) nucleasc-treated HeLa cell extract. Samples were taken for gel electrophoresis after 30, 50, 80. 200 and 300 rain incubation, shown from left to right in all cases. The two tracks labelled l show translation products in the HeLa cell system incubated for 100 rain in the absence (left-hand track) or presence (right track) of 40 p g / m l MS2 RNA, and the two tracks labelled (J) are the corresponding assays with the L-cell extract. Tracks ( K - N inclusive) are translation assays in the reticulocyte lysate incubated for 100 rain either under conditions normally used for HeLa eell-free systems (K, L), or under standard conditions with 100 mM KCI (M. N), in the absence (K, M), or presence (L, N) of 4 0 / z g / n d MS2 RNA. Each track was loaded with 5 pl diluted sample. corresponding to 0.5/tl assay mix: the dried gel was fluorographed for 48 h, except for panels A and B which were exposed for 16 h. The tracks labelled P show 14C-labelled poliovirus products labelled in infected HeLa cells. The arrow in the right margin shows the 42 kDa background band [13]. which was labelled in this experiment because [35S]methionine, grade SJ 204, was used (see Materials and Methods).

HeLa system (Fig. 10, panels D and H). Direct comparisons between the two types of cell-free system need to take account of the very much smaller pool of unlabelled methionine in the reticulocyte lysate (Lane, R.M. and Jackson, R.J., unpublished observations). When this is done, the conclusion is that poliovirus RNA is translated with much higher efficiency in the HeLa extract than the reticulocyte lysate, whilst the efficiency of EMCV RNA translation is similar in the two systems. It is also of interest to note that MS2 RNA was not translated by the L-cell and HeLa cell systems under conditions in which it was translatable in the reticulocyte lysate (Fig. 10, tracks I, J and L). Even in the presence of potassium acetate. HeLa and L-cell extracts therefore show a translational specificity which is certainly comparable in stringency to that of reticulocyte lysates incubated with high KCI, and may even be superior. Discussion

Although elevated K + concentrations are known to increase the rate of elongation of protein chains, and for

this reason may increase the yield of full-length products [32], most of the salt effects observed here appear to be related to initiation efficiency, as in previous studies [2-7], and especially initiation specificity. It is well established that the incorrect products of pofiovirus RNA translation result from initiation at internal sites in the genome [16], and the smaller polypcptides translated from TMV RNA from initiation within the reading frame encoding the 30 kDa protein [19-21]. Initiation at incorrect sites is almost certainly the cause, also, of the abnormal products of translation of EMCV and CPMV M RNAs, since the formation of these products showed similar characteristics as the incorrect poliovirus and TMV polypeptides. The results regarding cap-dependent translation of TMV RNA in the reticulocyte lysate conform closely to the generally held assumptions: (i) that the optimum concentration of added potassium acetate is higher than for KCI, and (ii) that the maximum incorporation obtainable with potassium acetate is greater. However, the increase in the optimum salt concentration and in the efficiency of translation when potassium acetate was substituted for KCI were both relatively modest. In our experience these findings are typical t,/capped mRNA

356 translation, and they are c~msistent with a number of previously published results [2,3,7]. Apart from EMCV RNA which is clearly atypical and needs to be discussed separately, the translation of uncapped RNAs also conforms, at least superficially, to these two assumptions. In fact, with these RNAs the choice of potassium salt made a larger difference to the optimum salt concentration and to the maximum amino acid incorporation than was observed with the cap-dependent translation. This is because the optimum KC! concentration for the uncapped RNAs was significantly lower than for TMV RNA, whereas this was not the case with potassium acetate, except when bacteriophage MS2 RNA was assayed. It is generally considered that uncapped or decapped mRNAs exhibit a lower optimum salt concentration than capped RNAs [5-7], although the translation of satellite tobacco necrosis virus RNA, and possibly decapped globin mRNA, in the wheat germ system did not [3]. The major difference between initiation on the two types of mRNA is that only with capped mRNAs is there an involvement of initiation factor elF-4F [33], which is thought to promote unwinding of the RNA from the 5'-end [34,35]. It is possible that the low salt optimum exhibited by some, but not necessarily all, uncapped RNAs reflects the requirement for sufficient destabilisation of secondary structure, unaided by eiF4F, to allow the scanning of the mRNA by 40S ribosomal subunits [36-38]. However, as secondary structure is affected by the catioh concentration rather than by the nature of the anion, this explanation does not account for the clear differences between the two potassium salts. Our results suggest that apart from any effect of cation concentration, initiation on uncapped RNAs is more susceptible to inhibition specifically by C1- than is the translation of capped messages. What is particularly significant is that the synthesis of incorrect or aberrant products was more susceptible to this inhibition than was the synthesis of correct products. Thus, the translation of bacteriophage MS2 RNA was strongly inhibited by even moderate concentrations of KCI, the synthesis of the incorrect products from CPMV M RNA and poliovirus RNA was more strongly inhibited by KCi concentrations above 80 mM than was the synthesis of the authentic products, and amongst the small products of TMV RNA translation the synthesis of the 30 kDa protein was more resistant to inhibition by KCI than was the synthesis of the smaller polypeptides. In contrast, increasing concentrations of potassium acetate showed a much less pronounced differential effect on the synthesis of authentic, as opposed to incorrect, products in the reticulocyte iysate system. However, HeLa and L-cell extracts have the capacity for such discrimination even in the presence of potassium acetate. They did not translate MS2 RNA, and

with poliovirus or EMCV RNA gave only the authentic products with no detectable incorrect polypeptides. It is also our experience that whilst such extracts synthesise the 30 kDa protein from TMV RNA, they produce very low yields of the smaller polypeptides even in the presence of potassium acetate (Lane, R.M. and Jackson, R.J., unpublished observations). The difference between the reticuiocyte lysate and the other .~?,'~temsmay be due to the partial loss of critical activitie:; with the ageing of the terminally differentiated cell. If this were the case we might expect some batch-dependen t variation, and it is notable that of the four batches tested, lysate II was somewhat exceptional and was closer to the HeLa cell extract in its characteristics. It might be argued that the differences between the two potassium salts could be easily explained if reticuIocyte lysates contain a ribonuclease activity which is absent from HeLa or L-cell extracts and which is inhibited by KCi at concentrations above 80-100 raM, but not by potassium acetate. The action of this nuclease at low KCI and at all potassium acetate levels could generate fragments of RNA which serve as templates for the synthesis of incorrect products by the normal scanning ribosome mechanism [38]. This explanation predicts that if an mRNA is first translated in the reticuloeyte lysate under conditions permissive for incorrect product synthesis (i.e., with potassium acetate or with low KCI concentrations), then re-extracted and translated again under restrictive conditions (reticulocyte lysate incubated with 100 mM KCI or HeLa and L-cell extracts incubated under standard conditions), a high yield of incorrect products would be synthesised in the second incubation despite the restrictive conditions. This prediction has been tested, particularly with poliovirus RNA, and has not been fulfilled. Moreover, the idea that potassium acetate might allow or promote more extensive RNA degradation than KC! is hardly compatible with the fact that it gives a higher yield of full-length products initiated at the 5'-proximal site of TMV RNA (Fig. 2) These results are of immediate importance from the operational standpoint of which potassium salts should be used for translation assays. With every RNA tested KCI gave the cleanest product pattern with the best signal to noise ratio, where signal is defined as the yield of authentic products and noise as the yield of incorrect products. Although our results imply that the choice of potassium salt will have little influence on the translation of an intact capped mRNA, this choice clearly becomes critical if fragmented RNA or uncapped RNA is assayed, and is therefore highly relevant to the increasingly common practice of studying the translation of RNAs syntbesised in vitro using bacteriophage SP6 or T7 RNA polymerase. Such RNAs are often transcribed and used in an uncapped form, and even if steps are taken to produce capped transcripts, the capping is

357 usually less than 100% efficient [39]. It is notable that in more than one recent report where uncapped in vitro transcripts were translated in the presence of potassium acetate, many incomplete products were synthesised from internal sites 140,41], and the recent criticism of the fidelity of the reticulocyte lysate s~stem [42] is based entirely on the results of assays carried out under these conditions. Our results show that high fidelity can be achieved, even with uncapped mRNAs, by reverting to the originally recommended use of KC! [1,13], particularly at concentrations about 20 mM above the level giving maximum incorporation. EMCV RNA clearly has many properties atypical of uncapped RNAs. In the first place it is translated with unusually high efficiency (provided KCI is used). Scanning densitometry was used to compare the total yield of labelled EMCV eapsid precursors and the combined yield of the TMV RNA 126 kDa and 183 kDa products synthesised by lysate l using equimolar amounts of the two RNAs. When the densitometry data were adjusted for the methionine content of the different products [19,30] it was found that the efficiency of EMCV RNA translation relative to TMV ~tNA translation was 128% at 100 mM KCI (the optimum for TMV RNA) and 181% at 120 mM KC! (the optimum for EMCV RNA). As TMV RNA is recognised to be among the more efficiently translated RNAs, initiation on EMCV RNA must therefore be exceptionally efficient. Although the translation of EMCV RNA in the reticulocyte lysate shows some properties in common with other uncapped RNAs iJ, that a number of incorrect products are synthesised at low KCI and at all potassium acetate concentrations, it is exceptional in the remarkably strong activation of authentic product synthesis specifically by KCi or low concentrations of KSCN. In control experiments we have found that the inhibitory effects of double-stranded RNA on reticulocyte lysate protein synthesis [43] are uninfluenced by the choice of potassium salt, or by low concentrations of KSCN, and we can therefore eliminate the possibility that our results are an artefact associated with contamination of the EMCV RNA with double-stranded RNA. It has recently been found that the translation of Theiler's routine encephalomyelitis virus RNA in a commercial reticulocyte lysate preparation (Promega) was activated by 5 mM KSCN (in the presence of 100 mM potassium acetate) even more dramatically than EMCV RNA translation [44]. Thus, the response to KSCN is not a peculiarity of lysates prepared in this laboratory, and it extends at least to RNAs from other cardioviruses. Recent evidence has shown that initiation on EMCV RNA takes place by a process of internal initiation which does not involve 40S ribosomal subunits scanning the RNA from the 5'-end [12]. It is tempting to speculate that the unique translation char-

acteristics may be related to this unusual mode of initiation, in which case activation by KSCN may prove to be a useful and simple diagnostic indicator for this type of initiation event. Initiation of poliovirus RNA translation is also believed to operate via a similar internal initiation process [45]. In our hands, KSCN does activate the synthesis of correct prodt:cts from poliovirus RNA to a certain extent (Fig. 6, panel G), but the magnitude of this activation is less than is observed with EMCV RNA. In comparison with EMCV RNA, poliovirus RNA is inefficiently translated in the reticulocyte lysate system, whereas the HeLa or L-cell systems translate the two RNAs with comparable efficiency (Fig. 10). HeLa or L-ceU extracts have been shown to contain one or more factors which, when added to the r.~ticulocyte lysate system, greatly stimulate initiation at the authentic site on poliovirus RNA and inhibit initiation at incorrect sites [10,16,17]. It may be that the rather modegt effect of KSCN in stimulating poliovirus RNA translation in the retic,,:locyte lysate is due to the lack of the limiting HeLa factor(s). In preliminary experiments we have found that KSCN does indeed activate poliovirus RNA translation more strongly if the reticulocyte iysate is supplemented with partially purified HeLa factors, even hough the stimulation is not yet as dramatic as we have observed using EMCV RNA. Acknowledgements I thank Mary Dasso and Tim Hunt for helpful suggestions, Ray Rots and Bert Semler for sharing their results prior to publication, and Peter Melton for his unlimited patience over the gel electrophoresis and densitometry. This work was supported by a grant from the Medical Research Council. References 1 Pelham. H.R.B. and J a c k , m , R J (1976) Eu~ J B~,~.hem 6 ,7. 247 --256. 2 Weber, L A . . Hicke~. E,D. Marone,,. P.A and Baghom, C. (1977~ J. Biol. Chem. 252. 40~)7-4012, 3 Kemper. B. and Stolarsky. L. (1977l Biochemistry, 16. 5676-5680. 4 Herson. D.. Schmidt, A., Seal. S.. Marcus, A. and Van Vloten-Doting. L. (1979) J. Biol. Chem, 254. 8245-8249 5 Chu, L-Y. and Rhoads. R.E. (19781 Bio~'hcmistr2, 17, 2450-.2455 6 Wc~lnar-Fihlmv.icz. A . Szczesna. E . Zan-Kowalczewska, M , Muthukrishnart, S., Szybiak, U., Legocki, A.B. and Fthpowicl, W. (1978) Eur. J. Biochem 92, 69-80. 7 Bergmann. J,F_.. and Lodish, H.F. (1979) J. Biol. Chem~ 254, 459-468. 8 Pelham. H.R.B. (1978) Eur. J~ Bioch-m. 85. 457-462. 9 Jackson, R_J. ¢19~1 Virologj 149. 114-127. 10 Jackson, R J . (1989) in Molecular Aspects of Ptcorna~,lrus Infection an,4 OetecUon (Ehrenfeld. E. and Semler. BL.. eds). pp. 51-71. ASM Pubhcations, Washington. 11 Jackson. R J . il989) Virology 172. 3 6 3 - 3 6 6

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