JOURNAL OF BACTERIOLOGY, Jan. 1991, p. 67-72 Vol. 173, No. 10021-9193/91/010067-06$02.00/0Copyright © 1991, American Society for Microbiology

Analysis of an mRNA Exhibiting AnomalousTranslational Specificity

ROBERT LUIS VELLANOWETHt AND JESSE C. RABINOWITZ*Department of Molecular and Cell Biology, Division of Biochemistry and Molecular Biology,

University of California, Berkeley, California 94720

Received 3 April 1990/Accepted 28 September 1990

Gene 6 mRNA of Bacillus subtilis phage +29 is inefficiently translated under standard in vitro conditions byEschenichia coli, while it is efficiently translated by the in vitro system derived from B. subtilis. This is a rareexample of the inability of E. coli to translate mRNA translated by B. subtilis. The ionic condition in thetranslation systems was the key component in the differential recognition of the gene 6 message by E. coli andB. subtilis ribosomes. Its translation by E. coli ribosomes was preferentially inhibitied by moderate levels ofKCl, while its translation by B. subtilis ribosomes was unaffected by these concentrations of salt. Thispreferential inhibition with E. coli ribosomes was observed in vitro as well as in vivo. While not influencing thegeneral phenomenon of preferential inhibition, anion-specific effects were observed in overall protein synthesis.Glutamate and acetate promoted efficient synthesis over a broad range of concentrations, whereas chloride wasinhibitory at all concentrations tested.

Messages derived from Escherichia coli are inefficientlytranslated by Bacillus subtilis both in vitro (16, 18, 22, 23, 38)and in vivo (1, 13, 35). Conversely, the E. coli translationalapparatus can efficiently translate B. subtilis mRNAs inaddition to its own. This translational specificity has beenobserved in a number of other gram-positive organisms (41).In vitro experiments have demonstrated that this transla-tional barrier to heterologous gene expression is a functionof the ribosomes and mRNAs and not the initiation factors,tRNAs, or amino acid-activating enzymes (11, 19, 23, 40).The 30S subunit rather than the 50S subunit is related totranslational specificity (33). Thus, the B. subtilis 30S ribo-somal subunit is unable to participate in the functionaltranslation initiation of E. coli messages either with its own50S subunit or with that of E. coli, while the B. subtilis 50Ssubunit does not prevent efficient initiation of E. coli mes-sages with the E. coli 30S subunit. Besides slight variationsin the 16S rRNA sequences, specific ribosomal proteindifferences are also found. The E. coli 30S subunit contains21 proteins, the largest of which, Si, is thought to play anintegral role in the normal initiation of protein synthesis in E.coli (45, 46). B. subtilis does not contain a counterpart to thisprotein, nor do any of the other gram-positive organisms thatexhibit translational specificity (33). Conversely, all gram-negative bacteria that have been shown to be relativelynonspecific in translation contain an S1-like protein. Con-cerning the mRNAs, all natural B. subtilis mRNAs haveShine-Dalgarno (S/D) sequences with predicted free energiesof binding to the 16S rRNA at least 6 kcal (ca. 25 kJ)/molstronger than that of the average E. coli message (11). Suchrelatively stable interactions appear to be a requirement forefficient translation in B. subtilis, although E. coli phagemessages containing very strong S/D sequences are nottranslated efficiently by B. subtilis ribosomes (10).While translational specificity as described above is ob-

served both in vitro and in vivo, at least one case is known

* Corresponding author.t Present address: Institute of Biological Chemistry, Washington

State University, Pullman, WA 99164-6340.

of a B. subtilis mRNA that is poorly translated in vitro by E.coli ribosomes. This unique case of anomalous specificity isthe translation of the gene 6 mRNA of the B. subtilis phage4)29. Under standard conditions of in vitro translation, it wasdemonstrated by using B. subtilis ribosomes that three major4)29 products are formed in response to 4)29 mRNA (22, 28).These proteins are the products of the early genes 5, 6, and17. Under similar conditions, E. coli ribosomes were foundto synthesize only protein 5 (p5) and p17; p6 was synthesizedvery poorly or not at all. The basis for this reverse specificityis unclear. Although the S/D region of gene 6 is ratherunusual in its potential ability to base pair with the extreme3' end of the B. subtilis 16S rRNA, the calculated free energyof binding to the E. coli 16S rRNA is well within the rangeapparently necessary for E. coli translation (28). In thisreport, we demonstrate that moderate levels of salt prefer-entially inhibit the in vitro synthesis of p6 by E. coliribosomes relative to other 4)29 products. On the other hand,B. subtilis ribosomes were unaffected by salt in their trans-lation of the gene 6 mRNA relative to the other mRNAs.While the substitution of various anions did not affect thegeneral observation that salt preferentially inhibited p6mRNA translation by E. coli ribosomes, large effects wereobserved on the extent of overall protein synthesis. Also,increases in the ionic strength of the intracellular milieu of anE. coli cell through increases in the osmolarity in the growthmedium resulted in the preferential inhibition of p6 synthesisin vivo identical to that observed in vitro.

MATERIALS AND METHODS

Bacterial strains, phage DNAs, and plasmids. B. subtilisW168 and E. coli MRE600 were common laboratory strains.E. coli K-12 Atrp(XN- c1857 AH1) was obtained fromMargarita Salas. 4)29 DNA was provided by Paul Hager.Plasmids pRP8 (30) and pGM26 (21) were kindly provided byMargarita Salas.

Reagents. Tris, sodium phosphoenolpyruvate, spermidinetrihydrochloride, trisodium ATP, trisodium CTP, cyclicAMP, all 20 amino acids (L form), potassium glutamate(K-glutamate), ammonium glutamate, rifampin, and rabbit

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muscle pyruvate kinase were purchased from Sigma; triso-dium UTP and trisodium GTP were from P-L Biochemicals;E. coli MRE600 tRNAs were from Boehringer Mannheim;[35S]methionine (1,426 Ci/mmol) and [et-32P]CTP (410 Ci/mmol) were obtained from Amersham; B. subtilis RNApolymerase (345 U/mg) was prepared as described previ-ously (6) and provided by Mark Roberts. All other chemicalsand reagents were of the highest grade available.

In vitro RNA synthesis. Transcripts from +29 DNA wereprepared with B. subtilis RNA polymerase in a 100-Ilvolume, using the following reaction conditions: 100 mMTris hydrochloride (pH 8.0), 10 mM MgCl2, 1 mM dithio-threitol, 1.6 mM spermidine, 160 mM KCl, 1 mM each ATP,GTP, CTP, and UTP, 0.1 mg of DNA per ml, and 0.72 mg ofRNA polymerase per ml. Reactions were started by theaddition of RNA polymerase and were incubated at 37°C for30 min. Polymerization was terminated by the addition ofEDTA to 10 mM and rifampin to 20 ,ug/ml. Incorporationwas quantified by performing parallel reactions in the pres-ence of [32P]CTP at 0.02 RCi/nmol. Reactions typicallyincorporated 4 to 12 nmol of trichloroacetic acid (TCA)-precipitable CMP, depending on the DNA used as a sub-strate.

In vitro protein synthesis. Protein synthesis was directed inan uncoupled manner in which aliquots of a stopped tran-scription reaction were added to a translation mix. Reactionswere carried out in 30-RI volumes under the followingconditions: 67 mM Tris hydrochloride (pH 8.0), 10 mMmagnesium acetate (MgOAc2), 0.1 mM EDTA, 12 mMP-mercaptoethanol, 6.25 mM phosphoenolpyruvate, 16.7 ,ugof pyruvate kinase per ml, 16 mM 10-formyltetrahydrofolate,0.17 mM amino acids (minus methionine), 10 ,uM [35S]methionine (25 pCi/nmol), 0.83 mg of E. coli tRNAs per ml,2 mM ATP, 0.5 mM GTP, S-150 fraction prepared asdescribed previously (39) from either B. subtilis (4.2 mg/ml)or E. coli (4 mg/ml), high-salt wash fraction prepared asdescribed from either B. subtilis (0.4 mg/ml) or E. coli (0.5mg/ml), sucrose-gradient purified vacant couple ribosomes(20 A260 units per ml) prepared as described previously (39)from either B. subtilis or E. coli, 20 pg of rifampin per ml, 50mM NH4X, and KX at the indicated concentration (whereX- is chloride, acetate, or glutamate). The reactions werestarted by the addition of 0 to 7.5 1LI of an in vitro transcrip-tion reaction mixture. All reactions were terminated by theaddition of an equal volume of 2x sodium dodecyl sulfate(SDS) sample buffer (50 mM Tris hydrochloride [pH 6.8], 3M urea, 0.7 M P-mercaptoethanol, 2% SDS, 20% glycerol[final concentrations]). For quantitation, 10-,I samples wereadded to 1 ml of 10% TCA, incubated on ice for 10 min,heated at 95°C for 10 min, cooled on ice for 10 min, andcollected on prerinsed Whatman GF/C filters. The filterswere then rinsed successively with 4 ml of 2% TCA-2 ml of100% ethanol, dried under an infrared heat lamp, andcounted in 4 ml of Scint-A liquid scintillation cocktail in aBeckman LS 8100 scintillation counter.

Plasmid-directed in vivo expression of protein products. Invivo labeling of 429 p6 in E. coli K-12 LAtrp(XN- c1857 AHI)from plasmid pRP8 was performed as described previously(8).

Electrophoresis, fluorography, and densitometry. Proteinproducts from in vitro translation reactions and in vivolabeling were separated on discontinuous SDS-polyacryl-amide gels (30:0.8, acrylamide/bisacrylamide) (14). Reactionmixtures were diluted 1:1 in 2x SDS sample buffer, heated at95°C for 5 min, cooled on ice, and loaded (5 to 15 ,ul) onto a15% polyacrylamide gel. Gels were run at 35-mA constant

A B

b c d e a b c d e

- p17 -

- p6 40M Amm -.

nn,.:.-::'

Ww-w ","?fi.

.4 ` tkF,::.rz

- p5 -

FIG. 1. Fluorogram of 4+29 translation products from uncoupledin vitro translation reactions at increasing concentrations of KCl.Samples (6 ,ul) were run through a 15% polyacrylamide gel; fluorog-raphy was for 15 h. (A) E. coli ribosomes; (B) B. subtilis ribosomes.Lane a, No added mRNA. Reaction mixtures in lanes b to econtained KCl concentrations of 25, 75, 125, and 175 mM, respec-tively.

current for 2.5 h, fixed for 30 min in 10% acetic acid-25%isopropanol, rinsed for 10 min in distilled H20, and soakedfor 60 min in 1 M sodium salicylate (3). After drying, the gelswere exposed to preflashed Kodak XAR film (15) at -80°Covernight. Protein bands were quantified from the fluoro-gram by densitometry, using an E-C apparatus densitometerconnected to an HP 3390A integrator.

RESULTS

Preferential inhibition of p6 synthesis. It was previouslyreported that the 4)29 gene 6 mRNA is the only known B.subtilis message that is inefficiently translated by E. coliribosomes under standard in vitro translation conditions(28). This phenomenon was found to be independent of thesource (E. coli or B. subtilis) ofRNA polymerase, S-150, andinitiation factor fractions. It could be demonstrated in bothcoupled and uncoupled translation systems and was depen-dent only on the source of ribosomes (data not shown). Onthe other hand, Pastrana et al. (30) reported that E. coli cellsharboring 4)29 gene 6 in an inducible expression vector couldproduce p6 to approximately 4% of the total cellular protein.Although this amount was considerably less than the quan-tities achieved with similar clones of other 4)29 genes (25),the extent of synthesis was unexpected in view of our invitro results. This contradictory result led us to reinvestigatethe conditions used for the in vitro translation system.An alteration of the ionic strength in the translation assay

revealed that the conditions previously used were subopti-mal specifically for p6 synthesis. The concentration of KClin uncoupled translation reactions was varied from 25 to 175mM, and labeled protein products were separated electro-phoretically (Fig. 1). At low concentrations of KCl, p6 waswell translated by E. coli ribosomes (Fig. 1A). However, asthe level of KCl was raised, p6 synthesis diminished rapidly.Figure 1B shows an identical experiment using B. subtilisribosomes. Although increased KCl also inhibited overallprotein synthesis, no preferential inhibition of p6 expressionwas observed as was with E. coli ribosomes. This preferen-tial inhibition is graphically illustrated in Fig. 2.

Influence of the anion on the preferential inhibition of p6synthesis and on overall protein synthesis. The vast majorityof bacterial in vitro systems described in the literature

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Co._4

0s

0

o

0.6

0.5

0.4

0.3

0.2

0.1

0.00 100 200

[KCI] mMFIG. 2. Expression of p6 as a function of KC1 concentration. The

fluorogram shown in Fig. 1 was scanned by densitometry. Thefraction of total synthesis in p6 was determined by dividing the areaof the scanned peak corresponding to p6 by the sum of all scannedpeaks. The curve fits are simple regressions of the data. Symbols: O,E. coli ribosomes; *, B. subtilis ribosomes.

A a b c d e f g h I

*~PU. :: ; 4 : : ::

* X .,s

B a b c d e

contain chloride-based salts in reaction buffers. However,the use of chloride is probably not justified on a physiologicalbasis since it is excluded from the cytoplasm in nonhalo-philic bacteria (32). Chloride anion has consistently beenfound to be much more inhibitory to biological processes

measured in vitro than are organic anions, especially thoseinvolving protein-nucleic acid interactions (2, 12, 17, 26, 29).To test whether the preferential inhibition of p6 synthesis byE. coli ribosomes was simply a Cl--mediated effect, we

examined the influence of other anions on the synthesis of4)29 products in uncoupled translation reactions. All chloridesalts and buffers were replaced with their acetate (OAc)counterparts (Tris-OAc, MgOAc2, NH4OAc, and KOAc),and the concentration of KOAc was varied from 44 to 300mM in uncoupled reactions using 429 mRNA (Fig. 3). Asobserved with chloride, E. coli ribosomes were preferen-tially inhibited by KOAc in the translation of the gene 6message relative to the other 4)29 products (Fig. 3, lanes a tof), while again B. subtilis ribosomes exhibited no preferentialinhibition (lanes g to 1). Complete inhibition of E. colisynthesis of p6, however, was not evident until the concen-

tration of KOAc was greater than 200 mM, more than twicethe concentration of KCl required for complete inhibition. Inan analogous experiment, glutamate anions were used inplace of chloride, and the extent of protein synthesis by E.coli and B. subtilis ribosomes was examined as a function of

a b c d e f g h i k

pl7 -

p6 -_ _ _

Ps aF.

FIG. 3. Fluorogram of 4)29 uncoupled in vitro translation prod-ucts at increasing concentrations of KOAc. Samples (8 ,ul) were run

through a 15% polyacrylamide gel; fluorography was for 16 h.Lanes: a to f, E. coli ribosomes; g to 1, B. subtilis ribosomes. Thefollowing millimolar concentrations of KOAc were used: lanes a andg, 44; lanes b and h, 100; lanes c and i, 150; lanes d and j, 200; lanese and k, 250; and lanes f and 1, 300.

.a4 -,, .. - ..

FIG. 4. Fluorogram of 4)29 uncoupled in vitro translation prod-ucts at increasing concentrations of K-glutamate. Samples were

resolved on a 15% polyacrylamide gel; fluorography was for 12 h.(A) E. coli ribosomes; (B) B. subtilis ribosomes. Lane a, No addedmRNA. Lanes b to j contained K-glutamate concentrations of 0, 50,100, 150, 200, 250, 300, 350, and 400, respectively.

K-glutamate concentration (Fig. 4). E. coli ribosomes (Fig.4A) efficiently expressed p5 and p17 from 429 mRNA evenat 400 mM K-glutamate. On the other hand, expression of p6was inhibited by increasing K-glutamate, but this salt was

much less inhibitory than acetate or chloride. The potassiumsalt of glutamate did not completely inhibit E. coli translationof p6 until the concentration was greater than 350 mM,compared with approximately 200 mM for acetate and 80mM for chloride. In contrast, p6 expression by B. subtilisribosomes (Fig. 4B) was not preferentially inhibited at all byK-glutamate but rather exhibited a very broad optimum.While the type of anion used in these translation experi-

ments had little influence on the preferential salt inhibition ofp6 synthesis by E. coli ribosomes other than to alter theconcentration of KX for complete inhibition, large anioneffects were observed for overall protein synthesis. Theamount of translation of 4)29 mRNA as a function of KXconcentration was quantified by TCA precipitation (Fig. 5).Translation by E. coli ribosomes (Fig. SA) was readilyinhibited by KCl; the optimum level for translation was

found to be the lowest level used. Acetate and glutamateexhibited broader optima, with glutamate slightly better athigher salt concentrations. The steepness of the E. colicurves at high salt concentrations, especially the acetate andglutamate curves, presumably reflects in large part theunusual salt sensitivity of gene 6 translation. Perhaps a more

accurate indication of the average influence of these anionson protein synthesis is exhibited by B. subtilis ribosomes(Fig. 5B). Chloride ion was the most inhibitory of the threetested at all concentrations. The largest difference could beseen at salt concentrations greater than 200 mM: glutamateenhanced synthesis to approximately 130% that of the initial

p6

g h I

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.200 'A ~~~~~~~~~~~040

o OAc-

*10 ~~0 OAcA 0

oVo20

E ~~~~~~~~~CLCL I

0 0~~~~~~~~00 100 200 300 0 00o 200 300[KX] (mM) [KX] (TM)

FIG. 5. Anion effect on 429 uncoupled translation. Incorporationof [35S]methionine into protein in the reactions analogous to thoseshown in Fig. 1, 3, and 4 was quantified by TCA precipitation. Totalincorporation as a function of the concentration of added potassiumsalt is shown. (A) E. coli ribosomes; (B) B. subtilis ribosomes.

value, acetate decreased synthesis to 70%, and chloridedecreased synthesis to 16%.

Effect of salt on in vivo expression of +29 p5 and p6 in E.coli. Intracellular ionic strength in E. coli, primarily in theform of potassium and glutamate, can be reproducibly variedfivefold by changing the osmolarity of the growth medium (7,32). This phenomenon has been exploited to compare salteffects on protein-nucleic acid interactions in vivo with thoseobserved in vitro (32). A similar approach was taken in thisstudy to examine salt effects on p6 translation in vivo. Twoplasmids were obtained from M. Salas that contain either4+29 gene 5 (pGM26) or gene 6 (pRP8) individually clonedunder the control of the thermoinducible A PL promoter (30).In vitro translation of mRNAs derived from these plasmidswas found to follow identically the pattern observed with thenative +29 polycistronic transcript: E. coli ribosomes pro-duced p6 from pRP8 in a salt-sensitive manner but wereunaffected by salt in the synthesis of p5 from pGM26.Similarly, B. subtilis ribosomes were relatively unaffected byincreasing salt concentrations in the translation of bothmessages (data not shown). In vivo labeling experimentswere also carried out with E. coli cells harboring plasmidpRP8 or pGM26. An autoradiogram of the products resolvedthrough a 15% polyacrylamide gel was scanned by densitom-etry to determine the amount of synthesis of p6 or p5 (Fig.6). The results in vivo were consistent with those in vitro;expression of p5 was not affected by changes in intracellularion levels, whereas p6 translation decreased as the intracel-lular ion concentration increased.

DISCUSSION

A major finding of this study was that anions play a role inthe overall efficiency of translation by both E. coli and B.subtilis ribosomes. The anions tested were inhibitory in theorder Cl- > acetate- > glutamate-. Inhibition by chloridewas evident at all concentrations tested from 44 to 350 mM,but efficient synthesis by both E. coli and B. subtilis ribo-somes was observed in the presence of nearly 0.5 M K-glu-tamate. The optimum concentration range of glutamate forefficient translation was also much broader than that ofchloride. These effects were general and did not affect thepreferential inhibition of gene 6 mRNA translation by E. coliribosomes; with each of the three anions, specific inhibitionof p6 synthesis with increasing salt concentration was ob-served. However, the fact that the species of anion deter-mined the concentration of its potassium salt at which

0

0. )

0I-

0

CU)00.

20 r

10 I

a

OH-

I . . I . I . a . I

0 1 2 3 4

%NaCI In Growth Medium

5 6

FIG. 6. Percentage of total in vivo protein synthesis of p6 (l)and p5 (a) as a function of growth medium salt concentration. E.coli K-12 Atrp(XN- c1857 AH1) cells containing either pRP8 orpGM26 were grown in labeling medium containing 0.5 to 5% NaCland labeled as described in Materials and Methods. Equal volumes(5 ,ul) were loaded onto a 15% SDS-polyacrylamide gel, and theresulting autoradiogram was scanned by densitometry. The curvefits were obtained by linear regression of the data.

complete inhibition was observed suggested that the lack ofefficient synthesis of p6 by E. coli ribosomes was not simplydue to some alteration of the mRNA structure, such as anenhanced stability of secondary structure by potassium ions.Rather, the involvement of the anion in the inhibition sug-gested that the inhibitory effect of salt was at the level ofprotein-nucleic acid interactions.

Specific anion effects on protein-nucleic acid interactionsin a number of different in vitro systems have been reported(2, 12, 17, 20, 26, 29). All of these protein-nucleic acidinteractions are strongly dependent on the nature and con-centration of electrolytes, and the interactions involved werefound to be more stable in either acetate or glutamate than inchloride or other halides. The general conclusion from thesestudies is that formation of the protein-nucleic acid complexinvolves the release of anions from tight binding sites on theprotein and different anions have different affinities for thosesites. Thus, the large anion effect on translation observed inthis study demonstrates the involvement of specific protein-nucleic acid interactions, including counterion release fromboth species (31), in the overall process of protein synthesis.Obviously such anion effects could reflect specific protein-nucleic acid interactions during initiation (Sl-mRNA, initia-tion factor-mRNA, ribosomal protein-rRNA, etc.) or duringelongation or both.Although chloride anion is extremely inhibitory to many

processes essential for growth and maintenance of a bacte-rial cell measured in vitro, the cytoplasmic level of chloridein many nonhalophilic bacteria under most conditions isnegligible (4, 5, 42). The major univalent anion in E. coli cellsis glutamate, which has been shown to increase in concen-tration to compensate for increased cation levels undervarious growth conditions (7, 24, 27, 32). Thus, the apparentabsence of chloride in the cytoplasm of E. coli and othernonhalophilic bacteria and the importance of glutamate asthe major univalent anion suggest a physiological relevanceof the in vitro effects of these anions.But how relevant are the extreme salt dependent effects

observed in vitro to a growing cell? Glutamate will enhanceprotein-nucleic acid interactions in vitro, but salt-dependenteffects are still observed, albeit at higher concentrations (17).Richey and co-workers (32) took advantage of the fact thatthe intracellular environment of an E. coli cell could be

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reproducibly varied to test the effects of such changes onprotein-nucleic acid interactions in vivo. They found thatwhile the equilibrium constant for the interaction of RNApolymerase with the A PR promoter varies as [KCI]-20 invitro (34), effects in vivo were no more than twofold. Thisfinding suggests that some mechanism (discussed by Richeyet al. [32]) to compensate for the increased ionic environ-ment inside the cell must exist, since high levels of K+, evenwith glutamate as the counterion, are expected to disruptprotein-nucleic acid interactions.While large salt effects on RNA polymerase-promoter and

repressor-operator interactions disappear in vivo, the samedoes not appear to be the case for the translation of the gene6 mRNA. Although we did not quantify the levels of K+ orglutamate inside the cell, the in vivo experiment shown inFig. 6 was an approximation of the method described aboveto vary these ions in vivo (2a). We observed that thetranslation of the gene 6 message was affected by salt toapproximately the same order of magnitude observed invitro. The control message, 4)29 gene 5, which was unaf-fected by salt in vitro, was similarly unaffected by changes inthe ionic environment in vivo. The significance of this in vivoobservation is attested to by the fact that while the system ofRichey and co-workers (32) was affected greatly by salt invitro but only negligibly in vivo, the translation of the gene 6message was affected to similar degrees both in vitro and invivo. Presumably then the compensation mechanisms serv-ing to buffer salt effects on protein-nucleic acid interactionsin vivo are not operative in translation.

In seeking a solution to the question of why B. subtilis andE. coli ribosomes are differentially affected by salt in thetranslation of the 4)29 gene 6 mRNA, we compared what isknown about the ionic compositions of various bacterialspecies. Such comparisons are complicated by the use ofdifferent growth conditions and different methodologies andassumptions. Nonetheless, both similarities and differencescan be deduced with respect to the two organisms of interesthere. As with most other nonhalophilic organisms, at leastone Bacillus species also excludes chloride from the cyto-plasm (5). However, the free pools of amino acids are 5- to20-fold higher in B. subtilis and other gram-positive organ-isms than in three gram-negative organisms tested (47).Glutamate is also the dominant amino acid in B. subtilis, butits concentration is 10-fold higher than that found in thegram-negative organisms. Differences are also evident uponosmotic upshift. Instead of glutamate, intracellular proline isaccumulated in B. subtilis in response to increased levels ofNaCl in the growth medium (24, 47). In fact, at 4% andgreater amounts of medium NaCl, proline becomes thedominant amino acid in B. subtilis, superceding glutamate.E. coli and B. subtilis have apparently evolved differentmechanisms for dealing with osmotic stress. E. coli cellsaccumulate charged species, primarily K+ and glutamate butalso putrescine (27), proline (24), and trehalose (44). B.subtilis cells have been shown to accumulate the neutralamino acid proline. Osmotic upshift effects on inorganic ionsor polycations in this organism are not known. Obviously theribosomes of E. coli and B. subtilis have separately evolvedto function properly in these different environments. Sincethe 4)29 phage normally infects B. subtilis, the sequence ofgene 6 presumably has evolved such that its message can betranslated under all conditions present in the B. subtilis cell.The E. coli ribosome, of course, has not had this advantage.The inability of the E. coli ribosome to translate the gene 6message at high salt concentrations must therefore be deter-mined by the sequence of the mRNA itself.

It is known that mRNA sequences of procaryotic ribo-some-binding sites are nonrandom throughout the entire40-nucleotide region bound by the ribosome (36, 37, 43). Ithas been proposed that this nonrandom distribution mayreflect additional contacts between the message and theribosome, other than S/D-16S rRNA duplex formation, thatare important for translation initiation (9). Since the pre-ferred positions outside the S/D sequence and the initiationcodon are not the same in E. coli or B. subtilis (11), theseadditional contacts may differ significantly. High salt con-centrations could alter the architecture of the gene 6mRNA-E. coli ribosome interaction such that these contactsare unfavorable and lead to diminished initiation. The use ofthe 4)29 gene 6 mRNA as a model system in a detailedanalysis of the salt dependencies of the many interactions ofE. coli and B. subtilis ribosomal particles with this messageis possible and could provide evidence for structural andfunctional differences between the two ribosomes.

ACKNOWLEDGMENTS

This work was supported by Public Health Service grant AM2109from NIAMDD. R.L.V. was supported by MARC predoctoralfellowship BM09094.

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