JOURNAL OF BACTERIOLOGY, June 1991, p. 3888-38930021-9193/91/123888-06$02.00/0Copyright © 1991, American Society for Microbiology

Vol. 173, No. 12

Growth Rate Regulation of Translation Initiation Factor IF3Biosynthesis in Escherichia coli

DIONYSIOS LIVERIS, ROBIN-ANN KLOTSKY, AND IRA SCHWARTZ*Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York 10595

Received 10 December 1990/Accepted 8 April 1991

infC, the gene encoding translation initiation factor IF3 in Escherichia coli, can be transcribed from threepromoters. Two of these promoters, P1, and PI2, are located in the upstream thrS sequence which codes forthreonyl-tRNA synthetase. Previous studies had shown that PI2 was the major promoter for infC. In the presentstudy, the extent of transcription from P1, and/or Pl2 at a variety of steady-state growth rates was analyzed bypromoter fusion studies. P12 was the more active promoter (two- to threefold stronger than PI1) at all growthrates tested. A fusion plasmid containing both PI1 and PI2 exhibited a transcription level approximately equalto the sum of those observed with the fusion plasmids containing the individual promoters. The transcriptionalactivities of P1, and PI2 did not change as the growth rate was varied from 0.3 to 1.7 doublings per h. Incontrast, a fusion plasmid carrying the rrnB PI promoter displayed the expected growth rate response. Thesteady-state concentrations of infC mRNA in cells grown at different rates were measured and found not tovary. These results indicate that the previously reported growth rate regulation of IF3 biosynthesis neither isaccomplished by transcriptional control nor is a result of differential mRNA stability. In view of these results,the steady-state levels of IF3 in cells grown at a number of different growth rates were determined byquantitative immunoblotting. IF3 levels were found to vary with growth rate in a manner essentially identicalto that observed for ribosomes. A model accounting for these results and describing a mechanism forcoordinate growth rate-regulated expression of ribosomes and IF3 is presented.

The initiation of translation in Escherichia coli requiresthe precise interaction of a 30S ribosomal subunit, initiatorfMet-tRNA, and a 60- to 80-nucleotide initiation region ofthe mRNA which includes both the Shine-Dalgarno se-quence and the initiation codon (4). Three translation initia-tion factors participate in this process. Translation initiationfactor IF3 binds directly to 30S subunits and ensures aconstant supply of free subunits for initiation complex for-mation (19). Recent evidence indicates that IF3 also plays arole in the kinetic selection of the correct initiation region ofmRNA (10) and destabilizes initiation complexes containingnoninitiator tRNAs (14).

infC, the gene encoding IF3, is located at 38 min on the E.coli chromosome and is clustered with the genes for severalother components of the translation machinery. These in-clude an upstream thrS gene which codes for threonyl-tRNAsynthetase and several downstream genes which code forribosomal proteins and phenylalanyl-tRNA synthetase. Thegenetic organization of this operon is unusual, and its patternof transcription is complex. The coding regions of thrS andinfC are separated by only 3 nucleotides, and infC is initiatedwith an AUU codon (20, 27). Transcription of infC isinitiated at three different promoters, PT, PI, and PI2 (30).PT is located upstream of thrS, and transcription from thispromoter results in an mRNA which codes for both threonyl-tRNA synthetase and IF3. In contrast, P1, and PI2 aresituated within the thrS coding region. A previous study hasshown that PI2 is the major promoter for infC (30).

Several regulatory mechanisms contribute to the mainte-nance of the steady-state levels of IF3 in the cell underdifferent physiological conditions. The in vivo level of IF3

* Corresponding author.

was shown to vary proportionately with cellular growth rate,resulting in an essentially constant IF3/ribosome ratio ofapproximately 0.15 (15). In addition, IF3 is translationallyautoregulated by a mechanism which appears to require anintact AUU initiation codon (2, 4). A model accounting forthis behavior has been proposed (9, 10).To ascertain the relative contributions of each of the three

infC promoters to the cellular levels of IF3, we measured theextent of transcription from Pj1 and PI2 at a variety of growthrates. In addition, the synthesis of IF3 as a function ofgrowth rate was reexamined. The results indicate thatgrowth rate regulation of IF3 is not exerted at transcriptioninitiation but is, rather, a posttranscriptional event.

MATERIALS AND METHODSPlasmid DNA and DNA fragment isolation. Large-scale

plasmid purification was carried out as previously described(7). Small-scale preparations of plasmid DNA were obtainedby the alkaline-sodium dodecyl sulfate method (1). Restric-tion endonuclease digestions were performed in accordancewith manufacturer directions. The enzymes were obtainedfrom Bethesda Research Laboratories (Gaithersburg, Md.),New England BioLabs (Beverly, Mass.), or BoehringerMannheim (Indianapolis, Ind.). DNA restriction fragmentswere purified from low-melting-temperature agarose gelsfollowing electrophoresis as described previously (24).Promoter fusion plasmid construction. Plasmid pAP1,

which contains thrS and infC, has been described previously(25). Plasmid pKK275-4, which contains the rrnB P1 pro-moter fused to cat, has been described previously (17) andwas kindly provided by Jurgen Brosius. The appropriatepromoter fusions were constructed by the insertion of vari-ous restriction fragments obtained by digestion of pAP1 intothe polylinker region of the promoter selection vector

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thrS

pT

infC

pl1 p12

pSK5

pSKB

pSK9

FIG. 1. Schematic diagram of the thrS-infC region of the E. colichromosome and origins of the promoter fragments used in thefusion plasmid constructions. The stippled and hatched rectanglesindicate the sizes and positions of thrS and infC, respectively. Thepositions of the three promoters are indicated. The extents of theDNA fragments used to construct pSK5, pSK8, and pSK9 are

indicated by the solid lines. For details of the fusion plasmidconstructions, see the text.

pKK232-8 (Apr Cms) (3), as described below. Followingtransformation into E. coli HB101 and the selection of AprCmr clones, the identities of the inserted fragments were

confirmed by restriction analysis. The appearance of Cmrcolonies indicates the fusion of an active promoter to cat.The fusion plasmids used in this study are depicted in Fig. 1.

(i) pSK5. pAP1 was digested with HpaI, and the resultant1.1-kb fragment was isolated. This fragment was furtherdigested with AluI, and the mixture of fragments was clonedinto the SmaI site of pKK232-8. The presence of a 215-bpinsert containing PI2 was confirmed by digestion of pSK5with EcoRI and HgiAI.

(ii) pSK8. pAP1 was digested with SmaI and PvuII, andthe 448-bp fragment, which includes P1,, was isolated afterelectrophoresis on low-melting-temperature agarose gels.Following ligation into the SmaI site of pKK232-8, thepresence of this fragment was confirmed by digestion withEcoRI and PvuI.

(iii) pSK9. pAP1 was sequentially digested with SmaI andAccI, yielding a 1,081-bp fragment containing both P1, andP12. The appropriate AccI site was selectively filled in byincubation with the Klenow fragment of DNA polymerase Iin the presence of dATP, dGTP, and dCTP. The resultantfragment was inserted into the SmaI site of pKK232-8, andits presence and orientation were confirmed by digestionwith EcoRI and PvuI.

Bacterial growth. Cells were grown in L broth (LB) or M9minimal medium supplemented with 0.4% glucose plus 0.4%tryptone (MGT), with 0.4% glucose (MGlc), or with 0.4%glycerol (MGly) to achieve steady-state growth at a varietyof different rates. Plasmid-transformed HB101 cultures werefurther supplemented with 1 ,ug of thiamine (B1), 17.4 pug ofproline, 17.4 ,ug of leucine, and 100 ,ug of ampicillin per ml.E. coli B/r cultures were only supplemented with 1 ,ug ofthiamine (B1) per ml in addition to the carbon sources.Overnight cultures grown in the corresponding media were

inoculated into fresh media to a starting A6. of 0.05 to 0.06and grown to the mid-log phase at 37°C. Cells were har-vested and processed as described previously (17).Promoter activity measurements. The transcriptional activ-

ities of promoter fragments cloned into pKK232-8 were

determined by measurement of chloramphenicol acetyltrans-ferase (CAT) and f-lactamase (BLA) activities as previouslydescribed (17). For each experiment, the individual datumpoints were determined in triplicate. Experiments were

repeated with duplicate aliquots obtained from the samecultures, which had been frozen and stored at -70°C.Promoter activities are presented as CAT/BLA ratios toaccount for variability in cell lysis and plasmid copy numberfrom experiment to experiment, as already described (17,30).

Determination of cellular IF3 levels by quantitative immu-noblotting. Cultures (100 ml) of E. coli B/r were grown to themid-log phase, harvested by centrifugation, washed oncewith ice-cold 10 mM Tris-HCl (pH 7.8)-10 mM MgCl2, andresuspended in 2.5 ml of the same buffer. Cells were lysed bysonication, and crude ribosome preparations were obtainedby centrifugation of the cell lysates at 30,000 x g for 45 minand at 150,000 x g for 2 h. The final, crude ribosome pellets(containing all of the IF3) were resuspended in 1.0 ml of 10mM Tris-acetate (pH 7.8)-10 mM magnesium acetate-30mM ammonium acetate-7 mM ,-mercaptoethanol-0.1 mMEDTA. Samples containing equal amounts of protein wereelectrophoresed on a 17% sodium dodecyl sulfate-polyacryl-amide gel and electroblotted to nitrocellulose. After transfer,the blots were pretreated with 0.5% dry milk in 0.5% sodiumazide-Tris-buffered saline (pH 7.4) for 6 h and incubated for1 h with 0.05% Tween in 0.5% sodium azide-Tris-bufferedsaline (pH 7.4). The blots were then incubated with a 1:5,000dilution of an anti-IF3 immunoglobulin G antibody prepara-tion (25), washed in 0.05% Tween, and further incubatedwith a 1:15,000 dilution of alkaline phosphatase-conjugatedmouse antirabbit immunoglobulin G for 1 h at 37°C. Thenitrocellulose blots were developed with the Protoblot sys-tem (Promega), with Nitro Blue Tetrazolium and 5-bromo-4-chloro-3-indolyl-phosphate as the substrates, essentially asdescribed by the manufacturer. The amount of IF3 in eachsample was determined by quantitative densitometry basedon a standard curve obtained with pure E. coli IF3 of knownconcentration run in tandem for each experiment. The datapresented represent the averages of three independent cellsamples.

Determination of IF3 mRNA content. The levels of infC-containing mRNA in cells grown at different rates weredetermined by dot blot hybridization. Total RNA was iso-lated from mid-log-phase cultures grown at different rates bythe hot phenol extraction method of Salser et al. (28). Equalamounts of RNA were slot blotted to nitrocellulose, and theblots were developed by hybridization to an infC-specificDNA probe. The probe used was a 1.1-kb HpaI fragmentfrom pAP1 which was labeled by nick translation (26). Theblots were incubated, washed, and processed by autoradi-ography as described previously (29).

RESULTS

Transcription from the infC promoters is not under growthrate regulation. It has been demonstrated previously thatseveral promoters contribute to the total cellular levels ofinfC mRNA (30). These include P1, and PI2 which aresituated within the upstream thrS coding region (Fig. 1). P12is the major promoter for infC (30). These findings suggestedthe possibility that utilization of the promoters may varyunder various physiological growth conditions. To quantita-tively assess the relative transcriptional activities of P1, andPI2 at a variety of steady-state growth rates, we fused DNAfragments containing these promoters to the reporter genecat in plasmid pKK232-8. We have already shown thatpromoter fusion constructs in this vector can be used forgrowth rate control studies (17). In these earlier experi-ments, it was demonstrated that BLA activity and plasmid

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TABLE 1. IF3 and ribosome contents in E. coli B/ra

Viableb Riooec IdMedium counts Ribosomes IF3b (molecules/ (molecules/

(100) ( 260) (ng) cell) cell)

MGly 7.55 12.5 997 2,429 390MGlc 7.63 24.3 1,645 4,777 652MGT 7.12 47.7 2,656 10,421 1,159LB 4.60 57.2 4,054 18,006 2,602a Cultures (100 ml) of E. coli B/r were grown in the indicated media, and

...../ . cells were harvested at the mid-log phase. Crude ribosome fractions were)- - -7 '' " -" *obtained as described in the text. The growth rates in MGly, MGlc, MGT, andI LB were 0.54, 1.12, 1.67, and 2.1 doublings per h, respectively. All values

presented are per 100 ml and are the averages of three independent experi-)- ments.

0.0 0.4 0.8 1.2 1.6 2.0 b The cellular content of IF3 was obtained by quantitative immunoblottingas described in the text. For each experiment, samples containing known

GROWTH RATE (p) amounts of pure IF3 were immunoblotted, and the values were used toconstruct a standard curve for the determination of IF3 in the crude ribosome

Transcription from PI, and P12 as a function of growth preparations.{i HB101 transformed with pSK5 (-), pSK8 (A), pSK9 C Values were calculated assuming an extinction coefficient for 70S ribo-K275-4 (0) was grown to the mid-log phase in LB, MGT somes of 24 pmol/A260 unit. The specific calculation was as follows: [A260 x4Gly. CAT and BLA activities were measured as de- (24 pmol/A260) x (6.02 x 10")J/viable count, where 6.02 x 1011 is Avogadro'she text. Results are plotted as the CAT/BLA ratio at each number (molecules per picomole).ietxt.esutsaepltte as he AT/BA raio t eah dValues were calculated with 20,537 as the molecular weight of IF3,relative to the CAT/BLA ratio observed at the slowest determined on the basis of the DNA sequence (27). The specific calculationThe latter values (CAT/BLA, x 1,000) were 192, 69, was as follows: [(ng of IF3/20,537) x (6.02 x 1014)]/viable count, where 6.02for pSK5, pSK8, pSK9, and pKK275-4, respectively. x io14 is Avogadro's number (molecules per nanomole).

The data are averages of six independent determinations, except forthose for MGly (two independent determinations).

copy number can vary over a fourfold range as a function ofgrowth rate, and BLA activity was an accurate indicator ofcopy number at all the growth rates tested. More impor-tantly, the growth rate responses of the rrnB PI promoterwere shown to be virtually identical when measured byCAT/BLA ratios and by actual transcript levels (17). Thus,CAT/BLA ratios accurately reflect promoter activities, evenunder conditions in which plasmid copy number may vary.The promoter fusion plasmids used in this study are shown

in Fig. 1. pSK8 contains PI1, pSK5 contains PI2, and pSK9contains both promoters. The promoter activities in HB101-transformed cells grown to the mid-log phase in LB weredetermined by a spectrophotometric assay of CAT and BLAactivities (measured in triplicate) in extracts prepared fromthese cells. For pSK8, pSK5, and pSK9, CAT/BLA ratios(x1,000) (averages of six separate experiments) were 9826, 196 + 24, and 311 + 24, respectively. These resultsindicate that PI2 is twice as active as is P1, under thesegrowth conditions, confirming earlier results obtained by Sinuclease mapping (30). The combination of transcriptionalactivities from both promoters (pSK9) is essentially the sumof the individual promoter transcriptional activities, suggest-ing that the region between the end of the PI1-containingfragment and the beginning ofPI2 does not contain additionalcontrol sequences which modulate the activity of P12 underthese conditions.HB101 cells harboring plasmids pSK5, pSK8, and pSK9

were grown to the mid-log phase in LB, MGT, MGlc, andMGly so as to achieve steady-state growth rates of between0.3 and 1.7 doublings per h. Transcriptional activities weremeasured in extracts prepared from these cells and were

expressed as CAT/BLA ratios. HB101 cells transformedwith plasmid pKK275-4 were used as a positive control. Thisplasmid contains the rrnB P1 promoter fused to cat. Theresults are presented in Fig. 2. Expression from P1, PI2, orthe combination of both promoters was essentially unaf-fected by variations in the steady-state growth rates of the

cells. In contrast, expression from rrnB PI varied signifi-cantly, increasing 4.5-fold at the fastest growth rate relativeto the slowest growth rate. The results demonstrate thattranscription from P1, and PI2 is not subject to growth rateregulation.To confirm the results obtained in promoter fusion studies,

we determined the steady-state levels of infC mRNA in E.coli B/r cells grown at different rates by dot blot hybridiza-tion with an infC-specific DNA probe. Relative infC mRNAlevels (determined by densitometry and reported in arbitrarydensitometry units) in cells grown in MGly, MGlc, MGT,and LB were 1.00, 0.85 + 0.13, 0.92 + 0.18, and 0.99 + 0.19,respectively (averages of four independent experiments); ineach experiment, the level of infC mRNA for each growthrate was normalized to that obtained for growth in MGly.The steady-state growth rates were 0.62, 1.1, 1.5, and 2.12doublings per h in MGly, MGlc, MGT, and LB, respectively.These results demonstrate that steady-state levels of infCmRNA do not vary appreciably during growth under avariety of different physiological conditions, suggesting thatneither the synthesis nor the stability of infC mRNA isdifferentially affected by variations in growth rate.Growth rate regulation of cellular IF3 levels. An earlier

study with two-dimensional gel analysis had shown that IF3synthesis was subject to metabolic regulation (15). In view ofthe results described above, which indicated that infC tran-scription is not growth rate regulated, the variations in thecellular levels of IF3 as a function of growth rate werereexamined by quantitative immunoblotting of crude ribo-somal fractions with a polyclonal antibody to IF3. E. coli B/rcells were grown at several different rates, and the totalribosome and IF3 contents per cell were determined. Theresults are presented in Table 1 and shown graphically inFig. 3. It is clear that the cellular levels of IF3 increase withincreasing growth rate. Furthermore, this increase parallelsthe increase in ribosome number in the cell (Fig. 3) such thatthe IF3/ribosome ratio is maintained at a relatively constantvalue of 0.11 to 0.17. These results are in agreement with

5.0 -

z0C')

-

wL

4.0 -

3.0 -

2.0 -

1.0

0.0

FIG. 2.rate. E. col(*), or pKIMGlc, or Ascribed in t}growth rategrowth rate237, and 92

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REGULATION OF IF3 BIOSYNTHESIS 3891

20000 -

16000

12000

8000-

04000-

0.0 0.5 1.0 1.5 2.0 2.5

GROWTH RATE (p)FIG. 3. Steady-state levels of ribosomes (-) and

function of growth rate. E. coli B/r was grown to theat the indicated rates, and the ribosome and IF3 coiwere determined as described in the text and Tableand d.

those of Howe and Hershey (15) and clearlythat cellular IF3 levels are subject to growth ratSince this control is not exerted at the level ofinitiation (Fig. 2) or by infC mRNA stability (sesteady-state concentrations of IF3 at all growtbe subject to posttranscriptional regulation.

DISCUSSION

A previous study had demonstrated that thepool of infC mRNA was derived by transcripticdifferent promoters, and S1 nuclease mappingthat PI2 was the major infC promoter (30). Istudy, quantitative assessment of the activities cinfC promoters was not done. The results prconfirm the earlier qualitative observation thaiactive than the other infC promoters, being twomore active than P1, at all growth rates tested. ]promoter fusion containing a fragment spannistream of P1, to a region downstream of P12 qscriptional activity that was approximately th4individual promoter activities, indicating that iacting sequence elements that modulate tranlocated in this area.The observation that a number of differer

could be used for the production of infC mRNthat these promoters may be differentially regaltered physiological conditions. Of particularthe response of the individual promoters togrowth at different rates. The number of ribos(in exponentially growing cells is roughly propogrowth rate (8, 18). The macromolecular nribosome requires that the gene products of t]protein and rRNA genes be coordinately expreaccomplished primarily by the regulation oftranscription and autogenous feedback regulasomal protein mRNA translation (16). The prnism for the regulation of rRNA gene transciknown, but Nomura and coworkers have suggecellular level of nontranslating ribosomes is Isignal which triggers growth rate control of rrn I

3000 16). In any event, the metabolic regulation of ribosomenumber is accomplished primarily through a transcriptionalcontrol mechanism. It was reported that the cellular levels of

2400 initiation factors are coordinately regulated with those of0 ribosomes such that the ratios are kept constant at a varietyI-

1800 m of growth rates (15). The results of the present promoterc: _ fusion experiments clearly demonstrate that expression fromm PI1 and PI2 is not under metabolic control. Furthermore, the

1200 cn steady-state levels of infC mRNA do not vary as a function° of growth rate, ruling out control by mRNA degradation.

In light of these results, it was important to reevaluate the600 ~- biosynthesis of IF3 as a function of the growth rate. Howeand Hershey (15) reported that IF2 and IF3 levels increased

0 coordinately with the level of ribosomal components as thegrowth rate was increased. This study was accomplished bya complex protocol involving the metabolic labeling of totalcellular protein with [35S]sulfate, two-dimensional gel elec-

IIF3 (A) as a trophoresis, and the measurement of radioactivity in individ-mid-log phase ual spots. In the present investigation, the amount of IF3 perntents per cell viable cell during exponential growth in a variety of media1, footnotes c was determined by quantitative immunoblotting. The results

in Fig. 3 definitively confirm those of the earlier study anddemonstrate that the cellular contents of IF3 and ribosomesincrease in parallel during growth in increasingly richermedia. Since the regulation of IF3 synthesis is not mani-

demonstrate fested at transcription initiation or by mRNA stability, thete regulation. control must be accomplished by a posttranscriptionaltranscription event.e above), the As already pointed out, infC-containing transcripts canh rates must also be produced by cotranscription from PT. The accumu-

lation of threonyl-tRNA synthetase is metabolically regu-lated, as is the case for all aminoacyl-tRNA synthetasesstudied (22). To date, quantitative measurements of PTactivity and the possible modulation of this activity as a

total cellular function of growth rate have not been reported. In the)n from three present study, plasmid fusions containing PT alone or ing established combination with PI, and PI2 were not analyzed. It isn the earlier therefore possible that the observed growth rate response off the various IF3 synthesis is modulated through expression from PTesented here Several facts make this unlikely. First, PI2 is by far the mostP12 is more active infC promoter at all growth rates (30). Second, the in

- to threefold vivo levels of infC mRNA are three- to fivefold higher thanIn addition, a are those of thrS mRNA, indicating that cotranscriptioning from up- from PT contributes a relatively small fraction of total infCshowed tran- transcripts (these differences are not due to enhanced stabil-e sum of the ity of the infC mRNA) (5). Finally, the reported increase inno other cis- threonyl-tRNA synthetase levels during growth in rich me-scription are dium is approximately 3.5-fold relative to that observed

during slow growth (22). This increase is about half thatit promoters shown here for IF3 (Fig. 3). Thus, while the precise mech-IA suggested anism governing the metabolic regulation of thrS is not,ulated under known, it is unlikely that regulation of transcription from PT,interest was even if it were to occur, would contribute to the largesteady-state changes in IF3 levels observed during growth at the slowestDmes present and fastest rates tested.rtional to the The coordinate expression of ribosomes and initiationature of the factors and the divergence in the molecular nature of thehe ribosomal growth rate response raise questions as to the mechanism for,ssed. This is achieving this coordinate expression. Gold et al. proposed arRNA gene model for the autogenous translational regulation of IF3tion of ribo- expression which is based on the unusual sequence of infCecise mecha- mRNA around the initiation codon (9, 10). The molecularription is not details of this regulatory event have not yet been fullysted that the elucidated; however, it is clear that this event is dependentthe feedback on the AUU initiation codon in infC (2, 4). Essentially, thepromoters (6, model predicts that high levels of cellular IF3 would prevent

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the translation of infC mRNA because IF3 itself causes thedissociation of mRNA-30S subunit complexes formed withmRNA containing poor translation initiation regions. Thiswould not occur with low IF3 concentrations; therefore,infC mRNA could be translated. Various aspects of thismodel have been supported by experiments (2, 4), and themodel is consistent with current notions regarding IF3 action(12, 13, 21). This model suggests a simple, passive mecha-nism for the coordinate regulation of IF3 and ribosomelevels. As growth rate increases, ribosome content is ele-vated by virtue of growth rate control of rrn gene transcrip-tion. This elevation leads to a transitory elevation in theribosome/IF3 ratio and a concomitant elevation in the trans-lation of infC mRNA by IF3-deficient ribosomes. Ulti-mately, the ribosome/IF3 ratio is reestablished (at approxi-mately 7:1) and the translation of infC mRNA is repressed.Thus, the key cellular parameter in the growth rate regula-tion of IF3 synthesis is the ribosome/IF3 ratio.The model predicts that any change which eliminates the

autoregulation of IF3 production (e.g., a mutation of theinitiation codon AUU to AUG) will also result in escapefrom growth rate regulation. Experiments to test this modelare currently in progress. Finally, it should be pointed outthat all three initiation factors appear to be coordinatelyregulated with growth rate (15). Studies indicate that inJBexpression is not autogenously regulated by its gene prod-uct, translation initiation factor IF2 (11, 23). The mechanismfor the growth rate control of infB expression has not beenelucidated, but on the basis of the model presented here forIF3 regulation, the expression of the two factors seems to becontrolled in different ways. Thus, at this stage it is not clearhow the cell achieves coordinate regulation of three initia-tion factors and couples this regulation to cellular ribosomelevels.

ACKNOWLEDGMENTS

We thank Jurgen Brosius for providing several of the plasmidsused in this study and Larry Gold for several helpful discussions.

This work was supported by Public Health Service grant GM-29265 from the National Institutes of Health.

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