Repression Is Relieved l3efore Attenuation in the trp Operon of

Transcription

1 JOURNAL OF BACTERIOLOGY, June 1984, p /84/ $02.00/0 Copyright 1984, American Society for Microbiology Vol. 158, No. 3 Repression Is Relieved l3efore Attenuation in the trp Operon of Escherichia coli as Tryptophan Starvation Becomes Increasingly Severe CHARLES YANOFSKY,* RICHARD L. KELLEY, AND VIRGINIA HORN Department of Biological Sciences, Stanford University, Stanford, California Received 10 January 1984/Accepted 21 March 1984 Expression of the tryptophan operon of Escherichia coli is regulated over about a 500- to 600-fold range by the combined action of repression and attenuation. Repression regulates transcription initiation in response to variation in the intracellular concentration of tryptophan. Attenuation regulates transcription termination at a site in the leader region of the operon in response to changes in the extent of charging of trnatit. We measured repression independently of attenuation to ascertain whether these regulatory mechanisms were used differentially by the bacterium as the severity of tryptophan starvation was increased. We found that repression regulated transcription of the operon over the range from growth with excess tryptophan to growth under moderate tryptophan starvation. By contrast, attenuation (termination control) was not relaxed until tryptophan starvation was in the moderate-to-severe range. Thus, attertuation and repression were used to regulate transcription in response to different degrees of tryptophan deprivation. Consistent with this conclusion is the observation that when tryptophan starvation was sufficient to relieve repression 50 to 60%, 65% of the trnatrp of the bacterium was charged. These fihdings provide a possible explanation for the existence of only two tryptophan codons in the coding region for the trp leader peptide of Enterobacteriaceae. Transcription of the tryptophan (trp) operon of Escherichia coli and other members of the Enterobacteriaceae is regulated by repression and attenuation (6, 24, 40). The combined action of these regulatory mechanisms permits expression of the operon to be varied over a 500- to 600-fold range, with repression and attenuation responsible for ca. a 80-fold and a 6- to 8-fold variation, respectively (2, 19). The two transcription regulatory mechanisms respond to sequentially related metabolic products: tryptophan and TrptRNATrP. Free tryptophan binds to the trp aporepressor to form the active trp repressor which then blocks transcription initiation at the trp promoter-operator (20). Charged trnatri is required for efficient translation of the leader peptide coding region (10, 43). Translation of this region allows formation of an RNA secondary structure that causes transcription termination at the trp attenuator (27, 32, 37, 45). When a cell is deficient in charged trnatrp, the ribosome translating the leader peptide coding region stalls over one of the Trp codons (7, 10, 43, 45). This stalling promotes formation of an alternate transcript secondary structure that prevents termination at the attenuator (27, 41, 45) Ȯperons concerned with the biosynthesis of Phe, His, Leu, Thr, Ile, and Val are regulated by attenuation in a manner nearly identical to attenuation control of the trp operon (1, 9, 12, 13, 15, 17, 21, 23, 25, 26, 30, 44). However, several of these operons, e.g. his, leu, thr, and ilv, do not appear to be regulated by repression (12, 14, 17, 21, 33). This possibility raises the following question: why do Enterobacteriaceae use two control mechanisms to regulate transcription of the trp operon if attenuation is sufficient as a regulatory mechanism for other amino acid biosynthetic operons? Three answers to this question have been considered. First, in E. coli, the trp repressor also regulates expression of aroh, the structural gene for one of three * Corresponding author isozymes that catalyze the initial reaction in the common pathway of aromatic amino acid biosynthesis (4, 16, 34, 46). It can be argued that in an ancestor of the Enterobacteriaceae, the trp operon was solely regulated by attenuation, and the aroh operon was regulated as it is now, exclusively by repression. The existence of a tryptophan-activated repressor within the cell then provided the selective opportunity for a segment of the trp promoter to evolve into an operator that could be regulated by the trp repressor. Perhaps the fact that the aroh and trp operon operators are at different locations in their respective promoters (16, 34, 46) implies that a particular segmnent of the ancestral trp operon promoter was moderately homologous to the aroh operator and therefore served as the locus for operator evolution. A second explanation for the existence of both repression and attehuation as transcription control mechanisrhs for the trp operon is that tryptophan and Trp-tRNATrP accumulation are sensed as signals of different metabolic events. Repression reflects changes in the intracellular tryptophan concentration. This is a function of the availability of tryptophan from the environment, the rate of tryptophan biosynthesis, and the rate of utilization of tryptophan in protein synthesis. Attenuation responds to changes in trna rp charging. This depends on the tryptophan pool concentration, the levels of trnatm and its cognate amino acyl-trna synthetase, and the overall rate of protein synthesis. Thus, it is reasonable to assume that repression is used primarily to sense the availability of tryptophan from the environment, whereas attenuation is used principally to respond to changes in the overall rate of protein synthesis. The existence of the two mnechanisins, therefore, would permit the cell to respond to the major external and internal events that are most relevant to regulation of trp operon expression. The third explanation, which is not exclusive of the other two, is that repression and attenuation play somewhat different roles in regulating trp operon transcription, i.e., their separate effects permit an expansion of the response of

2 VOL. 158, 1984 REPRESSION AND ATTENUATION IN E. COLI trp OPERON 1019 TABLE 1. Bacterial strains Strain Relevant genotype'a characteristics Relevant CY15050 trpr+ CY15051 trpr+ trpefbr Feedback-resistant trpe protein CY15052 trpr+ trpa234-ala trpa bradytroph (39, 42) CY15053 trpr+ trpa211-val trpa bradytroph (39, 42) CY15054 trpr+ trpl82 1 trp attenuator up mutation (36) CY15055 trpr+ trpl75 t trp attenuator down mutation (45) CY15056 trpr+ trpl82 t See CY15051 and trpefbr CY15054 CY15057 trpr+ trpl75s See CY15051 and trpefbr CY15055 CY15058 trpr Inactive repressor CY15059 trpr trpl82 T See CY15058 and CY15054 CY15060 trpr trpl75 r See CY15058 and CY15055 CY15061 trpr trpa234-ala See CY15058 and CY15052 CY15062 trpr trpa211-val See CY15058 and CY15053 "All strains are derivatives of W3110 tnaa2 AlacUJ69. ATL, trp-lac fusion phage (see text); all the strains employed are single lysogens. The trpefbr allele used is trpefbr-19 isolated in this laboratory. The trpa234- Ala and trpa2jj-val alleles have Gly - Ala and Gly -* Val replacements at positions 234 and 211, respectively, in the trpa polypeptide (39, 42). The trpr allele used is trpr2 isolated in this laboratory (see text). the operon as the severity of tryptophan starvation of a bacterial culture is increased. It is this latter possibility that is examined and supported by the present study. MATERIALS AND METHODS Strains. The bacterial strains used are listed in Table 1. These strains were constructed starting with the isogenic parents W3110 AlacU169 tnaa2 and W3110 trpr AlacUJ69 tnaa2 (24), each of which was lysogenized with the repression indicator phage XTL. The DNA used for XTL construction was derived from a trp plasmid containing the internal trp deletion AtrpLEJ413 (29, 31) which fuses codon 9 of the leader region to codon 338 of trpe, thus completely removing the attenuator. A 355-base-pair Sau3A restriction fragment containing this trppo"le" region was fused in frame to codon 8 of lacz in prlk15 (R. L. Kelley, unpublished data), itself a derivative of pmc931 (5). The resulting 8.7-kilobase Sal fragment carrying the trppo L-ElacZ Y fragment was ligated into the adjacent Sal sites of Xgt4 (8) to yield Lac+ recombinant phage hereafter referred to as XTL (Fig. 1). The recombinant phage XTL was used to prepare the single-copy lysogens, CY15050 and CY Low multiplicities of infection were used in lysogenization, and several lysogens of each type were picked and assayed. An occasional lysogen had double the lacz enzyme level; the others were assumed to be single lysogens. To be certain that CY15050 and CY15058 both had the same number of XTL genomes the trpr alleles were exchanged, and the resulting strains were assayed for P-galactosidase. The trpr allele used throughout, trpr2, is a nonsequenced frameshift mutant. Strains with this allele had trp enzyme levels indistinguishable from those of trpr amber and trpr deletion strains, indicating that the trpr2 allele did not specify a functional repressor. Media, growth conditions, and enzyme assays. The minimal medium of Vogel and Bonner (38) was used throughout and was supplemented with L-tryptophan (20 to 50,ug/ml) or acid-hydrolyzed casein (0.2%), as indicated, plus 0.2 to 0.3% glucose. All cultures were grown with shaking at 30 to 31 C. For enzyme assays, cultures were grown overnight and for several generations during the following day in supplemented or unsupplemented minimal medium. Generally, three cultures were grown with different dilutions of the same inoculum, and the culture which would reach the desired density (3 x 108 to 4 x 108 cells per ml) within 4 h was used. Cell samples were taken, processed, and assayed for I- galactosidase as described by Miller (28). Samples taken for anthranilate synthetase (trpe protein) assays were sedimented, washed with saline, sedimented, and frozen. The sedimented cells were permeabilized by Triton X-100 treatment and assayed for anthranilate synthetase as described previously (24). Labeling and quantifying trpe mrna and lac mrna. RNA was isolated from 10-ml cultures which had been pulse labeled with [3H]uridine for 1-min periods as described previously (43) and hybridized to denatured DNA of phages 4)80 trpe7 and A plac5 (35) immobilized on nitrocellulose filters (BA85; Schleicher & Schuell Co.). Hybridization was for 16 h at 66 C. The filters were washed, treated with ribonuclease, washed again, dried, and counted. Phage 4)80 trpe7 carries the initial segment of the trp operon up to and including about the first one-third of trpd. A 10-p.g amount of denatured DNA was immobilized on each 13-mm filter. Hybridizations were performed in duplicate, and the counts on control filters containing 4)80 or A c1857 DNA were subtracted. The cultures used for mrna labeling and isolation were also assayed for,-galactosidase and anthranilate synthetase activities. These cultures were grown at 31 C. Determining charged trnatfp. Cultures (500 ml) were grown with shaking at 31 C. The cultures were killed, and charging was instantaneously stopped by addition of warm trichloracetic acid and sodium dodecyl sulfate as described by Folk and Berg (11). RNA was isolated, and the extent of charging of trnatip was determined by periodate treatment (11). Highly purified tryptophanyl-trna synthetase generously provided by Karl Muench was used in the determination of the fraction of trnatrp that was charged (22). RESULTS Construction of a repression indicator. To measure repression of the trp operon of E. coli independently of attenuation, we constructed an operon-gene fusion in which the trp promoter-operator regulates,-galactosidase formation. In this construct, we fused the trp promoter-operator-leadertrpe region of deletion strain AtrpLE1413 (29, 31) to lacz at the BamHI site at codon 8 of lacz (5). Since the 1413 deletion fused the first nine codons for the trp leader peptide to a distal segment of trpe immediately preceding a BglII site, the fusion had five trpe codons between the first nine codons of the trp leader peptide coding region and the lacz coding region (Fig. 1). This entire fusion was introduced into K trp P/O trpl'- trpe'- lacz' 1 acy K AtrpLEl43 FIG. 1. Structure of the trp-lac gene fusion phage XTL. The AtrpLEJ413-lacZ gene fusion joins the first 9 codons of trpl to 5 codons of trpe and then to codon 8 of lacz.

3 1020 YANOFSKY, KELLEY, AND HORN phage lambda which was subsequently integrated in singlecopy form into the E. coli chromosome (see above). Chromosomal mutations were then introduced to give homologous strains with various mutations that influence expression of the operon (See Table 1). Using these strains, we measured the level of P-galatosidase produced from the X trp-lac fusion as an indicator of repression and the level of anthranilate synthetase produced from the resident trp operon as an indicator of the combined effects of repression and attenuation. The trpe and lacz enzyme activities of the trpr+ strain (CY15050) grown in minimal medium are 9 and 15%, respectively, of those of the repressor-deficient (trpr) reference strain (CY15058) (Table 2). The latter strain overproduced tryptophan and had identical enzyme levels when grown with or without added tryptophan. In trpr strains which have trpl mutations that decrease and increase termination at the attenuator, CY15059 and CY15060, trpe enzyme activity was increased or decreased without influencing P-galactosidase activity. These control analyses established that 3-galactosidase levels reflected repression at the trp operator, whereas trpe enzyme levels reflected the combined effects of repression and attenuation. Expression in tryptophan bradytrophs growing in minimal medium. The trpa234-ala and trpa2jj-val alleles specified defective but functional trpa proteins. Strains with the trpa234-ala mutation (such as CY15052) grow at the wildtype growth rate in minimal medium (39), but to do so they produce elevated trp enzyme levels that are 60 to 80% of the trpr level. Strains with the trpa211-val mutation (such as CY15053) grow slightly more slowly than wild type in minimal medium (39) and produce trp enzyme levels twofold higher than those of the trpr trp+ strain (see Table 2). When trpr strains with these altered trpa alleles (CY15061 and CY15062) were grown in the presence of tryptophan, the trpe and lacz enzyme levels were identical to those of the trpr trp+ reference strain (see Table 2). The lacz enzyme measurements in Table 2 demonstrated that the response of strain CY15052 [trpr+ trpa234-ala ] was largely due to repression relief, since the lacz enzyme level was 54% that of the reference strain and similar to the trpe enzyme level, which was 67%. Had attenuation been relieved as well, the trpe enzyme levels would have been much higher. The trpr+ trpa2jj-val strain (CY15053) showed complete repression relief (96% lacz level). The higher trpe enzyme levels in this strain indicate that attenuation relief contributed an additional twofold elevation in trp operon expression. These findings suggest that repression was relieved but attenuation was not relieved in the trpa234- Ala strain growing in minimal medium. Also relevant are the trpe and lacz enzyme levels of strain CY15050, the trpr+ control culture, grown in minimal medium. These levels were 9 and 15%, respectively, of those of the trpr reference strain (Table 2). This result indicates that attenuation (termination) was maximal in CY15050 growing in minimal medium. Note that the trpe and lacz enzyme levels of CY15050 are almost a factor of two apart relative to those of the comparable trpr strain. The explanation for this difference is unknown; however, it could not be due to attenuation because it was opposite to the direction of difference expected if attenuation were partially relaxed in CY The relative lacz mrna and trpe mrna levels in this strain were identical (Table 3). Comparison of enzyme and mrna levels. To verify that our results reflect changes in transcript levels, we compared trpe mrna and lacz mrna levels and trpe and lacz enzyme levels in four test strains and in the trpr reference J. BACTERIOL. TABLE 2. Effect of mild tryptophan starvation on repression and attenuation Enzyme levels (% of control) Strain Relevant genotype Medium' withb: trpe lacz CY15058 trpr Trp CY15059 trpr trpl82 t Trp CY15060 trpr trpl7s l Trp CY15050 trpr+ Min 9 15 CY15052 trpr+ trpa234-ala Min 67C 54C CY15053 trpr+ trpa211-val Min CY15061 trpr trpa234-ala Trp CY15062 trpr trpa211-val Trp Media: Trp, tryptophan; Min, minimal. b The values are the percent of the values obtained with the trpr control reference strain, CY15058, which were set at 100% (see the first line of table). C These values are the averages of eight cultures each. strain. It is evident that the wild-type strain CY15050 has trpe and lacz enzyme and mrna levels 10 to 20% that of the reference strain (Table 3). The trpa234-ala strain had comparable relative levels of lacz enzyme and mrna and trpe enzyme and mrna, but the latter values were slightly higher than the lac values, indicating that in these particular experiments there was slight relief of attenuation even though repression was not totally relieved. In the trpa211- Val strain lacz enzyme and mrna levels were identical to those of the reference strain and, as expected, both the trpe enzyme and mrna levels were twofold higher than the reference strain. The twofold increase in expression therefore must be due to partial attenuation relief superimposed on the absence of repression. In the repressor-deficient strain trpr trpl82 (CY15059) which has an attenuator alteration, the trpe enzyme and mrna levels were circa threefold higher than those of the reference strain. These findings established that the increase in trp operon expression observed in the trpa234-ala bradytroph was mainly due to repression relief. Nutritional and genetic effects on expression. To extend these comparative measurements of repression and attenuation over a greater range of levels of trp operon expression, we measured trpe and lacz enzyme activities in a variety of strains both in minimal medium and under mild tryptophan starvation conditions imposed by growth in a tryptophanfree, acid-hydrolyzed casein medium (Table 4). It was apparent that the trpe and lacz enzyme levels increased in parallel until severe starvation was imposed by growing the trpa234- Ala strain in casein hydrolysate. Note that strains with a feedback-resistant trpe protein produced lower enzyme levels than their feedback-sensitive homologs. As has been shown by Bliss et al. (3), lower trp enzyme levels are needed for maximum growth rates in cultures which produced a feedback-resistant trpe protein (Table 4). When a trpe feedback-resistant allele was combined with either attenuator up (L82) or attenuator down (L75) mutations (strains CY15056 and CY15057, respectively), trpe and lacz enzyme levels were both lower than in the feedback-sensitive homologs (strains CY15054 and CY15055) (Table 4). However, the

4 VOL. 158, 1984 REPRESSION AND ATTENUATION IN E. COLI trp OPERON 1021 TABLE 3. Determination of enzyme and mrna levels in two bradytrophs % of trpr control of the followingb: Strain genotype Mediuma lacz lacz mrna trpe trpe mrna activity levels activity levels CY15058 trpr Trp CY15050 trpr+ Min CY15052 trpr' Min trpa234- Ala CY15053 trpr+ Min trpa211- Val CY15059 trpr trpl82 t Trp a Media: Trp, tryptophan; Min, minimal. b Each value is the average of four cultures; the average of the values obtained with strain CY15058 was set at 100%o. relative trpe and lacz levels were up or down depending on whether the L82 or L75 allele was present (Table 4). These latter relative values are extremely important; they establish that trpe and lacz enzyme levels deviated from those of the reference strain when attenuation was affected. trnatfp charging in a Trp bradytroph. Since attenuation was not relieved in the trpa234-ala bradytroph CY15052, it seems likely that moderate tryptophan starvation did not deplete the pool of charged trnatrp. To explore this possibility directly, we determined the fraction of trnatnp that was charged in growing cultures of the trpa234-ala bradytroph, by the procedure of Folk and Berg (11). As controls, charging was determined in a trpr+ wild-type strain (CY15050) and a trpr prototroph (CY15059). It was found that in strains CY15050 and CY15059 grown in minimal and minimal-plus-tryptophan medium, respectively, 105 and 83% of the trnatrp was charged. In the bradytroph CY15052 grown in minimal medium 65% of the trnatrp was charged. These findings establish that under conditions where repression of the trp operon was relieved 55% (Tables 2 and 3), 65% of the trnatrp was charged. This finding explains the observation that attenuation is not relieved under such conditions. The fact that the trnatrp of the trpr+ culture (CY15050) was fully charged is also relevant. In minimal medium, the trp enzyme levels of such cultures are about 10- fold higher than the levels observed in cultures growing with excess tryptophan (19). Thus, repression was relieved about 10 to 15% under conditions in which the trnatrp of the culture was fully charged and attenuation was maximal. DISCUSSION Both repression and attenuation are used to regulate transcription of the trp operon of E. coli. Although the signals sensed by these regulatory mechanisms, tryptophan and charged trnatrp, are metabolically sequentially related, it did not follow that the extent of trnatrp charging varied directly with the intracellular tryptophan concentration. Thus, as the tryptophan concentration dropped within the cell at the onset of starvation, tryptophanyl-trna synthetase activity might still be adequate to charge much of the cellular trnatrp. If this were the case, then attenuation (termination) in the trp operon would not be relaxed until tryptophan starvation was severe. We examined this possibility by analyzing the effects of tryptophan starvation on repression and attenuation. This was accomplished by measuring enzyme and mrna levels in lysogenic strains containing an engineered lambda repression indicator. This indicator phage carries a trp promoter-operator trp-lacz gene fusion. In lysogens containing this phage, P-galactosidase activity and lacz mrna levels reflected exclusively trp repressor control at the trp operator of the trp-lac fusion. In parallel with P-galactosidase measurements, we determined trpe enzymatic activity or trpe mrna levels or both, products of the resident trp operon, to assess the combined regulatory contributions of repression and attenuation. We then calculated the effect of attenuation by subtracting the observed effect of repression. The values obtained by using these observations and calculations are presented in Tables 5 and 6. The values in Table 5 are idealized values based on numerous determinations with control cultures. These values show that repression regulated expression of the operon over about an 80-fold range and attenuation contributed an additional 6-fold. Mutations that alter repression (trpr) or attenuation (trpl) increased or decreased operon expression accordingly. Addition of the tryptophan analog indole-3- acrylic acid relieved repression and termination. The values in Table 6 are the averages of those determined experimentally in this study. The contribution of attenuation was calculated from the observed lacz (repression) and trpe TABLE 4. Attenuation relief as a function of tryptophan limitation and feedback sensitivity Enzyme levels (% of control) Strain Relevant genotype Mediuma withb: trpe lacz CY15051 trpr+ trpefbr Min 3 3 CY15050 trpr+ (ATL) Min 8 17 CY15051 trpr+ trpefbr ACHa CY15050 trpr+ ACH CY15052 trpr+ trpa234-ala Min CY15052 trpr+ trpa234-ala ACH CY15056 trpr+ trpl82 Min 8 <2 trpefbr CY15054 trpr+ trpl82 Min 15 4 CY15057 trpr+ trpl75 Min 4 10 trpefbr CY15055 trpr+ trpl75 Min 8 33 a Media: Min, minimal; ACH, acid hydrolyzed casein. b Values for the control strain trp were set at 100%.

5 1022 YANOFSKY, KELLEY, AND HORN TABLE 5. Idealized reference control valuesa Relevant *% Attenuation Expression genotype Mediumb Repression (fold (% of trpr relief' multiplier) level) trpr Min or Trp trpr+ Trp trpr trpl Trp trpr+ trpl Trp trpr trpl I Trp trpr+ trpl l Trp trpr +1- IA a % Repression relief x attenuation = expression. A value of 1 is taken as the extent of read-through transcription in trpr and trpr+ cultures growing in a tryptophan-containing medium. We estimate that a value of 1 corresponds to one molecule of polymerase reading through the attenuator for every four to six molecules that terminate transcription. b Media: Min, minimal; Trp, tryptophan; IA, indole-3-acrylic acid. ' This value is lacz or trpe enzyme level in test strain relative to level in a trpr strain (The 3-galactosidase and trpe enzyme levels of the trpr strain are arbitrarily set at 100). (repression plus attenuation) enzyme activities. It is apparent that when there was greater than 50% repression, i.e., lacz activity was less than 50%, termination at the attenuator was maximum. Only when repression was essentially eliminated, presumably owing to an appreciably reduced intracellular tryptophan concentration, was there significant relaxation of attenuation. Consistent with this observation is the finding that trnatrp was 65% charged in cultures in which repression was 55% relieved. Also consistent with these observations is the fact that the growth rate of various trp bradytrophs in minimal medium is identical to that of the wild-type strain until trp enzyme levels approach or exceed those of a repressor-deficient (trpr) strain (39). Apparently, trnatrp charging did not become growth limiting until the intracellular tryptophan concentration was so low that it was inadequate to activate the trp aporepressor. These findings and considerations therefore suggest that repression and attenuation come into play independently in regulating trp operon transcription. This conclusion has been anticipated on the basis of rigorous considerations of growth rate J. BACTERIOL. balance as affected by amino acid deprivation and operon regulation (18). Our findings with feedback-resistant strains support the conclusion of Bliss et al. (3) that in a minimal medium trp operon expression is generally elevated because the feedback-sensitive trpe protein is partially inhibited by tryptophan. When we compared any pair of trpr strains producing feedback-sensitive or -resistant trpe proteins, operon expression was invariably lower-but obviously sufficientwhen the trpe protein was feedback resistant. Thus, although E. coli employs two mechanisms to control trp operon transcription, thereby implying that economy of RNA and protein synthesis is important to the bacterium, it produces double the level of trp polypeptides that it needs so that feedback control can be employed to regulate the flow of carbon into the tryptophan pathway. The conclusion that attenuation is used to regulate trp operon expression in response to severe tryptophan starvation has implications for the organization of the trp operon leader region. It has been noted that the leader peptide coding region of the trp operon is atypical in comparison with peptide coding regions of other amino acid biosynthetic operons (25, 40a). Only two Trp codons are present in the trp coding region, whereas in other amino acid biosynthetic operons regulated by attenuation there are four or more regulatory codons. Comparative studies with seven species of Enterobacteriaceae have shown that the two-trp-codon arrangement is conserved (40a) and therefore presumably is functionally significant. The two-codon arrangement can explain the finding that attenuation regulated trp operon expression primarily under severe tryptophan starvation. Only when an appreciable fraction of the cellular trnatrp was uncharged would the translating ribosome in the leader peptide coding region be expected to dwell for an extended period over one of the Trp codons. Thus the two-codon arrangement may determine that attenuation was relaxed only upon severe tryptophan starvation. In other operons regulated by attenuation, particularly in those regulated by attenuation alone, a slight reduction in charging of the appropriate trna(s) would have a profound effect because a TABLE 6. Observed and calculated values Strain Relevant Mediuma Observed Attenuation genotype lacz activity multiplier Observed trpe activity CY15051 trpr+ trpefbr Min CY15050 trpr+ Min (?) 8 CY15052 trpr+ trpa234- Min Ala CY15053 trpr+ trpa2jj- Min Val CY15051 trpr+ trpefbr ACH CY15050 trpr+ ACH CY15052 trpr+ trpa234- ACH Ala CY15054 trpr+ trpl82 Min (ATL) CY15055 trpr+ trpl75 Min (ATL) Cy15056 trpr+ trpl82 T Min <2 >4 8 trpefbr CY15057 trpr+ trpl75 l Min trpefbr (ATL) a Media: Min, minimal; ACH, acid-hydrolyzed casein.

6 VOL. is8, 1984 REPRESSION AND ATTENUATION IN E. COLI trp OPERON 1023 modest translational delay at each regulatory codon would be amplified by a comparable delay at each succeeding codon (18). Thus, the two-trp-codon arrangement in the trp operon may represent an optimal accommodation to physiological need. ACKNOWLEDGMENTS We are indebted to Valley Stewart for comments on the manuscript. The studies described in this paper were supported by a grant from the National Science Foundation (PCM ) and the American Heart Association (69-015). C.Y. is a Career Investigator of the American Heart Association, and R.L.K. is a predoctoral trainee of the U.S. Public Health Service. LITERATURE CITED 1. Bauer, C. E., J. Carey, L. Kasper, S. Lynn, D. Waechter, and J. Gardner Attenuation in "prokaryotic gene expression", p Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 2. Bertrand, K., and C. 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