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Internal promoter of the tryptophan operon of Escherichia coli is located in a structural gene
J. Mol. Bid. (1972) 69, 307313
Internal Promoter of the Tryptophan Operon of Escherichia cdi is Located in a Structural Gene The constitutive low-efficiency promoter site (P.J near the middle of the tryptophan operon of Eacherichb co2i has been mapped by analysis of short deletions internal to the trp operon. Comparison of deletions which remove this internel psomoter with those which retain it show that Pa is located within trpD, the region coding for phosphoribosyl anthranilate transferase. Pa maps near the operatordistal end of trpD, on the operator-proximal side of two trpD point mutants. Comparisons of strains with and without the P2 site indicate that initiations at this promoter are responsible for synthesis of 80% of the trpC, trpB and trpA polypeptides present in repressed cells.
The tryptophan operon in both Salwwnellu typhimurium and Escherichia wli contains five contiguous structural geneswhich are transcribed into a polycistronic messenger RNA (Yanofsky, 1967). The map of the E. coli operon shown in Figure l(a), showsthe order of the genesthe enzymes which they specify, and the reactions directed by the gene products. The order of the genes and the reactions catalyzed are the same in S. typhimurium and in E. coli although the letter designationsfor the genesare different. The letter designations we will use throughout are those employed for the E. coli trp genes. Synthesis of polycistronic trp mRNA is subject to repressionwhich is dependent on excess tryptophan, the presence of the product of the tryptophan repressor gene (tqpR), and an intact trp operator region (Yanofsky, 1971). When the operon is derepressed,transcription begins at the E gene end of the operon and proceeds sequentially along the operon, and results in the co-ordinate synthesis of the five tryptophan biosynthetic proteins (Yanofsky, 1967). The presence of a constitutive low-eficiency promoter site, termed P,, near the middle of the S. typhimurium trp operon, at which transcription can also begin, was demonstrated by Bauerle & Margolin (1966, 1967) and Margolin & Bauerle (1966). Morse & Yanofsky (1968) subsequently inferred the presenceof an analogousP, in the trp operon of E. wli. This promoter results in low-level, non-repressible synthesis of the trpC, trpB and trpA gene products. The level of this P,-initiated synthesis is about 3% of the derepressedlevel and so doesnot contribute significantly to it. P, is presumably located somewherebefore trpC, the genefor indoleglycerol phosphate synthetase, and, in S. tryphimurium, it is between the ends of two deletions, one ending late in the PRAt transferase gene, trpD, and one ending early in the IGP synthetase gene, trpC (Bauerle & Margolin, 1967). No physiological function has been suggestedfor P,. Direct proof for the presenceof P, in E. wli is provided by the studies described here. Comparisonsof short deletions internal to the trp operon which remove P, with 7 Abbreviations used: PRA transferase, phosphoribosyl glycerol phosphate. 307 21
anthranilate
transferam;
IGP, indole
308
E.
N.
JACKSON
AND
C.
YANOFSKY
those which retain it show that P, is located within the region coding for PRA transferase. This location raises the possibility that P, may serve no useful function but may exist simply as a consequence of the presence of a nucleotide sequence necessary to code for an essential region of a functional enzyme. The deletions studied are shown diagrammatically in Figure l(b). The specific activities of tryptophan synthetase A and B for each deletion strain are shown in Table 1. The presence or absence of P, should be revealed by the tryptophan synthetase A and B derepression ratios (the ratio of tryptophan synthetase activity in a tryRculture to the activity in a repressed trpR + culture), since deletion of P, should significantly decrease the repressed synthesis of tryptophan synthetase A and B but should not detectably alter the derepressed synthesis of these polypeptides. Thus deletion of Nucleotide porn CyS8 ___.._-__
y
1000
2000 :
3000 --t----trp D
@O
IrrpE
4000 5000 +--t------l fr,C
6OCC
trp5
,+?!+.~
fOr7B
I I i
Phosphorlbosyl anthranllate IsomeroseAnthronilote synthetose
Phosphorlbosyl anthrmlate transferase
lndole gl cerol phosp Ylate synthetose
/3
Tryptophan ’ synthetase
(0)
(b)
FILL 1. (a) The tryptophan operon of E. coli. The length of the structuml genes is drawn to scale. (b) Genetic map of trp internal deletions. The length of the tquE and tqwD genes and the distribution of point mutant sites (shown by dashed vertical lines) is to scale (Yanofsky et al., 1971). The deletions were mapped by recombination with the tqn point mutants shown as described elsewhere (Jackson & Yanofsky, manuscript in preparation). A horizontal bar on the map represents the extent of the deletion. A dotted extension to a horizontal bar indicates that the extent of the deletion in that region of the map has not been defined. A vertical bar at the end of a dotted extension de6nes the maximum extent of the deletion.
+ + + + + + + + +
trpR trpR
trpR trpR
trpR trpR
trpR trpR
trpR trpR
trpR trpR
trpR trpR
trpR trpR
trpR trpR
trpADClO3
trpADC82
trpAED21
trpAED24
trpD1383
trpD1431
trpADCl81
trpAED53
trpADC104
trpR allele +
or
trpR trpR
deletion mutant
(0.015 -
-
<0.015 -
-
-
-
-
<0.015 -
(0.015 -
2.3 0.031
PRATase @pD)
levels in trpR-
Wild type W-P + 1
Internal trp point
Enzyme
1
5.8 0.08
11 0.15
9.0 0.12
9.2 0.68
9.8 0.68
13 0.61
17 0.66
5.0 0.18
11 0.78
11-14 0.5-0.7
6.3 0.12
13 0.19
10 0.16
12 0.96
14 0.91
19 0.96
21 0.72
5.4 0.24
11 0.80
12-15 O.CO.8
TSase (WA)
A
55
83
a5
80
85
100
100
45
100
100
53
88
79
90
100
100
100
42
100
100
o/c of trp+ enzyme levelf: T&we B T&se A
trpR+ cells grown in the presence of tryptophm
Specific aotivityt 11 TSase B VvB)
and
TABLE
73
73
75
14
14
21
26
53
69
63
13
15
20
29
22
14
14 28
15-25
16-28
Dorepression ratio 0 T&se T&se B
A
l-continued
t Specific activity is units of enzyme activity per mg of protein (Creighton & Yanofsky, 1970). $ Per cent of wild-type activity is calculated relative to a wild-type culture grown in parallel st the same time. $ The derepression ratio for a given pair of trpR + and trpRisogenic strains is the ratio of the enzyme specific activity in the trpRthat in the trpR + background. 11Abbreviations used PRATase, phosphoribosyl anthranilate transfcrase; T&se A, tryptophsn synthetase a; T&se B, tryptophan
to &.
background synthetase
In order to compare the function of a trp operon containing. sn internal deletion when repressed and derepressed, each tTp operon internal deletion was transferred by bacteriophage Plkc transduction into the same pair of strains which me isogenic except at the trpR locus (trpR+trpB9579oc tnuor trpR-trpR9579oc tna-). Recombinants were selected which carried the trp intern& deletion, were trpB+ trpA +tnoand were either trpR+ or trpR-. The same trpRallele was used throughout these studies; it results in constitutive synthesis of all trp operon polypcptides in the presence of excess tryptophan. The t7ta- mutation results in inability to synthesize tryptophanase. The tna- mutation in all strains allows accurate measurements of low levels of tryptophan synthetase activity without errors introduced by the activity of tryptophanase. Each trpR+ and trpRisogenio pair carrying a given trp internal deletion wss grown with vigorous aeration in excess tryptophan, harvested during logarithmic growth, sonic&ted, and assayed for tryptophan synthetase (T&se) B and A and PRA transferam activities as described elsewhere (Jackson & Yanofsky, manuscript in preparation). We term derepreseed the level of trp operon enzymes measured in trpRstrains grown in the presence of excess tryptophsn, and repressed the level of trp operon enzymes found in trpR + strains grown under identical conditions. Protein concentration was determined by the method of Lowry, Rosebrough, Farr & Randall (1961). We consider that deletions in strains having derepressed T&se levels equal to or greater than 80% of the trp+ control are not significantly polar.
trpAEClO1
+-
trpAEC81
trpR trpR
trpR trpR+
trpADC71
TD~
LETTERS
TO
THE
EDITOR
311
P, will lead to a derepression ratio higher than that found in the wild type. Therefore, the derepression ratio should show whether a given deletion removes P,, and location of the deletion termini on the genetic map of the operon can be used to gain information about the location of P,. Inspection of the derepression ratios in Table 1 shows that the deletion strains fall into two distinct classes. Deletion strains trpADC103, trpADC82, trpAED21, and trpAED24 have derepression ratios in the range 14 to 30, as does the trp+ control which contains P,. These deletion strains therefore retain normal P, function. The second class, trpADCl81, trpAED53, trpADC104, trpADC71, trpAEC81 and trpAEC101 have derepression ratios in the range 70 to 90. We expect deletion of P, to increase the derepression ratio, since P, function adds to the tryptophan synthetase B and A levels measured in the repressed trpR + background but does not contribute significantly to the trpR- tryptophan synthetase B and A levels. Thus, we conclude that all the strains having a derepression ratio for tryptophan synthetase B and A greater than 70 carry deletions which cover P,. Indeed, we would predict that deletions trpAEC81 and trpAEClO1 must remove P, since they both delete all of trpD and most of trpC. We know that P, must precede trpC but not trpD, since IGP synthetase but not PRA transferase synthesis can originate at P,. Furthermore, by analogy with the data from Salmonella, deletion mutants trpAEC81 and trpAEC101 should lack Pp. Deletion mutants trpAEC81 and trpAEClO1 have derepression ratios in the range 70 to 90, thus contlrming our conclusion that a derepression ratio in this range is characteristic of a deletion which removes the P, site. We can now use these deletions to locate precisely the P, site in the trp operon. Deletions trpADClO3 and trpADC82 both remove a portion of trpD since neither corresponding strain has PRA transfersse activity (see Table 1). Deletion mutant trpADClO3 fails to recombine with the last known trpD point mutant, trpD1537, but recombines with all other trpD point mutants, while deletion mutant trpADC82 recombines with all known trpD point mutants (see Fig. l(b)). Yet deletion strains trpADClO3 and trpADC82 both have P, function as judged by their derepression ratios. Therefore the P, site must lie within trpD. Inspection of the properties of strain trpAED53 reinforce this conclusion. Strain trpAED53 recombines with the last two tvpD point mutants known, and so retains a small segment of trpD. Since trpAED53 deletes P,, the P, site occurs before the end of .trpD. The locations of the deletions on the genetic map (see Fig. l(b)) a 11ow us to conclude that the P, site maps to the left of the altered site in trpD1537 (since trpADClO3 deletes trpD1537 but not Pa) and to the right of the mutation in trpD1300 (since trpAED21 and trpAED24 delete trpD1300 but not Pa). That trpADCl81 covers P, but does not delete the trpD1300 site is further evidence that P, is on the C gene side of the trpD1300 site. In addition, Pa must be located to the left of the mutational site in trpD1431 because trpAED53 deletes Pz but not the tvD1431 site. Thus PI must be located between the altered sites in the trpD point mutants trpD1300 and trpD1431. There is one known trpD point mutant, trpD1383, which maps in the region in which we have shown Pa is located. trpD1383 was transduced into the same isogenio trpR+ or trpR- tna- backgrounds as the internal deletions and assayed for tryptophan synthetase B and A (Table 1). Values for trpD1431, a trpD point mutant mapping very close to trpD1383, are given for comparison. The derepression ratios obtained with trpD1383 are 14 to 16 (see Table l), indicating that trpD1383 has a functional P, site. Scrutiny of the trpR- levels of tryptophan synthetase A and B shown in Table 1
312
E.
N.
JACKSON
AND
C. YANOFSKY
reveals that some of the deletion strains, such as trpR- trpADC82, trpR- trpADC71, and trpR- trpADC104 have less tryptophan synthetase A and B than the isogenic trpR- trp+ control strain (see Table 1, per cent of trp + enzyme level). We believe that the fusions resulting from these deletions end out of phase for proper translation, thus bringing a nonsense codon into phase just past the deletion terminus in trpC and resulting in a polar effect on tryptophan synthetase B and A synthesis. The amount of tryptophan synthetase B and A measured in each of these three trpR- deletion strains is about the same as observed in a trpR- strain carrying a known trpC nonsense point mutation at the position in trpC where the three deletions end (Jackson & Yanofsky, manuscript in preparation). Since the nonsense codon would be located after the P, site, polarity would decrease P,-governed and operator-governed synthesis of tryptophan synthetase B and A equally. Therefore, the presence of a nonsense codon to the right of P, would reduce the levels of tryptophan synthetase B and A activity observed but should have no effect upon the derepression ratio calculated by comparing levels in trpR+ and trpR- backgrounds. This conclusion is justified by the finding that the derepression ratio for one of the polar deletion strains, trpADC82, is in the range characteristic of strains having P,, while two of the polar deletion strains, trpADC71 and trpADC104 have derepression ratios in the 70 to 90 range, indicating that P, has been deleted. Prom our data it is now possible to estimate what fraction of the trpR+ trp+ repressed levels of tryptophan synthetase B and A in E. coli is due to transcription initiations at P,. The tryptophan synthetase B and A levels in non-polar deletion strains trpADC181, trpAED53, trpAEC81, and trpAEClO1 represent the amount of these enzymes made under repressed conditions in the absence of P,. This level is about 20 to 30% of the repressed trpR+ trp+ level. Thus P, in the wild type accounts for about 70 to 80% of the tryptophan synthetase B and A seen under repressed conditions. This is exactly the P,-initiated level of synthesis seen in S. typhimurium (Bauerle & Margolin, 1967 ; Margolin & Bauerle, 1966). Our studies therefore establish that a nucleotide sequence near the trpC end of trpD functions both to code for a sequence of amino acids in the carboxy-terminal region of the trpD polypeptide and to promote initiation of transcription. It follows that P, may not have a physiologically important function as a transcription initiatior but may exist simply as a consequence of the presence of a nucleotide sequence essential for functional PRA transferase. In this regard it is of interest that promoter-like initiator sequences have been induced by 2-aminopurine treatment in the first gene of the trp operon of S. typhimurium and one such initiator sequence does not interfere with the production of a functional gene product (Wuesthoff & Bauerle, 1970). In view of the genetic location of P, it should be possible to select point mutants in trpD which have altered P, function and lack active PRA transferase. The one known tqwD point mutant, trpD1383, which maps in the region where P, is located, has normal P, function. However, experiments to select point mutations in trpD which alter P, function are in progress. If several such mutants of the missense type were available, it might be possible to deduce the nucleotide sequence of the P, region from the ammo-acid sequence in the corresponding segment of the trpD polypeptide. The authors are indebted to Virginia Horn and Miriam Banner for their assistance. The studies described in this paper were supported by grants from the National Sciences Foundation (GB6790), the U.S. Public Health Service (GM-09738) and the American Heart Association.
LETTERS
One of us (E. N. J.) is a predoctoral author (C. Y.) is a Career Investigator Department of Biological Sciences Stanford University Stanford, Calif. 94305, U.S.A. Received
28 February
TO
THE
EDITOR
313
trainee of the U.S. Public Health Service. The other of the American Heart Association. E.N. JACKSON C.YANOFSKY
1972
REFERENCES Bauerle, R. H. & Margolm, P. (1966). Proc. Nat. Acad. Sci., Wash. 56, 111. Bauerle, R. H. & Margolin, P. (1967). J. Mol. Biol. 26, 423. Creighton, T. E. & Yanofsky, C. (1970). In Methods irz Enzymology, ed. by S. P. Colowick & N. 0. Kaplan, vol. l?A, p. 365. New York: Academic Press. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). J. Biol. Chem. 193, 265. Margolin, P. & Bauerle, R. H. (1966). CoZd Spr. Ha&. Syv. Quunt. Biol. 31, 311. Morse, D. E. & Yanofsky, C. (1968). J. Mol. Biol. 38, 447. Wuesthoff, B. & Bauerle, R. H. (1970). J. Mol. BioZ. 49, 171. Yanofsky, C. (1967). Harerey Lect. 61, 146. Yanofsky, C. (1971). J. Amer. Med. Aseoc. 218, 1026. Yanofsky, C., Horn, V., Bronner, M. L Stasiowski, S. (1971). Genetics, 69, 409.
Internal Promoter of the Tryptophan Operon of Escherichia cdi is Located in a Structural Gene The constitutive low-efficiency promoter site (P.J near the middle of the tryptophan operon of Eacherichb co2i has been mapped by analysis of short deletions internal to the trp operon. Comparison of deletions which remove this internel psomoter with those which retain it show that Pa is located within trpD, the region coding for phosphoribosyl anthranilate transferase. Pa maps near the operatordistal end of trpD, on the operator-proximal side of two trpD point mutants. Comparisons of strains with and without the P2 site indicate that initiations at this promoter are responsible for synthesis of 80% of the trpC, trpB and trpA polypeptides present in repressed cells.
The tryptophan operon in both Salwwnellu typhimurium and Escherichia wli contains five contiguous structural geneswhich are transcribed into a polycistronic messenger RNA (Yanofsky, 1967). The map of the E. coli operon shown in Figure l(a), showsthe order of the genesthe enzymes which they specify, and the reactions directed by the gene products. The order of the genes and the reactions catalyzed are the same in S. typhimurium and in E. coli although the letter designationsfor the genesare different. The letter designations we will use throughout are those employed for the E. coli trp genes. Synthesis of polycistronic trp mRNA is subject to repressionwhich is dependent on excess tryptophan, the presence of the product of the tryptophan repressor gene (tqpR), and an intact trp operator region (Yanofsky, 1971). When the operon is derepressed,transcription begins at the E gene end of the operon and proceeds sequentially along the operon, and results in the co-ordinate synthesis of the five tryptophan biosynthetic proteins (Yanofsky, 1967). The presence of a constitutive low-eficiency promoter site, termed P,, near the middle of the S. typhimurium trp operon, at which transcription can also begin, was demonstrated by Bauerle & Margolin (1966, 1967) and Margolin & Bauerle (1966). Morse & Yanofsky (1968) subsequently inferred the presenceof an analogousP, in the trp operon of E. wli. This promoter results in low-level, non-repressible synthesis of the trpC, trpB and trpA gene products. The level of this P,-initiated synthesis is about 3% of the derepressedlevel and so doesnot contribute significantly to it. P, is presumably located somewherebefore trpC, the genefor indoleglycerol phosphate synthetase, and, in S. tryphimurium, it is between the ends of two deletions, one ending late in the PRAt transferase gene, trpD, and one ending early in the IGP synthetase gene, trpC (Bauerle & Margolin, 1967). No physiological function has been suggestedfor P,. Direct proof for the presenceof P, in E. wli is provided by the studies described here. Comparisonsof short deletions internal to the trp operon which remove P, with 7 Abbreviations used: PRA transferase, phosphoribosyl glycerol phosphate. 307 21
anthranilate
transferam;
IGP, indole
308
E.
N.
JACKSON
AND
C.
YANOFSKY
those which retain it show that P, is located within the region coding for PRA transferase. This location raises the possibility that P, may serve no useful function but may exist simply as a consequence of the presence of a nucleotide sequence necessary to code for an essential region of a functional enzyme. The deletions studied are shown diagrammatically in Figure l(b). The specific activities of tryptophan synthetase A and B for each deletion strain are shown in Table 1. The presence or absence of P, should be revealed by the tryptophan synthetase A and B derepression ratios (the ratio of tryptophan synthetase activity in a tryRculture to the activity in a repressed trpR + culture), since deletion of P, should significantly decrease the repressed synthesis of tryptophan synthetase A and B but should not detectably alter the derepressed synthesis of these polypeptides. Thus deletion of Nucleotide porn CyS8 ___.._-__
y
1000
2000 :
3000 --t----trp D
@O
IrrpE
4000 5000 +--t------l fr,C
6OCC
trp5
,+?!+.~
fOr7B
I I i
Phosphorlbosyl anthranllate IsomeroseAnthronilote synthetose
Phosphorlbosyl anthrmlate transferase
lndole gl cerol phosp Ylate synthetose
/3
Tryptophan ’ synthetase
(0)
(b)
FILL 1. (a) The tryptophan operon of E. coli. The length of the structuml genes is drawn to scale. (b) Genetic map of trp internal deletions. The length of the tquE and tqwD genes and the distribution of point mutant sites (shown by dashed vertical lines) is to scale (Yanofsky et al., 1971). The deletions were mapped by recombination with the tqn point mutants shown as described elsewhere (Jackson & Yanofsky, manuscript in preparation). A horizontal bar on the map represents the extent of the deletion. A dotted extension to a horizontal bar indicates that the extent of the deletion in that region of the map has not been defined. A vertical bar at the end of a dotted extension de6nes the maximum extent of the deletion.
+ + + + + + + + +
trpR trpR
trpR trpR
trpR trpR
trpR trpR
trpR trpR
trpR trpR
trpR trpR
trpR trpR
trpR trpR
trpADClO3
trpADC82
trpAED21
trpAED24
trpD1383
trpD1431
trpADCl81
trpAED53
trpADC104
trpR allele +
or
trpR trpR
deletion mutant
(0.015 -
-
<0.015 -
-
-
-
-
<0.015 -
(0.015 -
2.3 0.031
PRATase @pD)
levels in trpR-
Wild type W-P + 1
Internal trp point
Enzyme
1
5.8 0.08
11 0.15
9.0 0.12
9.2 0.68
9.8 0.68
13 0.61
17 0.66
5.0 0.18
11 0.78
11-14 0.5-0.7
6.3 0.12
13 0.19
10 0.16
12 0.96
14 0.91
19 0.96
21 0.72
5.4 0.24
11 0.80
12-15 O.CO.8
TSase (WA)
A
55
83
a5
80
85
100
100
45
100
100
53
88
79
90
100
100
100
42
100
100
o/c of trp+ enzyme levelf: T&we B T&se A
trpR+ cells grown in the presence of tryptophm
Specific aotivityt 11 TSase B VvB)
and
TABLE
73
73
75
14
14
21
26
53
69
63
13
15
20
29
22
14
14 28
15-25
16-28
Dorepression ratio 0 T&se T&se B
A
l-continued
t Specific activity is units of enzyme activity per mg of protein (Creighton & Yanofsky, 1970). $ Per cent of wild-type activity is calculated relative to a wild-type culture grown in parallel st the same time. $ The derepression ratio for a given pair of trpR + and trpRisogenic strains is the ratio of the enzyme specific activity in the trpRthat in the trpR + background. 11Abbreviations used PRATase, phosphoribosyl anthranilate transfcrase; T&se A, tryptophsn synthetase a; T&se B, tryptophan
to &.
background synthetase
In order to compare the function of a trp operon containing. sn internal deletion when repressed and derepressed, each tTp operon internal deletion was transferred by bacteriophage Plkc transduction into the same pair of strains which me isogenic except at the trpR locus (trpR+trpB9579oc tnuor trpR-trpR9579oc tna-). Recombinants were selected which carried the trp intern& deletion, were trpB+ trpA +tnoand were either trpR+ or trpR-. The same trpRallele was used throughout these studies; it results in constitutive synthesis of all trp operon polypcptides in the presence of excess tryptophan. The t7ta- mutation results in inability to synthesize tryptophanase. The tna- mutation in all strains allows accurate measurements of low levels of tryptophan synthetase activity without errors introduced by the activity of tryptophanase. Each trpR+ and trpRisogenio pair carrying a given trp internal deletion wss grown with vigorous aeration in excess tryptophan, harvested during logarithmic growth, sonic&ted, and assayed for tryptophan synthetase (T&se) B and A and PRA transferam activities as described elsewhere (Jackson & Yanofsky, manuscript in preparation). We term derepreseed the level of trp operon enzymes measured in trpRstrains grown in the presence of excess tryptophsn, and repressed the level of trp operon enzymes found in trpR + strains grown under identical conditions. Protein concentration was determined by the method of Lowry, Rosebrough, Farr & Randall (1961). We consider that deletions in strains having derepressed T&se levels equal to or greater than 80% of the trp+ control are not significantly polar.
trpAEClO1
+-
trpAEC81
trpR trpR
trpR trpR+
trpADC71
TD~
LETTERS
TO
THE
EDITOR
311
P, will lead to a derepression ratio higher than that found in the wild type. Therefore, the derepression ratio should show whether a given deletion removes P,, and location of the deletion termini on the genetic map of the operon can be used to gain information about the location of P,. Inspection of the derepression ratios in Table 1 shows that the deletion strains fall into two distinct classes. Deletion strains trpADC103, trpADC82, trpAED21, and trpAED24 have derepression ratios in the range 14 to 30, as does the trp+ control which contains P,. These deletion strains therefore retain normal P, function. The second class, trpADCl81, trpAED53, trpADC104, trpADC71, trpAEC81 and trpAEC101 have derepression ratios in the range 70 to 90. We expect deletion of P, to increase the derepression ratio, since P, function adds to the tryptophan synthetase B and A levels measured in the repressed trpR + background but does not contribute significantly to the trpR- tryptophan synthetase B and A levels. Thus, we conclude that all the strains having a derepression ratio for tryptophan synthetase B and A greater than 70 carry deletions which cover P,. Indeed, we would predict that deletions trpAEC81 and trpAEClO1 must remove P, since they both delete all of trpD and most of trpC. We know that P, must precede trpC but not trpD, since IGP synthetase but not PRA transferase synthesis can originate at P,. Furthermore, by analogy with the data from Salmonella, deletion mutants trpAEC81 and trpAEC101 should lack Pp. Deletion mutants trpAEC81 and trpAEClO1 have derepression ratios in the range 70 to 90, thus contlrming our conclusion that a derepression ratio in this range is characteristic of a deletion which removes the P, site. We can now use these deletions to locate precisely the P, site in the trp operon. Deletions trpADClO3 and trpADC82 both remove a portion of trpD since neither corresponding strain has PRA transfersse activity (see Table 1). Deletion mutant trpADClO3 fails to recombine with the last known trpD point mutant, trpD1537, but recombines with all other trpD point mutants, while deletion mutant trpADC82 recombines with all known trpD point mutants (see Fig. l(b)). Yet deletion strains trpADClO3 and trpADC82 both have P, function as judged by their derepression ratios. Therefore the P, site must lie within trpD. Inspection of the properties of strain trpAED53 reinforce this conclusion. Strain trpAED53 recombines with the last two tvpD point mutants known, and so retains a small segment of trpD. Since trpAED53 deletes P,, the P, site occurs before the end of .trpD. The locations of the deletions on the genetic map (see Fig. l(b)) a 11ow us to conclude that the P, site maps to the left of the altered site in trpD1537 (since trpADClO3 deletes trpD1537 but not Pa) and to the right of the mutation in trpD1300 (since trpAED21 and trpAED24 delete trpD1300 but not Pa). That trpADCl81 covers P, but does not delete the trpD1300 site is further evidence that P, is on the C gene side of the trpD1300 site. In addition, Pa must be located to the left of the mutational site in trpD1431 because trpAED53 deletes Pz but not the tvD1431 site. Thus PI must be located between the altered sites in the trpD point mutants trpD1300 and trpD1431. There is one known trpD point mutant, trpD1383, which maps in the region in which we have shown Pa is located. trpD1383 was transduced into the same isogenio trpR+ or trpR- tna- backgrounds as the internal deletions and assayed for tryptophan synthetase B and A (Table 1). Values for trpD1431, a trpD point mutant mapping very close to trpD1383, are given for comparison. The derepression ratios obtained with trpD1383 are 14 to 16 (see Table l), indicating that trpD1383 has a functional P, site. Scrutiny of the trpR- levels of tryptophan synthetase A and B shown in Table 1
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reveals that some of the deletion strains, such as trpR- trpADC82, trpR- trpADC71, and trpR- trpADC104 have less tryptophan synthetase A and B than the isogenic trpR- trp+ control strain (see Table 1, per cent of trp + enzyme level). We believe that the fusions resulting from these deletions end out of phase for proper translation, thus bringing a nonsense codon into phase just past the deletion terminus in trpC and resulting in a polar effect on tryptophan synthetase B and A synthesis. The amount of tryptophan synthetase B and A measured in each of these three trpR- deletion strains is about the same as observed in a trpR- strain carrying a known trpC nonsense point mutation at the position in trpC where the three deletions end (Jackson & Yanofsky, manuscript in preparation). Since the nonsense codon would be located after the P, site, polarity would decrease P,-governed and operator-governed synthesis of tryptophan synthetase B and A equally. Therefore, the presence of a nonsense codon to the right of P, would reduce the levels of tryptophan synthetase B and A activity observed but should have no effect upon the derepression ratio calculated by comparing levels in trpR+ and trpR- backgrounds. This conclusion is justified by the finding that the derepression ratio for one of the polar deletion strains, trpADC82, is in the range characteristic of strains having P,, while two of the polar deletion strains, trpADC71 and trpADC104 have derepression ratios in the 70 to 90 range, indicating that P, has been deleted. Prom our data it is now possible to estimate what fraction of the trpR+ trp+ repressed levels of tryptophan synthetase B and A in E. coli is due to transcription initiations at P,. The tryptophan synthetase B and A levels in non-polar deletion strains trpADC181, trpAED53, trpAEC81, and trpAEClO1 represent the amount of these enzymes made under repressed conditions in the absence of P,. This level is about 20 to 30% of the repressed trpR+ trp+ level. Thus P, in the wild type accounts for about 70 to 80% of the tryptophan synthetase B and A seen under repressed conditions. This is exactly the P,-initiated level of synthesis seen in S. typhimurium (Bauerle & Margolin, 1967 ; Margolin & Bauerle, 1966). Our studies therefore establish that a nucleotide sequence near the trpC end of trpD functions both to code for a sequence of amino acids in the carboxy-terminal region of the trpD polypeptide and to promote initiation of transcription. It follows that P, may not have a physiologically important function as a transcription initiatior but may exist simply as a consequence of the presence of a nucleotide sequence essential for functional PRA transferase. In this regard it is of interest that promoter-like initiator sequences have been induced by 2-aminopurine treatment in the first gene of the trp operon of S. typhimurium and one such initiator sequence does not interfere with the production of a functional gene product (Wuesthoff & Bauerle, 1970). In view of the genetic location of P, it should be possible to select point mutants in trpD which have altered P, function and lack active PRA transferase. The one known tqwD point mutant, trpD1383, which maps in the region where P, is located, has normal P, function. However, experiments to select point mutations in trpD which alter P, function are in progress. If several such mutants of the missense type were available, it might be possible to deduce the nucleotide sequence of the P, region from the ammo-acid sequence in the corresponding segment of the trpD polypeptide. The authors are indebted to Virginia Horn and Miriam Banner for their assistance. The studies described in this paper were supported by grants from the National Sciences Foundation (GB6790), the U.S. Public Health Service (GM-09738) and the American Heart Association.
LETTERS
One of us (E. N. J.) is a predoctoral author (C. Y.) is a Career Investigator Department of Biological Sciences Stanford University Stanford, Calif. 94305, U.S.A. Received
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trainee of the U.S. Public Health Service. The other of the American Heart Association. E.N. JACKSON C.YANOFSKY
1972
REFERENCES Bauerle, R. H. & Margolm, P. (1966). Proc. Nat. Acad. Sci., Wash. 56, 111. Bauerle, R. H. & Margolin, P. (1967). J. Mol. Biol. 26, 423. Creighton, T. E. & Yanofsky, C. (1970). In Methods irz Enzymology, ed. by S. P. Colowick & N. 0. Kaplan, vol. l?A, p. 365. New York: Academic Press. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. (1951). J. Biol. Chem. 193, 265. Margolin, P. & Bauerle, R. H. (1966). CoZd Spr. Ha&. Syv. Quunt. Biol. 31, 311. Morse, D. E. & Yanofsky, C. (1968). J. Mol. Biol. 38, 447. Wuesthoff, B. & Bauerle, R. H. (1970). J. Mol. BioZ. 49, 171. Yanofsky, C. (1967). Harerey Lect. 61, 146. Yanofsky, C. (1971). J. Amer. Med. Aseoc. 218, 1026. Yanofsky, C., Horn, V., Bronner, M. L Stasiowski, S. (1971). Genetics, 69, 409.