Promoter occlusion: Transcription through a promoter may inhibit its activity

Cell, Vol. 29, 939-944,

Promoter Promoter

July 1982,

Copyright

0 1982

by MIT

Occlusion: Transcription May Inhibit Its Activity

Sankar Adhya and Max Gottesman Laboratory of Molecular Biology National Cancer Institute Bethesda, Maryland 20205

Summary Induction of prophage lambda inhibits the expression of the gal operon from its cognate promoters. The effect is observed only in cis, and is due to frequent transcription of the gal promoter region by RNA polymerase molecules initiating upstream at the prophage fL promoter. The frequency of transcription initiation at R is some 30 times greater than that at the gal promoter, Pgl. PL is one of the strongest procaryotic promoters. This “promoter occlusion” is essentially complete when the distance between gal and R is small (510 kb); and when PL is fully active (that is, in the absence of the cl or cro repressors). We discuss the possibility that promoter occlusion at two lambda promoters, PI,, and P.4, might play a role in the sequential expression of viral functions. Introduction The gal operon of Escherichia coli can be transcribed from one of two overlapping promoters (Muss0 et al., 1977; Adhya and Miller, 1979). The first, Pg,, depends on cyclic AMP and its receptor protein, CRP, for activity, while the second, Pg2, is inhibited by CAMPCRP. Both are regulated by the gal repressor (Adhya and Miller, 1979). In addition, gal can also be transcribed from the prophage lambda promoter, PL, under the control of the lambda repressor (Adhya et al., 1974). The PL promoter is considerably more active than either f,, or Pg2 (Merril et al., 1978; see below). There are now numerous examples in procaryotes in which a gene or genes can be transcribed from more than one promoter. The bacteriophage T7 early region is transcribed from three distinct promoters (Dunn and Studier, 1973; Minkley and Pribrow, 1973). The late gene region of bacteriophage lambda is transcribed principally from PR’, but can also be transcribed weakly from PF1(Couturier et al., 1973). The lambda int gene is transcribed from IP,,~ or PL (Singer, 1970; Katzir et al., 1976). E. coli ribosomal RNA synthesis can derive from either of two closely linked promoter regions (Glaser and Cashel, 1979). Certain bacterial operons, such as trp and his, carry internal promoters, resulting in dual transcription origins for a portion of the operon (Bauerle and Margolin, 1967; C. Bruni, personal communication). In some of these cases, the expression of a gene depends on the source of transcription. Thus the P,nr promoted transcript is efficiently translated, while the transcript emanating from PL yields very little Int pro-

through

a

tein (Katzir et al., 1976). For gal, the first cistron of the operon, galE, is expressed from P,, or P,,, but not from PL, whereas the promoter-distal cistron ga/K can be expressed efficiently from the phage promoter (Merril et al., 1978). The failure to translate the ga/E sequences of the PL transcript has been shown to be due, in part, to the formation of secondary structures that block the access of ribosomes to the ShineDalgarno site of ga/E (Merril et al., 1981). It is commonly believed that promoters transcribing in the same direction act independently, whereas promoters that initiate convergent transcription interfere with each other (Ward and Murray, 1979). Recently, however, evidence for functional interference between the two trp promoters, both of which transcribe the sense strand of WpAB, has been reported (Hausler and Somerville, 1979). At the same time, we were finding indications of a similar negative interaction between the gal promoters and PL. These experiments, which we report here, are based on our ability to distinguish, by determining the ratio of the galK and ga/E products, between the gal mRNA initiated at the gal promoters and gal mRNA initiated at PL. We find that activation of the PL promoter abolishes the activity of the downstream gal promoters. This phenomenon of “promoter occlusion” may, we believe, play a role in gene regulation, particularly in the development of the bacteriophage lambda. Results Occlusion of gal Promoters by a Lambda Promoter The gal operon of E. coli consists of three structural genes, galK, galT and galE, encoding galactokinase, galactose-1 -phosphate uridyltransferase and uridinediphosphogalactose-4-epimerase, respectively. The operon is transcribed from promoters located at the galE end that are activated by the addition of o-galactose or o-fucose to growing cultures. In wild-type cells, the active promoter is P,,; transcription from the CAMP-CRP-dependent PSI promoter results in equimolar synthesis of the three gal enzymes (coordinate expression) (Wilson and Hogness, 1969; Merril et al., 1981). Transcription of gal can also initiate at the PL promoter of prophage lambda, integrated nearby, when the latter is induced. In our experiments, this is accomplished by thermal inactivation of the lambda c/857 repressor (Adhya et al., 1974). In contrast with transcription from Ps,, P,-promoted transcription of gal leads to grossly discoordinate expression of the Gal enzymes, with extensive synthesis of kinase and essentially no synthesis of epimerase. In Table 1, we show the expression of the gal operon in strain N4830 from P,, or PL. Activation of P,, results in a 10 to 15 fold increase in kinase or epimerase levels. Induction of PL raises the kinase levels over lOO-fold, while epimerase activity remains at the basal level. Our ability to distinguish between the P,, transcript,

Cell 940

Table

1. Occlusion

of gal Promoters induced Promoter’

Galactokinase LeveP

Uridinediphosphogalactose4-epimerase LevelE

None P 01 P‘ p,, + PL

1.1 15.0 144 180

3.0 36 4.0 5.0

None pL?2 PL pg2 + PL

0.5 7.0 104 116

9.0 57 13.0 7.7

None P Wl PL p,, + 6

2.4 20.0 138 128

9.0 63 12.0 42

Nam7,53

None P *l PL p,, + &

1.3 12.0 1.9 14.0

2.5 32 4.0 30

N5179

nutL -

None P 01 6 p,, + PL

0.9 14.0 1.3 15.0

2.0 28 3.0 31

N4837

Nam7,53

None P gl PL pg, + PL

2.4 13.1 51 .l 62.8

2.2 35 6.3 10

SA1999

p‘-

None P *l 6 pg, + s

1.0 10.0 22.0 20.0

3.0 32 12.0 22

SA1986

ABAM AH1

None P gl 9 p,, + PL

1.7 17.0 45.0 50.0

3.1 34.0 5.1 15.0

N5045

AH1

None P 91 s P,, + PL

1.7 11.0 26.0 43.0

3.2 30.0 5.0 34

N5413

cro-

PL p,, + 6

N5412

cro+

PL p,, + PL

Strain

Relevant

Genotype

N4630

gal+ A8 c/85 7 ABAM

SA1994

cya -

N5276

F’gal*/N4630

N4631

ga/T-

rho1 5

Cell growth, induction of gal cistrons * P,, is the CAMP-dependent. and lambda. Pg,, Pg2 or P, was induced b Units of kinase are nanomoles of c Units of epimerase are nanomoles

AH1

293 261 63.0 67.0

.

4.0 3.0 5.0 14.1

and enzyme assays were carried out as described in the Experimental Procedures. PQp is the CAMP-independent, promoter of the E. coli gal operon; P‘ is the leflward for 30 min. ‘%-galactose-1 -phosphate formed per minute by 1 OS cells. of UDP-galactose converted to UDP-glucose per minute by 1OS cells.

which expresses epimerase, and the PL transcript, which does not, permitted us to ask if activation of PL affected the function of P,,. As shown in Table 1, dual induction of the lambda lysogen N4830 with o-galactose and by a temperature shift to 42°C resulted in the extensive synthesis of kinase without a corresponding increase in epimerase. The CAMP-CRPindependent gal promoter Pg2 is also inhibited by prophage induction (Table 1, strain SA1994). In this experiment, a cya derivative of N4830, SA1994, was induced with o-galactose, at 32°C or at 42”C, and the kinase and epimerase activities were determined. The

early

promoter

of phage

high levels of kinase relative to the basal epimerase levels at 42°C indicate that both P,, and Pg2 are inactive at that temperature. These results are most easily interpreted by a model of “promoter occlusion.” When a promoter is transcribed from another highly active, upstream promoter, it can no longer initiate transcription. RNA polymerase molecules initiating upstream inhibit other RNA polymerase molecules from gaining access to downstream promoters. Evidence for this notion is presented in the remainder of Table 1. First, inhibition of P,, is a cis effect. A second copy

Promoter 941

Occlusion

of the gal operon, unlinked to PL, was introduced into a galT- derivative of N4830 (N5278) to test the effect of PL activation on gal induction in trans. The high level of epimerase seen after dual induction indicates that the episomal gal operon is not inhibited in trans by prophage induction. Other unlinked cistrons such as /acZ are likewise not inhibited by induction of the prophage in N4830 (data not shown). Second, for transcription emanating from PL to enter gal, it must be modified by the lambda regulatory function N (Adhya et al., 1974). The interaction between the P,-transcription complex and N most likely occurs at the prophage n&L site, and creates a “juggernaut” that is resistant to transcription termination signals in phage or bacterial DNA. When prophages were mutant in either N (N4831) or n&L (N5179; Salstrom and Szybalski, 19781, thermal induction of kinase synthesis did not occur. At the same time, there was no inhibition of epimerase synthesis after prophage induction. This finding is consistent with the promoter occlusion model, and eliminates the possibility that the prophage N function is directly responsible for the discoordination effect. Third, that N function plays no direct role in the inhibition of P,, by P,-promoted transcription is also shown by the induction of strain N4837, a rho15 derivative of N4831 (Das et al., 1978, Gottesman et al., 1980). In this mutant strain, defective in transcription termination, the requirement for prophage N product for P,-promoted gal transcription is largely eliminated. Thermal induction of N4837 provoked the synthesis of 51 units of kinase, compared with 144 units for N4830, and partially blocked the synthesis of epimerase (10 units versus 30 units for N4831). Fourth, the above observation suggests that the activity of P,, is inversely related to the frequency with which it is transcribed from Pt. Because N4830 synthesizes kinase but not epimerase at 42% it is sensitive to galactose. We isolated 117 spontaneous galactose-resistant, Gal+ mutants from N4830 as described in the Experimental Procedures. Of these, 115 were prophage mutants: 112 were N- and 3 were b _----_ --- -_-- 9Gls_--

--_____

PL-. The former, when tested, acted like N4831; at 42°C they neither synthesized kinase nor blocked epimerase expression from P,, (data not shown). The results of induction of a P,-defective mutant (SA1999) are shown in Table 1. Thermal induction led to the synthesis of about 20 kinase units. The inhibition of Pgr was correspondingly less: about 22 units of epimerase relative to 30 units were made. Fifth, three additional experiments indicated that the inhibition of P,, activity is proportional to the intensity of readthrough transcription of P,, from PL. Transcription of gal from PL decreases with increasing distance between the two elements. Strains N4830, SA1986 and N5045, otherwise isogenic, vary in the amount of DNA between the PL promoter and galthat is, 6.5 kb, 18.9 kb and 22.6 kb, respectively (Figure 1). Prophage-induced kinase activity in the three strains was 180, 50 and 43 units, respectively, in inverse relation to epimerase activities of 5, 15, and 34 units, respectively (Table 1). The activity of PL is repressed by lambda cro gene product. Strains N5412 and N5413 are derivatives of N4830 that carry the A443 deletion in place of AH1 (see Figure 1). N5413 bears the cfo mutation crol0 (Franklin, 1971); N5412 is cfo+. As expected, N5413 showed a high kinase level and a basal epimerase level when both 9 and P,, were induced (Table 1). In contrast, the cro+ strain, N5412, displayed one fourth as much kinase, but significant epimerase activity (14.1 units), after dual induction (Table 1). The PL promoter is also negatively regulated by the phage cl repressor. In these experiments, we have used the reversibly denaturable repressor, c/857, which is active at 32°C inactive at 42°C and partially active at intermediate temperatures (Folkmanis et al., 1977; see below). To modulate the activity of PL, we grew N4830 to mid-logarithmic phase in LB broth and then, where indicated, added o-galactose and shifted it to 42°C (Table 2). After 10 min, portions of the growing cultures were cooled to 40X, 38.5% or 32°C and growth was continued for an additional 50 min. Enzyme assays demonstrated that as PL activity

4

Al79 c - - - - - -. - - - ,

+-------------,

A312

k--p_??-

4

,2?!L-----------------

A482

+-----------------A

+-AS- - -( KC

chlD

blu

A HI --------~-------__---__

+A%-, 8rr int

B(M) kg CM

P

tL1

biol0

Deletions

1. Schematic are indicated

Map of ProphaQe by broken

P**G

a

OP

QP~~

SRA

PL

I

I Figure

d

N

Lambda

and Adjacent

lines, substitutions

Genetic

Regions

by solid lines. Sizes

have not been drawn

to scale.

J

8fr bio

Cell 942

increased with temperature (note increasing kinase levels), the activity of Pgj was correspondingly reduced (note decreasing epimerase levels when both promoters were induced).

Second, the average speed of transcription from PL to galK has been determined, based on the data of Figure 2. As the distance between PL and galK decreases, the lag between the time the culture is shifted to 42% and the appearance of kinase is reduced. If we assume a constant rate of translation of galK mRNA of 1000 nucleotides/min (Michaelis and Starlinger, 19671, the speed of transcription from PL for wild-type, chlD and prophage deletion lysogens varies from 33 bases/set to 52 bases/set. Although the differences between the strains are reproducible, the significance of these results is not obvious.

Studies on the PL Promoter There are two characteristics of the lambda PL that distinguish it from most, if not all, bacterial promoters, and that are probably relevant to its capacity to occlude downstream promoters. First, the frequency of transcription initiation at PL is higher than that of many known bacterial promoters (see below; J. Salstrom, personal communication). To obtain an estimate of the strength of PL, we have measured the expression of galK in a set of chlD deletion strains (Figures 1 and 2). As mentioned above, the synthesis of kinase is inversely related to the distance between PL and galK. In strain A482, PL lies some 2.2 kb from ga/K, without intervening terminators. In the absence of Cro and at 42X, the rate of kinase synthesis is 9.0 units/min. In terms of kinase polypeptides, this represents 31 polypeptides/min/ cell. In contrast, expression of ga/K from P,, is 1 polypeptide/min/cell (M. lrani and S. Adhya, manuscript in preparation). These calculations indicate that PL initiates transcription some 30 times more efficiently than Pg,.

Table 2. Restoration

of gal Promoter

Activity

Promoter

Temperature

by cl Repressor

Discussion We have shown that activation of the PL promoter of bacteriophage lambda interferes with the activity of two nearby downstream promoters, P,, amd Pg2, of the E. coli gal operon. We propose that this interference is the result of “promoter occlusion,” in which RNA polymerase molecules initiating upstream block the access of other RNA polymerase molecules to downstream promoters. This might result from direct steric hindrance or from distortion of DNA structure. Consistent with this model are the following observations. First, the occlusion is entirely in cis. Second, tran-

of Lambda Galactokinase Level

Induced’

32’C

Uridinediphosphogalactose+epimerase

P gl

1.5 12.2

42’C.

then 32’C

PL P‘ + p,t

38.0 46.9

42%.

then 38.5”C

9 PL + pg,

135 132

7.8 32.3

42°C

then 40%

PL P‘ + p*7

351 331

7.8 12.7

P‘ PL + p*,

520 524

6.2 8.7

42°C

Cell growth, induction of gal cistrons and enzyme promoters are described in the text and in Table 1. ’ Pg, was induced by the addition of o-galactose.

assays

were

carried

out as described

3.3 30.6 3.2 29.8

in the Experimental

Procedures

Level

and Results.

The

Figure 2. Kineticsof Galactokinase Synthesis from Lambda PL Promoter in Various Deletion The extent of respective prophage deletions are shown at top and in Figure 1. The genotypes are described in Table 3. Cells were grown at 32°C and the PL promoter was induced by a shift to 42°C at 0 min. Aliquots were taken at various time intervals and assayed for galactokinase. Other details are described in the Experimental Procedures. 0

10

2s

30

40

as -10 0 10 2s J) 40 50 -10 TIME IN MIN AFTER INDLKYllON

0

10

20

30

40

50

Promoter Occlusion

943

scription from PL per se appears to be responsible. No known phage function, including the lambda transcription antitermination function, the N gene product, plays a direct role. Third, the extent of occlusion is directly related to the frequency with which P,, is transcribed from PL. Thus, as the distance between PL and P,, increases, the level of P,-promoted gal transcription fails; the occlusion of P,, activity is correspondingly less. The rate of transcription initiation at PL is modulated by two phage repressors, cl repressor and Cro. Although the former is usually an “all or none” regulator, we were able to establish intermediate levels of PL activity by the use of the thermosensitive cl857 repressor. Cultures lysogenic for cl857 prophage, grown at intermediate temperatures, displayed partial P, activity and partial occlusion of P,,. Lambda cro+ lysogens showed less PL activity (see Figure 2) and less promoter occlusion than lambda cro- lysogens. Although instances of clustered promoters, like the case described for gal, are also described in other procaryotic systems, only one other case of promoter occlusion -in the trp operon systems-has been reported. Thus the conditions appropriate for promoter occlusion may not be common. First, the frequency of transcription initiation at the upstream promoter must far exceed that at the downstream promoter. We have calculated the relative strengths of PL and P,, to be on the order of 3O:l. With the possible exception of some ribosomal RNA promoters, for example, Prrne (M. Cashel, personal communication), no bacterial promoter is of comparable strength. Second, there must be a sufficient “dwell time”; that is, there should be an extended period during which an RNA polymerase molecule initiating upstream dwells on and occludes a downstream promoter sequence. The more complex the downstream sequence, the longer the dwell time. For Pg,, which requires CAMP-CRP for activity, sequences required for promoter activity extend as far as -50 from the start site of Pg, mRNA (Busby et al., 1982). However, the CAMP-CRP-independent promoter Pg2 is also inhibited by transcription from PL. Furthermore, the rate of P,-promoted mRNA elongation is abnormally high, 30-50 nucleotides/set, thus reducing the dwell time. It is therefore possible that this second factor may not play a primary role in the occlusion of the gal promoters by P,-initiated transcription. Promoter occlusion may play a role in the regulation of the bacteriophage lambda life cycle. Aspects of the phenotype of the cro- mutants are consistent with this notion, although it should be stressed that no direct evidence is available at present. First, hcroforms stable lysogens at reduced frequency (H. Echols, personal communication). We believe that when PL is fully active, transcription initiation at Pint is blocked, and the synthesis of Int is prevented. In Cro+ phage, PL activity is inhibited by a factor of 4 to 5 after

a few minutes (see Figure 2), permitting Pin, to function. This mechanism may serve to avoid the premature synthesis of Int. Second, hero- also fails to propagate lytically (Folkmanis et al., 1977).,There are two components of this phenotype: a trans component, eliminated by deletion of the P,-operon function, EalO; and a cis component, which is alleviated by mutations partially inactivating PR (Georgiou et al., 1979). It has been suggested that transcription initiating at PR must be reduced by Cro for normal phage DNA replication to occur (Folkmanis et al., 1977). However, it is also possible P,-initiated transcription occludes the downstream P,’ promoter. The lambda late-gene region is normally transcribed from the P,’ promoter, under the influence of the phage-positive regulatory gene, Q. Defects in phage endolysin synthesis, a late-gene product, in Xcro- mutants are consistent with this hypothesis. Temporary occlusion of PR’ might be important in ensuring the sequential appearance of viral functions. Experimental Procedures The E. coli K12 strains used in this study are listed in Table 3. Standard microbiologibal techniques were used for construction of the strains. Cells were grown in LB media at 32X for induction. The P,, and Pgz promoters were induced by the addition of 0.3% D galactose. We induced the PL promoter of the lambda prophage by shifting the cultures to 42’C. unless otherwise stated. Galactokinase and uridinediphosphogalactose-4-epimerase were assayed as described previously (Merril et al., 1978).

Table 3.

E. coli Ki 2 Strains

Used Source or Reference

Strain

Genotype

SA1615

Fsuhis- strR WAga/OP3::/S2

N4830

SAl815

gal+ A8 (k/857

re/A

ABAM

AHl)

0. Reyes Gottesman

et al..

1980

N5276

F,‘ga/+/N4830

SA1994

N4630

i/v+ cya -

This study

N4831

N4830

Nam7,53

Gottesman 1980

N5179

N4830

nutL33

Salstrom balski,

galTam

This study

N4837

N4831

rho1 5

This study

SA1999

N4830

PL-

This study

M72

f -su-

sb’

N3754

F- W857

SA1316

N3754

A313

trpam lacam red3

At/l)

M. Shulman This study

SAl308

N3754

A482

This study

SA1615(Xc/857ABAMAHf)

This study

N5045

SAl615&~/857bio70AH1) SA1615

N5413

N5412

gal+ i/v+ A8 (Xc1857

and Szy. 1978

l-l. Eisen

SA1966

N5412

et al.,

This study This study

ABAM A443) ~010

This study Franklin, 1971

Cdl 944

Isolation of GaP Mutants Strain N4830 is insensitive to galactose at 42°C and grows poorly on MacConkey galactose plates at 42’C (see Results). GalR strains of N4830 were isolated as Gal+, rapidly growing red colonies after overnight incubation at 42% on MacConkey galactose plates. Such colonies arose at a frequency of about 1O-7. They were screened for mw- prophage mutations by their ability to support the growth of 41 ‘C of superinfecting Aimm434 phages but not Aimm434 Nam7.53 phages. GalR mutants were classified as fL- (for example, SAI 9991, if they complemented poorly a Aimm434 Nam7,53 phage and showed reduced galactokinase synthesis from the prophage PL promoter (compare the nutL- N5179 strain). The mutations were also shown to be linked with the gal operon by phage Pl cotransduction experiments.

Katzir, N., Oppenheim, A., Belfort, M. and Oppenheim. A. (1976). Activation of the lambda int gene by the cll and clll gene products. Virology 74, 324-331.

Acknowledgments

Musso. R. E.. Di Laura. R.. Adhya, S. and de Crombrugghe. B. (1977). Dual control for transcription of the galactose operon by cyclic AMP and its receptor protein at two interspersed promoters. Cell 12, 847-854.

We thank Susan Garges for helpful discussions and Annette Kuo for typing our manuscript. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received

January

11, 1982:

revised

April 8, 1962

Merril. C., Gottesman. M. and Adhya, operon proteins made after prophage 147, 875-887. Michaelis, galactose

S. (I 981). Escherichia co/i gal lambda induction. J. Bacterial.

G. and Starlinger, P. (1967). Sequential appearance of the enzymes in E. co/i. Mol. Gen. Genet. 100, 21 O-21 5.

Minkley, E. and Pribnow, D. (1973). Transcription of the early region of bacteriphage T7: selective initiation with dinucleotides. J. Mol. Biol. 77, 255-277.

Salstrom. J. and Szybalski. W. (1978). Coliphage butt-: a unique class of mutants defective in the site of N product utilization for antitermination of leftward transcription. J. Mol. Biol. 724, 195-221. Singer, E. (I 970). On the control 624-633.

of lysogeny

in phage X. Virology

Ward, D. and Murray, N. (I 979). Convergent transcription riophage h: interference with gene expression. J. Mol. 249-266.

References Adhya. S. and Miller, W. (1979). Modulation’of the two promoters the galactose operon of Escherichia co/i. Nature 279, 492-494.

of

Adhya. S., Gottesman. M. and de Crombrugghe, B. (1974). Release of polarity in Eschefichia co/i by gene N of phage X: termination and antitermination of transcription. Proc. Nat. Acad. Sci. USA 71, 25342538. Bauerle. Ft. and Margolin, P. (1967). Evidence for two sites for initiation of gene expression in the tryptophan operon of Salmonella typhimurium. J. Mol. Biol. 26, 423-436. Busby, S., Aiba, H. and de Crombrugghe, B. (1982). Mutations galactose operon that define two promoters and the binding the CAMP receptor protein. J. Mol. Biol. 754, 21 l-227. Couturier, M.. Dambly, C. and Thomas, R. (1973). opment in temperate bacteriophages. V. Sequential viral functions. Mol. Gen. Genet. 720, 231-252.

in the site of

Control of develactivation of the

Das. A., Court, D. and Adhya, S. (1976). Isolation and characterization of conditional lethal mutants of fscherichia co/i defective in transcription termination factor Rho. Proc. Nat. Acad. Sci. USA 73, 19591963. Dunn, J. and Studier, site-specific cleavages.

Merril. C.. Gottesman. M., Court, D. and Adhya. S. (1978). Discoordinate expression of the Eschefichia co/i gal operon after prophage ; lambda induction. J. Mol. Biol. 118, 241-245.

W. (1973). T7 early RNAs are generated by Proc. Nat. Acad. Sci. USA 70, 1559-l 563.

Folkmanis, A., Maltzman, W.. Mellon, P., Skalka, A. and Echols, H. (1977). The essential role of cro gene in lytic development by bacteriophage A. Virology 81, 352-362. Franklin, N. (I 971). The N operon of lambda: extent and regulation as observed in fusions to the tryptophan operon oof Escharichia co/i. In The Bacteriophage Lambda, A. D. Hershey, ed. (Cold Spring Harbor, New York: Cold Spring Harbor Laboratory), pp. 621-638. Georgiou, M.. Georgopoulos, C. and Eisen. H. (1979). An analysis the tro phenotype of bacteriophage X. Virology 94, 36-54.

of

Glaser, G. and Cashel. M. (1979). In vitro transcripts from the rrn B ribosomal RNA cistron originate from two tandem promoters. Cell 76, 111-121. Gottesman. M., Adhya, S. and Das. A. (1980). Transcription antitermination by bacteriophage lambda Ngene product. J. Mol. Biol. 140, 57-75. vausler, B. and Somerville. R. (1979). Interaction in viva between strong closely spaced constitutive promoters. J. Mol. Biol. 727, 353356.

46,

in bacteBiol. 133,

Wilson, D. and Hogness, D. (1969). The enzymes of the galactose operon in Escherichia co/i. IV. The frequencies of translation of the terminal cistrons in the operon. J. Biol. Chem. 244, 2143-2148.