Proc. Nat. Acad. Sci. USA Vol. 71, No. 6, pp. 2534-2538, June 1974

Release of Polarity in Escherichia coli by Gene N of Phage x: Termination and Antitermination of Transcription (escape synthesis/juggernaut model/gal operon) SANKAR ADHYA, MAX GOTTESMAN*, AND BENOIT DE CROMBRUGGHE Laboratory of Molecular Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20014 Communicated by Allan Campbell, March 1, 1974

ABSTRACT The induction of X prophage provokes the was to antagonize the action of rho (6). N-product would constitutive expression of the adjacent gal operon in E. serve as an antiterminator, permitting transcription to extend coli. This "escape synthesis" can result from transcription that initiates at a phage promoter and extends into the beyond tL and tR. gal operon. The effect requires the product of the X gene The gal operon of E. cali is transcribed in the same direction N. N-mediated transcription not only fails to terminate as the adjacent prophage X genes N through int (7). Although at the prophage-bacterial junction and at the ends of it is separated from the prophage by about 10 bacterial genes bacterial operons, but ignores termination signals caused by polar insertions or ochre mutations within gal. Suppres- (8) (Fig. 1), prophage induction results in the derepression sion of polarity by N-function is a cis-effect; only trans- of the gal operon (9, 10). We believe that this "escape syn- cription initiated at the phage promoter is influenced. thesis" could result from the extension of transcription ini- We propose that the transcription complex is influenced tiated in X into the gal operon. The suppression of normal by N-product to become termination-resistant at a site in bacterial termination mechanisms might result from the in- the phage genome (juggernaut model). This site appears to be at or near the phage promoter. hibition of rho by the X N gene product. We have previously demonstrated that rho is effective in terminating transcrip- The expression of the bacteriophage X genome is almost en- tion on bacterial DNA templates (11). tirely dependent on the product of X gene N (1). In the ab- We report that (1) transcription initiating at the prophage sence of N product, the N and tof genes, but not the distal sex promoter does, in fact, extend into the bacterial chromo- genes, are transcribed at wild-type levels (see Fig. 1) (2-4). some at least as far as the gal operon; (2) the effect is mediated When N-product is present, transcription of the distal genes by N-product; and (3) under the influence of N-product, sex- results from the elongation of the N and tof messages. If X promoted transcription also fails to terminate at polar muta- DNA is used as a template in a purified in vitro transcription tions in gal, suggesting that polarity is the result of rho action. system, N, tof, and distal genes are all transcribed. The addi- The suppression of polarity during escape synthesis is limited tion of an Escherichia coli protein, rho, terminates transcrip- entirely to this phage-promoted transcript. A preliminary ac- tion at sites to the left of N (tL) and to the right of tof (tR), count of some of these results has been reported elsewhere analogous to the pattern seen in vivo in the absence of N (12). product (5). Roberts proposed that the function of N product MATERIALS AND METHODS Strains. The wild-type E. coli K-12 strain used is SA500. * Biochemical Genetics Section. Its genotype is F- gal+su-his-str-r. The strain N2853 is a

pi p p

K T E (OP) chID (blu phr) int xis exo dll tL N sxV2 cI

I ....._... . . , . . . . ro I I I -1 1 11 1 11 * I III I I I I I I I I II MredII -xvlv3 of tR 0 P a T76 t 256 8146 243 10

11 t 69 p 386- FIG. 1. Partial genetic map of prophage X and adjacent bacterial chromosome. x and sex are the promoters for rightward and leftward transcriptions in X. tL and tR designate the two rho-sensitive transcription termination signals. The gal operon consists of three structural genes E, :T and K, E being located at the operator-promoter (OP) end. The wavy lines with arrowheads show the direction and extent of various transcription processes discussed in the paper. 2534 Downloaded by guest on September 26, 2021 Proc. Nat. Acad. Sci. USA 71 (1974) Polarity and Transcription Termination 2535

derivative of SA500 lysogenic for XcI857int29xl3. All other TABLE 1. Escape synthesis ef galactokinase strains used are also derived from SA500 and are described in Tables 1-3 and Fig. 2. Bacteriophage Xi434cI-int6b2S7 was Addi- Galacto- used in superinfection experiments described in Tables 2 and tion of kinase gal 1mM specific 3. Strain genotype mutation fucose Temp activity Media. Cells were grown in minimal medium M56 (13) sup- 1. K+T+E+ plemented with 0.3% (v/v) glycerol and 0.1% casamino acids (XcI857) Wild-type - 320 1.1 for galactokinase assay and in Medium A (14) supplemented 2. " " + 320 3.2 with 0.5% glycerol and 50 ;Ag/ml of histidine for RNA mea- 3. " - 410 7.3 surements. 4. " " + 410 10.4

assayed using D-['H] 5. K+T+Es,4 Rho-sensitive galactose as described by Wilson and Hogness (15). Specific (XcI857) insertion - 320 0.06 activity of the enzyme is defined as nmole of D-['H]galactose 6. " " + 320 0.07 7. " - 410 3.9 1-phosphate formed per min/ml of cells of OD590,. = 1.0. 8. "" + 410 4.1 Phage Infection. Cells were infected by phage at a multi- 9. +TNioE + Rho-insensitive plicity of infection of 5. MgCl2 (7 mM) was present in the (XcI857) insertion - 320 0.07 media all throughout the superinfection experiments. 10. " " + 320 0.08 " " - 410 4.1 Chemicals. D- ['H ]galactose was purchased from Interna- 11. 2. " " + 410 4.2 tional'Chemical and Nuclear Corp. [3H]uridine came from Schwarz-Mann. All other chemicals were from Sigma Chem- 13. K+T+E,5 ical Co. (XcI857) Ochre - 320 0.04 14. " " + 320 0.20 RNA Synthesis Studies. Cells at a density of 108/ml were 15. " " - 410 5.5 labeled with ['HJuridine (20 Ci/mmole; 0.25 mCi/10' cells). 16. I It + 410 5.6 At the end of the labeling period the cells were cooled in the 17. K+T+E+ presence of 0.02 M sodium azide, centrifuged, lysed, and their (Xi21clts) Wild-type - 410 5.9 was as Varmus et al. (16). Total RNA extracted described by 18. K+T+ES143 Rho-sensitive labeled RNA and gal-specific RNA was measured as described (xi'lcIts) insertion - 410 5.4 by Guha et al. (7). RESULTS All prophages carried a replication-defective mutation, P3, and an excision-defective mutation, xis6 in the case of XcI857 or The expression of the gal operon of E. coli is under the negative int6 in the case of Xi21lIs. gal operon or XcI857 prophage were control of the gal repressor, the product of the unlinked galR induced for 30 minm, Xi2lcts prophage, for 50 min. gene, and under the positive control of cyclic AMP and the cyclic AMP receptor protein (CRP) (17-20). The operon inhibited by gal repressor. It is known from in vitro studies is induced by D-galactose or D-fucose, which inhibits the bind- that gal repressor inhibits gal transcription only at the level ing of gal repressor to gal operator DNA (21). The induction of initiation (22). of prophage X, which is inserted near the gal operon, overrides We next tested the effects of prophage induction in a lyso- these normal control mechanisms, resulting in the constitu- gen carrying a polar mutation in galE, the operator-proximal tive, cyclic AMP-independent synthesis of the gal enzymes gene of the operon (Fig. 1). S148 consists of an insertion of a (refs 9 and 10; see below). This phenomenon, known as "es- foreign DNA fragment containing a rho-dependent transcrip- cape synthesis," is the result of two independent processes: tion termination site (11). The polarity of the S148 insertion (1) X-induced replication of the gal operon which leads to is clearly suppressed by X induction (Table 1, lines 6-8). In titration of gal repressor. This effect is eliminated by the use addition an entirely different type of insertion, N102, in of replication-defective X mutants (x-, 0-, or P-) or by the galT, and a polar ochre mutation in galE, 95, are suppressed addition of 5-fluorodeoxyuridine. (2) Extension of transcrip- as well (Table 1, lines 9-16). Neither N102 nor 95 is polar in tion initiating at the prophage sex promoter into neighboring a purified in vitro transcription system containing rho. It bacterial DNA, including the sense strand of the gal operon should be noted that it is the polarity and not the mutation (G. Buttin, unpublished; also see below). In the experiments that is suppressed; escape synthesis of UDP-galactose-4- reported below, escape synthesis due to replication has been epimerase, the prQduct of galE, observed with a wild-type gal blocked, while that due to transcriptional read-through has operon is not found with the ochre 95 mutant (data not been amplified by mutations which prevent prophage excision. shown). Suppression of polarity of nonsense mutations in the The escape synthesis of galactokinase, the product of the trp operon by infection of a Xtrp phage, in which N is cis to galK gene, is shown in Table 1 (lines 1-4). The addition of the trp operon, has been observed independently by Franklin fucose to a strain carrying a wild-type gal operon increases (23). galactokinase activity 3-fold in 30 min. The strain is lysogenic The results obtained with the insertion and ochre strains for a X prophage whose repressor is reversibly thermolabile are, in fact, very similar: (1) Extensive suppression of polarity (XcI857). At 410, the prophage is induced. After 30 min of when the prophage is thermally induced. (2) The inability prophage induction the level of galactokinase has risen 7-fold, to augment escape synthesis by inducing the gal operon with even though fucose has not been added to the medium. The fucose. (In the case of the N102 insertion in gaiT, the escape effects of fucose and prophage induction are additive, sug- synthesis of UDP-galactose4-epimerase, coded by the pre- gesting that X-promoted transcription of the gal operon is not ceding galE gene, but not that of galactokinase is augmented Downloaded by guest on September 26, 2021 2536 Biochemistry: Adhya et al. Proc. Nat. Acad. Sci. USA 71 (1974)

TABLE 2. Role of sex and gene N in 11 escape synthesis of galactokinase

Super- Galacto- infection Addition kinase with of 1 mM specific Strain genotype Xj434 fucose Temp activity 1. K+T+ES148 (XcI857) + + 320 0.05 2. - - 410 5.8 3. + - 410 3.3 4. K+T+E81s, (XcI857sexl) - - 410 0.30 5. " + - 410 0.10 6. K+T+Es148 MRAITES (XcI857N7,53) - - 410 0.04 7. + - 410 3.4 FIG. 2 (left). Kinetics of escape synthesis of the gal operon. At various times after induction aliquots of induced cells were The bacterial strain carries the galEs148 insertion mutation. chilled and assayed for galactokinase. Open circles represent a The full genotypes of the prophages were: XcI857P3int6, in lines bacterial strain carrying the 8148 insertion mutation in galE 1-3; XcI857sexlint29S7b515b519 in lines 4 and 5; and XcI857N7,53 lysogenic for XcI857int29xl3 and filled circles a strain lysogenic in lines 6 and 7. Escape synthesis of galactokinase due to pro- for Xc1857int29P3. phage replication was prevented by adding 20 /Ag/ml of uracil FIG. 3 (right). Comparison of the sizes of fucose-induced gal and 40 ug/ml of 5-fluorodeoxyuridine to cultures 5 min before RNA and prophage-induced gal RNA. E. coli N2853 were grown induction. Where indicated, Xi434N + phage was added at a at 320 in medium A plus glycerol (0.5%) and histidine (50 ,g/ multiplicity of infection of 5. Cells were induced for 50 min. ml) to a density of 108 cells per ml. Part of the culture (panels A and C) was maintained at 320 and supplemented with fucose to a final concentration of 25 mM. Temperature of the other by fucose; data not included.) We conclude that these muta- part of the culture (panels B and D) was raised to 420. After 25 tions are at the same time polar to transcription initiating at min of either fucose or heat induction 10-ml samples were pulse initiating in X. labeled with 3[Hluridine for 30 sec. RNA was extracted and re- the gal promoter and nonpolar to transcription suspended in 0.6 M NaCl-0.06 M Na citrate, pH 7 (4 X SSC). Induction of a X-21 hybrid phage also provokes gal escape An aliquot of this RNA was put on top of a 5-20% sucrose synthesis and polarity suppression (Table 1, lines 17 and 18). gradient in 1 X SSC and centrifuged at 40 for 150 min at 56,000 This hybrid phage is nonhomologous to X in a phage region rpm in an SW56 rotor. The fractions were assayed for total required for these effects (see Discussion). labeled RNA (A and B) and for gal-specific labeled RNA (C and In the preceding section we assumed that galactokinase es- D). Centrifugation is from right to left. The numbers on the cape synthesis was due to extension of transcription initiated ordinates are the product of the indicated factor and the ex- at the X sex promoter into the gal operon. This assumption is perimental values. based on the following evidence. (1) Escape synthesis of galactokinase occurs in cyclic- Induction of a prophage bearing a sexl mutation, which re- AMP-deficient cells and the enzyme level is not increased duces leftward transcription about 10-fold (24), results in only by the addition of 2 mM cyclic AMP (data not shown). low levels of galactokinase synthesis (Table 2, lines 4 and 5). Cyclic AMP stimulates transcription from the gal promoter Superinfection with Xi4i4 does not increase these levels; the but not from the X sex promoter (S. Nakanishi, S. Adhya, M. sexl mutation is cis-dominant. Gottesman, and I. Pastan, in preparation), suggesting that (3) Galactokinase appears about 12 min after prophage in- actiyation of the gal promoter is not required for escape syn- duction, consistent with the distance between the sex pro- thesis. moter and the gal operon (Fig. 2; refs. 8 and 25). By contrast, (2) Activation of the sex promoter in cis is required for es- exonuclease, the product of the X exo gene (Fig. 1) would appear cape synthesis. Infection of a repressed X lysogen with the in about 2 min after induction (26). Like X exonuclease, ga- Xi4i4 does not stimulate lactokinase escape synthesis is under the negative control of heteroimmune phage galactokinase shuts synthesis although N and other phage functions are present the X tof gene product (26, 27). Galactokinase synthesis (Table 2, line 1). Note that transcription has been initiated off about 40 min after induction of a Xx+ prophage, but con- carries an x13 muta- at the gal promoter by fucose; evidently this transcription is tinues at a linear rate when the prophage terminated at the S148 polar insertion. This result is consis- tion. Xx13 is defective in all rightward functions, including tentwith the data presented in Table 1, where we showed that tof. insertion and ochre mutations still exerted polar effects to (4) Escape synthesis is under the positive control of the X initiated at the despite the presence gene N product. Induction of a XN - prophage does not stimu- transcription gal promoter lines 6 and of X furiction.t late galactokinase synthesis (Table 2, 7). Escape synthesis is restored by superinfection of the induced N- lysogen with Xi4i4, which carries the N gene of X. t An alternate explanation, that superinfection by Xj434 inhibits that has been eliminated. (5) We have shown above galactokinase escape syn- fucose-induced gal operon expression, is under the same controls as the of the int- of a strain induced with fucose at 320 pro- thesis expression Superinfection gal+ the to this duced less than a inhibition of galactokinase synthesis cIII region of A. Since transcript corresponding 50% to (data not shown). region initiates at the sex promoter, it is reasonable suppose Downloaded by guest on September 26, 2021 Proc. Nat. Acad. Sci. USA 71 (1974) Polarity and Transcription Termination 2537

that the gal escape transcript does so as well. The following terial DNA. To determine whether other phage functions or analysis of mRNA made after prophage induction is consistent sites are required, we compared lysogens in which portions with this interpretation. of the X genome to the left of sex have been deleted and re- (A) The synthesis of X mRNA precedes the synthesis of gal placed with bacterial DNA. Prophages deleted for sequential mRNA (in preparation); this is expected for a message origi- portions of their genome, from int to within N, still provoke nating at an internal prophage promoter and extending to escape synthesis (Table 3). Xbio256 and XbioT76 must be com- gal. plemented with N+ superinfecting phage. The deletion in (B) A transcript with an origin at sex and extending into Xbio256 is thought to remove the carboxy-terminal end of the the gal operon should be very large. Although failure to find N gene (30) and the deletion in XbioT76 ends in the middle of such a molecule would not invalidate our hypothesis (degrada- it (31). We conclude that there can be no function to the left tion could accompany synthesis), we have, in fact, been able of the bio243 deletion and no site to the left of N required for to recover this large transcript (Fig. 3). For this experiment, escape synthesis (see Fig. 1). Moreover, the bacterial DNA strain N2853 was grown in the presence of fucose at 320 or present in the various Xbio's does not block the N-mediated induced at 410 in the absence of fucose. At 25 min a 30-sec transcription from the sex promoter. pulse of ['H ]uridine was administered and the RNA extracted DISCUSSION and centrifuged in a 5-20% sucrose density gradient. Samples of the gradient were analyzed for labeled total and gal mRNA. We have shown that transcription which originates at the sex Note that (i) the size of the general RNA population in the promoter of prophage X can extend into the adjacent region two cultures is roughly the same (Fig. 3A and B); (ii) the gal of the bacterial genome, resulting in the constitutive synthesis mRNA made after fucose induction is heterogeneous and of the enzymes of the gal operon. This gal "escape synthesis" falls in a range of 12-22 S (Fig. 3C), consistent with complete is controlled by the prophage rather than by the bacterial gal and partial transcripts of the operon (11, 28); and (iii) the repressor and cyclic AMP and its receptor protein (CRP), gal mRNA synthesized as the result of prophage induction ap- which normally modulate the expression of the gal operon. pears considerably larger and, under these centrifugation Unlike normal mRNA synthesis, the transcription responsi- conditions, sediments to the bottom of the tube (Fig. 3D). ble for gal escape synthesis does not terminate at the ends of A gal-containing transcript with similar properties was re- genes or operons. It is also insensitive to the abnormal termina- covered after induction of a Xx13 lysogen carrying the S148 tion induced by polar mutations. The polarity of all the polar insertion in gal. An analysis of the molecular weight of these mutations that we tested was suppressed by induction of a transcripts, under conditions where possible RNA aggregation XN+ prophage in cis. Analysis of phage deletion mutants is excluded, remains to be done. suggests that no diffusible function other than N gene-product The finding of what appears to be a sex-gal transcript sug- is likely to be involved. If the only role of N-function is to gests that, as long as X functions are made, this species is antagonize the bacterial transcription termination factor, resistant to degradation. In fact, this mRNA molecule is de- rho, then it follows that all polarity is the consequence of pendent for its stability on the continuous expression of X rho-mediated termination. In the case of one class of polar functions (manuscript in preparation). We are determining mutations, consisting of insertion of foreign DNA segments, whether the antitermination effects of N can be dissociated S148 and 490, rho-mediated polarity has been demonstrated from the abnormal stability of the sex-promoted RNA. in vitro (11). Another class of insertion mutation, represented We have shown above that gal escape synthesis depends by N102, is polar only in a crude transcription system (high upon transcription which initiates at the X sex promoter and, salt ribosomal wash), suggesting that factors in addition to under the influence of the N gene product, extends into bac- rho are required for this mutation. A role for rho in the po-

TABLE 3. Site of recognition of gene N product Superinfection Galactokinase Prophage strain Prophage genes deleted Bacterial genes added with Xii" specific activity 1. XcI857att2 none bioA +B +C+D +uvrB + - 2.0 2. Xc1857bio386 (int-xis) bio4 - 4.2 3. XcI857bio69 (int-exo) bioA +B + - 3.4 4. XcI857bioll (int-gdm) bioA +B + - 2.0 5. XcI857biolO (int-cldI) bioA +B + - 3.3 6. Xc1857bio232 (int-cIII) bioA +B + - 4.6 7. Ac1857bio233 (int-cIII) bioA +B+C+D+uvtB - 2. 1 8. Xc1857bio242 (int-cI) bioA +B + - 6.1 9. Ac1857bio243 (int-cill) bioA +B + - 3.0 10. AcI857bio256 (int-cIII) bioA +B +C+D +uvtB - 0.10 11. Xc1857bio256 (int-cIII) bioA +B +C+D+uvtB + 2.0 12. )cI857bioT76 (in-N) bioA +B +C+D + - 0.05 13. XcI857bioT76 (int-X) bioA +B +C +D + + 3.3 The bacterial strain carries the galEs18 insertion mutation. Replicational escape synthesis was abolished by 5-fluorodeoxyuridinie as in Table 2. Cells were induced for 50 min. A slash in the genotype designates that the deletion ends within the indicated gene. The original Xbio256 strain reported by Signer et al. (29) was found to carry a sex type of mutation, which was crossed out from the transducing phage before constructirng the Xbio256 lysogen used in the experiments of lines 10 and 11. The XbioT76 lysogen, used in the experiments of lines 12 and 13, carries an N-independent (nin) mutation (38). The latter has been found not to influence escape synthesis. Downloaded by guest on September 26, 2021 2538 Biochemistry: Adhya et al. Proc. Nat. Acad. Sci. USA 71 (1974) larity produced by nonsense mutations is indicated by the 1. Campbell, A. (1961) Virology 14, 22-32. in vivo observation that these mutations prevent transcription 2. Kourilsky, P., Marcaud, L., Sheldrick, P., Luzzati, D. & Gros, F. (1968) Proc. Nat. Acad. Sci. USA 61, 1013-1020. of the adjacent promoter-distal gene, although the mutant gene 3. Heineman, S. F. & Spiegelman, W. G. (1970) Cold Spring itself is transcribed beyond the mutation (32). The presence of Harbor Symp. Quant. Biol. 35, 315-318. a rho-sensitive transcription termination signal at the end of 4. Kumar, S., Calef, E. & Szybalski, W. (1970) Cold Spring galE has been demonstrated in vitro (11). We propose, there- Harbor Symp. Quant. Biol. 35, 331-339. fore, when translation terminates (at a nonsense codon), 5. Roberts, J. (1969) Nature 224, 1168-1174. 6. Roberts, J. (1970) Cold Spring Harbor Symp. Quant. Biol. transcription can proceed only to the first rho-termination 35, 121-126. site, i.e., to the 3'-OH end of the mutant gene. Alternatively, 7. Guha, A., Tabuczynski, M. & Szybalski, W. (1968) J. Mol. it is possible that N-product has functions other than to an- Biol. 35, 207-213. tagonize rho. We hope to resolve these hypotheses by isolating 8. Kayajanian, G. & Campbell, A. (1966) Virology 30, 482-492. 9. Buttin, G. (1963) J. Mol. Biol. 7, 610-631. a rho-deficient mutant. 10. Yarmolinsky, M. & Wiesmayer, H. (1960) Proc. Nat. Acad. Our data also demonstrate that, while the prophage-ini- Sci. USA 46, 1626-1645. tiated transcript propagates indefinitely, other messages in il. de Crombrugghe, B., Adhya, S., Gottesman, M. & Pastan, the cell appear to terminate normally. This implies that anti- I. (1973) Nature New Biol. 24i, 260-264. termination by N-function requires a prophage site in cis to 12. Adhya, S., Gottesman, M. & de Crombrugghe, B. (1973) Control of Transcription, ed. Hollaender, A. (Plenum Press, gal. In agreement with the conclusions of Friedman et al. (33), New York-London). we propose that the transcription complex is influenced at a 13. Gottesman, M. E. & Yarmolinsky, M. (1968) J. Mol. Biol. phage site, either directly or indirectly, by N-function, so as 31, 487-505. to become resistant to termination (juggernaut model). This 14. Perlman, R. & Pastan, I. (1968) Biochem. Biophys. Res. Commun. 30, 656-664. site of recognition of N-function is distinct from the sites 15. Wilson, D. & Hogness, D. (1966) in Methods in Enzymology, where N-function acts. The latter represent the loci where eds. Neufield, E. & Ginsburg, V. (Academic Press, New transcription termination can occur, i.e., at the ends of op- York), Vol. VIII, pp. 229-240. erons, the ends of genes or within insertion mutationg.t The 16. Varmus, H. E., Perlman, R. L. & Pastan, I. (1970) J. Biol. location of the N recognition site appears to be at or near the Chem. 245, 2259-2267. 17. Buttin, G. (1973) J. Mol. Biol. 7, 183-205. sex promoter. Evidence for interaction between RNA poly- 18. Adhya, S. & Echols, H. (1966) J. Bacteriol. 92, 601-608. merase and N product has been reported (34, 37). 19. Saedler, H., Gullen, A., Feithern, L. & Starlinger, P. (1968) Friedman (33) has proposed that phlge 21 has an N-like Mol. Gen. Genet. 102, 79-88. function, and our data are consistent with this notion. The 20. Miller, Z., Varmus, H. E., Parks,. J. S., Perlman, R. L. & Pastan, I. (1971) J. Biol. Chem. 246, 2898-2903. failure of phage 21 to complement a X N-mutant (24, 35) may 21. Nakanishi, S., Adhya, S., Gottesman, M. & Pastan, I. (1973) reflect the fact that N recognition site is in the region of non- Proc. Nat. Acad. Sci. USA 70, 334-338. homology between the two phages. We assume that the two 22. Nakanishi, S., Adhya, S., Gottesman, M. & Pastan, I. (1973) N functions are specific for their corresponding recognition J. Biol. Chem. 248, 5937-5942. sites. 23. Franklin, N. (1974) J. Mol. Biol., in press. 24. Gottesman, M. & Weisberg, R. (1971) in The Bacteriophage Suppression of polarity caused by insertions within the A A, ed. Hershey, A. D. (Cold Spring Harbor Laboratory, Cold genome by N function has already been reported by Brachet Spring Harbor, N.Y.), pp. 113-138. et al. (36). Furthermore, it is known that nonsense mutations 25. Geiduschek, E. P. & Haselkorni, R. (1968) Annu. Rev. in X genes directly under N control are nonpolar (1, 39). It Biochem. 38, 647-676. mutations would be in the 26. Luzzati, D. (1970) J. Mol. Biol. 49, 515-519. is likely that some of these polar 27. Pero, J. (1971) in The Bacteriophage A, ed. Hershey, A. D. absence of N-functiot. The isolation of N-independent X (Cold Spring Harbor Laboratory, Cold Spring Harbor, variants permits a direct test of this hypothesis. N.Y.), pp. 599-608. These data show how control of the expression of an operon 28. Hill, C. & Echols, H. (1966) J. Mol. Biol. 19, 38-51. can be exerted by a positive control function interacting with 29. Signer, E. R., Manly, K. F. & Brunstetter, M. A. (1969) Virology 39, 137-141. its specific recognition site to prevent transcription termina- 30. Greenblatt, J. (1973) Proc. Nat. Acad. Sci. USA 70, 421-424. tion. Whether this mechanism is utilized by other operons 31. Cohen, S. N. & Hurwitz, J. (1968) J. Mol. Biol. 37, 387-406. under positive control remains an interesting possibility. 32. Hiraga, S. & Yanofsky, C. (1972) J. Mol. Biol. 72, 103-110. 33. Friedman, D., Wilgus, G. & Mural, R. (1973) J. Mol. Biol., We are indebted to Don Court and Daavid Friedman for many in press. suggestions and fruitful criticisms, to Helen Greer for gift of 34. Ghysen, A. & Pironio, M. (1972) J. Mol. Biol. 65, 259-272. several phage strains, and Naomi Franklin for communication of 35. Couturier, M., Dambly, C. & Thomas, R. (1973) Mol. Gen. unpublished results. We thank M. Harshman for preparation of Genet. 120, 231-252. the manuscript. 36. Brachet, P., Eisen, H. & Rambach, A. (1970) Mol. Gen. Genet. 108, 266-276. 37. Georgopoulos, C. P. (1971) Proc. Nat. Acad. Sci. USA 68, 2977-2981. 38. Court, D. & Campbell, A. (1972) J. Virol. 9, 938-945. t Philippe Kourilsky (see ref. 33) also suggested a distinction 39. Scandella, D. (1972) Ph. D. Dissertation, Stanford Univer- between the sites of action and recognition of X to! gene product. sity, Stanford, Calif. Downloaded by guest on September 26, 2021