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Evidence of read-through at the termination signal for transcription of the trp operon
VIROLOGY
70, 181-184 (1976)
SHORT Evidence
of Read-Through
TAKASHI Department
of Microbial
COMMUNICATIONS
at the Termination of the trp Operon SEGAWA*
AND
Signal for Transcription
FUMIO IMAMOTO
Genetics, Research Institute for Microbial Yamada-kami, Suita, Osaka, Japan
Diseases, Osaka University,
Accepted September 24, 1975
We reported earlier that the polarity induced by nonsense mutations in the tFp operon is relaxed when the trp mRNA is part of a long transcript from the phage h promoter P,. Here we report that in addition, the transcription initiated at the PL promoter of phage A continues into the region beyond the operator-distal end of the translocated trp operon in htrp phage.
We have directly shown that a nonsense mutation in the trp operon can either express polarity or not when the operon is translocated into the early region of bacteriophage A. When the operon is transcribed by “read-through” from the P, promoter of the N gene, the mutation does not express polarity; but when the operon is transcribed from the authentic Ptrp promoter, it shows polar effects (1). This may correlate with the fact that when transcription of the trp operon originates at the PL promoter, it is insensitive to the blockage of translation by antibiotics, in contrast to the sensitivity of synthesis initiated at the Ptrp promoter (2). These findings support the hypothesis that the synthesis of some species of mRNA, including trp mRNA, depends on functional translational machinery, while that of other species of mRNA does not (3-6); and it suggests that somehow the promoter, or other region(s) located at the beginning of the operon, determine(s) whether there is “coupling” 17) of RNA polymerase to the translational machinery in. uiuo. In the analysis of PL transcripts from h&p&Asir@3 phages, we observed that the amount of mRNA hybridizable to
+8OtrpC-A DNA was significantly higher than that of mRNA hybridizable to 48OtrpED DNA (Ref. (I), first and second rows of Table 4, B). This could result from leftward transcription that continued into the region beyond the operator-distal end of the translocated trp operon in Atrp phages. Both htrpE-A and +8OtrpC-A phages include some parts of the bacterial chromosome beyond the trp region (Fig. 11, which would detect such transcription as “extra” hybridization. Consistent with those notions were the sucrose gradient sedimentation profiles for the majority of the trp mRNA molecules synthesized by transcription from the P, promoter: They sedimented significantly faster than did &p-promoted mRNA (Figs. 5, b and 4, b, respectively, in Segawa and Imamoto (1)). It was difficult to interpret the differences in mRNA sizes only by the covalent fusion of the trp mRNA to the sequence containing the iV mRNA (121, since the size of the latter is only about 12 S or so (13; also, Yamamoto and Imamoto, unpublished results), far too small to yield the observed differences. In this paper, we will report direct evidence indicating that the transcriptional stop signal located at the end of the trp * Present address: Research Unit of Molecular Genetics, School of Medicine, Keio University, 35- operon fails to terminate transcription Shinanomachi, Shinjuku, Tokyo. originated at the P, promoter of htrpE-A. 181
Copyright 8 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
182
SHORT
COMMUNICATIONS
The trp mRNA synthesized specifically from the translocated trp operon of XtrpEA phage was assayed after infection of either WDB558groP (14) or W3llO trpAEl(X) (2), both hosts in which Atrp cannot replicate. The trpAE1 deletion mutation removes the whole trp operon. In order to demonstrate uniquely P,-promoted or Ptrp-promoted synthesis of trp mRNA, strain WDB558groP (which retains a tryptophan regulator (trpR) gene) or trpAEl(A), lysogenic for A and therefore possessing h repressor, was infected with htrpE-A in the presence or absence of Ltryptophan, respectively. In all the experiments, DNA of @Otrp phages carrying various trp gene segments was used as a DNA complement in DNA-RNA hybridization assays to determine trp mRNA, and l-strand DNA of himm2’ phage was employed to assay A mRNA from the b2-J region, which is located downstream from the translocated trp operon in the AtrpE-A. The genetic maps of these phages are shown in Fig. 1. Production of the oop RNA (II) was negligible as compared to that of the b2-J mRNA in the present system: The hybridization value of [3HlRNA from trpAE1 infected with AtrpE-A in the presence of L-tryptophan with 1 -strand DNAs of himm2’, hatt80immR0 (15) and 480 were 0.49, 0.54 and 0.06% of total input, respectively, and the difference of the last two values was 0.48%. Thus, the hybrid value of the oop mRNA (0.490.48%) was very small compared with that of b2-J mRNA. In Fig. 2a it is shown that, after infection of WDB558groP with AtrpE-A , the P,.promoted transcription proceeded sequentially in the order of trpED, trpC-A and the b2J regions. The synthesis of mRNA reached a maximum during the initial period after infection and then declined until it reached a steady state (16’). The decline in the rate of synthesis seen after several minutes of infection is thought to be caused by the function of the tof gene whose product acts at the 0, operator of the N gene and substantially reduces transcription of the l-strand of the early region of A soon after the initiation of phage development (17). The reduction in the rate
_.-_-_--_--_- -_..... :. __.__-_--_- __.__ __._
__ __- __ ______._. _____6aot,,c-A
FIG. 1. Simplified molecular maps of relevant coliphage. The relative size of trp genes carried by each 48Otrp phage is estimated from the molecular weight of the corresponding polypeptides (23). The location of termini of trp operon segment carried by sites has each @Otrp relative to known mutation been described previously (241. The genetic map of AtrpE-A is based on the data of Nishimune (25) and Fiandt et al. (26). Location of the right endpoint of the bacterial substitution in the phage and that of the left endpoint of the imm” substitution in himm” (261 are represented aligned on a scale relative to A chromosome length (27). Solid, broken, zigzag and double lines indicate the region of A, 480, 21 and bacterial chromosome segments, respectively. The length of dual transcription of the translocated trp operon of AtrpE-A is represented by two open arrows, according to the conclusions inferred in this paper.
of synthesis of trpED, trpC-A and b2J mRNA was also sequential in that order as was the appearance of those mRNAs, though the decline of b2-J mRNA was rather slower than those of the other two trp mRNA species probably because of a gradual magnifying of heterogeneity in the bacterial population of the rate of transcription seen on the region far from the initiation site. These results strongly support the notion that the transcription of the b2-J region is controlled by the P, promoter and the O1. operator. (For those cells, any transcription of the trp operon from the host bacterial chromosome was repressed by the trp repressor.) An argument that the effect observed might be due to some physiological feature related to phage growth is exluded by using a host bearing the groP mutation (14, 18). An
SHORT COMMUNICATIONS
FIG. 2. Comparison of the time course of transcription originated at the P, (a) and Ptrp (b) promoters. Strains WDB558groP (14) (a), kindly supplied by Dr. T. Ogawa, or W3110 trpAE1 (h) (b) grown at 30” were infected with htrpE-A and incubated in the presence or absence of L-tryptophan (50 pg/ml), respectively. pulse-labeling was carried out with 100 PCi of 13HJuridine (19 Ci/mmole) for successive 1-min periods after infection. Other conditions and procedures for bacterial growth, pulse-labeling, preparation of RNA, and assay of trp mRNA by hybridization with 480, (bSodrpE0 and #KkrpC-A DNAs were described previously (I). Hybridization of pulse-labeled RNA (a, 6690 pg; b, 22-26 pg) with l-strand DNA of himm” (0.25 pg) was carried out in 50 ~1 of 2x SSC solution for 4 hr at 66”. Hybrids were collected on a Millipore filter by filtration and washed with 100 ml of 2x SSC. From each hybrid value, background values (a, 0.024%; b, 0.080%) of 13HlRNA from uninfected cells with the I-strand DNA of himm” were subtracted, respectively. Relatively high background values seen with the RNA from trpAEl(h) may be attributed to constitutive but weak expression of the int gene on the prophage (28). Between 105 and 143 gg of 13HlRNA (sp act, 4.9 x 1V-4.7 x 103 cpm/pg) (a) or 6.1 and 7.2 pg of VHIRNA (sp act, 1.2 x lff-1.8 x 104 cpm/gg) (b) were used for each of trp mRNA assay. Data are plotted in the middle of each period of pulse-labeling. (O), trpED mRNA; CO),trpC-A mRNA; (O), b2J mRNA.
essentially similar effect was also observed in trpAE1 infected with htrpE-A in the presence of tryptophan, despite the fact that replication of phage DNA could occur (data not shown). In contrast, during transcription initiated at the Ptrp promoter, no significant increase in the rate of transcription of the b2J region was observed (Fig. 2b). In the host employed, trpAE1 (A), replication of the phage DNA was repressed by the X
183
repressor to the same extent as in the WllB558g~0P host. The amount of trp mRNA hybridizable to @0t~pED DNA was approximately two times higher than that hybridizable to ~#&OtrpC-ADNA. This would be due to relatively high production of trpE mRNA (19, 20). Although this much inequality of the mRNA synthesized at opposite ends of the trp operon has been consistently observed under the conditions used, the amount of trpD, trpCB and trpA mRNAs produced correlates with the relative length of those segments of the trp operon (19,20). These results indicate that the transcription initiated at the P, promoter by Escherichia coli RNA polymerase is so modified that the signal for terminating transcription at the end of the trp operon is ignored. Thus, the transcription of the trp operon as a result of read-through from the P,-promoter shows at least three major differences from Ptrp-promoted transcription: (i) The transcription fails to terminate at a normal stop signal; (ii) the translational termination at nonsense mutation sites of the P,-promoted transcript fails to produce polarity (1,9,10>; and (iii) the trp mRNA as a part of P,-promoted transcript is chemically stable (16). The phenomenon of the relief of polarity of nonsense mutations has also been found in studies of the trp (8,9) orgu2 (10) operon translocated into phages $80 or A. Franklin (9) and Adhya et al. (10) have suggested that transcription initiated at the P, promoter by E. coli RNA polymerase is modified by interaction with the N protein in such a way that normal transcription stop signals in fused E. coli genes are ignored; however, the nature of the tx termination signal (9) and of a transcriptional termination signal at the end of the bioA gene (10) are still obscure (9, II 1. These disparate changes are difficult to rationalize with a single model. At least the first phenomenon must presumably occur at the level of RNA polymerase. It might arise through the interaction of a phage product with RNA polymerase, and Richardson et al. (21) have suggested that a change in polymerase that makes it insensitive to the terminating effect of rho
184
SHORT
COMMUNICATIONS
factor might account for the failure of both termination and polarity (effects 1 and 2). However, such a mechanism has several drawbacks: (i) It requires the act hoc assumption that the phage-modified polymerase binds only at some promoters, since Ptrp-promoted transcripts are normal; (ii) it is not clear how a modified polymerase could affect mRNA decay; and (iii) it is not clear why expression and functional inactivation of P,-promoted mRNA remain normal. It seems reasonable that, as an alternative, there is a more general mechanism which couples transcription, polarity, and chemical degradation but not functional inactivation, to the translational machinery (cf. 22). A phage-specific protein could then affect the coupling mechanism, perhaps through an interaction with the ribosome. ACKNOWLEDGMENTS We wish to express appreciation to Dr. D. Schlessinger for his critical reading of the manuscript. REFERENCES
1. SEGAWA, T., and IMAMOTO, F., J. Mol. Biol. 87, 741-745 (1974). 2. IIKAMOTO, F., and TANI, S., Nature New Biol. 240, 172175 (1972). 3. IMAMOTO, F., and KANO, Y., Nature New Biol. 232, 169-173 (1971). 4. SCHLESSINGER, D., In “Stadler Symposia, University of Missouri, Columbia,” Vol. 3, pp. I12. 1971. 5. CRAIG, E., Genetics, 70, 331-336 (1972). 6. IMAMOTO, F., J. Mol. Biol. 74, 113-136 (1973). 7. STENT, G. S., Science, 144, 816820 (1964). 8. ZALKIN, H., YANOFSKY, C., and SQUIRES, C. L., J. Biol. Chem. 249, 465-475 (1974). 9. FRANKLIN, N. C., J. Mol. Biol. 89, 33-48 (1974). 10. ADHYA, S., G~TTESMAN, M., and DE CROM-
BRUGGHE, B., Proc. Nat. Acad. Sci. USA 71, 2534-2538 (1974). HAYES, S., and SZYBALSKI, W., Mol. Gen. Genet. ll. 126, 275-290 (1973). S., and IMAMOTO, F., J. Mol. Biol. 92,30512. TANI, 309 (1975). 13. ROBERTS, J. W., Cold Spring Harbor Symp. Quant. Biol. 35, 121-126 (1970). 14, OGAWA, T., J. Mol. Biol. 91, in the press (1975). 15. FRANKLIN, N. C., DOVE, W. F., and YANOFSKY, C., B&hem. Biophys. Res. Commun. 18, 91@ 923 (1965). 16. YAMAMOTO, T., and IMAMOTO, F., J. Mol. Biol. 92, 289-304 (1975). 17. SZYBALSKI, W., BQVRE, K., FIANDT, M., HAYES, S., HRADECNA, Z., KUMAR, S., LOZERON, H. A., NIJKAMP, H. J. J., and STEVENS, W. F.,
Cold Spring Harbor Symp. Quant. Biol. 35, 341-353 (1970).
18. GEORGOPOIJL~~, C.
P., and HERSKOWITZ, I., In “The Bacteriophage Lambda” (A. D. Hershey, ed.), pp. 553-564. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y., 1971. 19. IMAMOTO, F., J. Mol. Biol. 43, 51-69 (1969). 20. IMAMOTO, F., Mol. Gen. Genet. 106, 123-138 (1970). 21. RICHARDSON, J. P., GRIMLEY, C., and LOWERY,
C., Proc. Nat. Acad. Sci. USA 72, 17251728 (1975). 22. IMAMOTO, F., and SCHLESSINGER, D., Mol. Gen. Genet. 135, 29-38 (19’74). 23. IMAMOTO, F., and YANOFSKY, C., J. Mol. Biol. 28, l-23 (1967). 24. IMAMOTO, F., KANO, Y., and TANI, S., Cold
Spring Harbor Symp. Quant. Biol. 35,471-490 (1970). 25. NISHIMUNE, Y., Virology, 53, 236246 (1973). 26. FIANDT, M., SZYBALSKI, W., and IMAMOTO, F., Virology, 61, 312-314 (1974). 27. DAVIDSON, N., and SZYBALSKI, W., In “The Bacteriophage Lambda” (A. D. Hershey. ed.), pp. 45-82. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. 1971. 28. SHIMADA, K., and CAMPBELL, A., Proc. Nat. Acad. Sci. USA 71, 237-241 (1974).
70, 181-184 (1976)
SHORT Evidence
of Read-Through
TAKASHI Department
of Microbial
COMMUNICATIONS
at the Termination of the trp Operon SEGAWA*
AND
Signal for Transcription
FUMIO IMAMOTO
Genetics, Research Institute for Microbial Yamada-kami, Suita, Osaka, Japan
Diseases, Osaka University,
Accepted September 24, 1975
We reported earlier that the polarity induced by nonsense mutations in the tFp operon is relaxed when the trp mRNA is part of a long transcript from the phage h promoter P,. Here we report that in addition, the transcription initiated at the PL promoter of phage A continues into the region beyond the operator-distal end of the translocated trp operon in htrp phage.
We have directly shown that a nonsense mutation in the trp operon can either express polarity or not when the operon is translocated into the early region of bacteriophage A. When the operon is transcribed by “read-through” from the P, promoter of the N gene, the mutation does not express polarity; but when the operon is transcribed from the authentic Ptrp promoter, it shows polar effects (1). This may correlate with the fact that when transcription of the trp operon originates at the PL promoter, it is insensitive to the blockage of translation by antibiotics, in contrast to the sensitivity of synthesis initiated at the Ptrp promoter (2). These findings support the hypothesis that the synthesis of some species of mRNA, including trp mRNA, depends on functional translational machinery, while that of other species of mRNA does not (3-6); and it suggests that somehow the promoter, or other region(s) located at the beginning of the operon, determine(s) whether there is “coupling” 17) of RNA polymerase to the translational machinery in. uiuo. In the analysis of PL transcripts from h&p&Asir@3 phages, we observed that the amount of mRNA hybridizable to
+8OtrpC-A DNA was significantly higher than that of mRNA hybridizable to 48OtrpED DNA (Ref. (I), first and second rows of Table 4, B). This could result from leftward transcription that continued into the region beyond the operator-distal end of the translocated trp operon in Atrp phages. Both htrpE-A and +8OtrpC-A phages include some parts of the bacterial chromosome beyond the trp region (Fig. 11, which would detect such transcription as “extra” hybridization. Consistent with those notions were the sucrose gradient sedimentation profiles for the majority of the trp mRNA molecules synthesized by transcription from the P, promoter: They sedimented significantly faster than did &p-promoted mRNA (Figs. 5, b and 4, b, respectively, in Segawa and Imamoto (1)). It was difficult to interpret the differences in mRNA sizes only by the covalent fusion of the trp mRNA to the sequence containing the iV mRNA (121, since the size of the latter is only about 12 S or so (13; also, Yamamoto and Imamoto, unpublished results), far too small to yield the observed differences. In this paper, we will report direct evidence indicating that the transcriptional stop signal located at the end of the trp * Present address: Research Unit of Molecular Genetics, School of Medicine, Keio University, 35- operon fails to terminate transcription Shinanomachi, Shinjuku, Tokyo. originated at the P, promoter of htrpE-A. 181
Copyright 8 1976 by Academic Press, Inc. All rights of reproduction in any form reserved.
182
SHORT
COMMUNICATIONS
The trp mRNA synthesized specifically from the translocated trp operon of XtrpEA phage was assayed after infection of either WDB558groP (14) or W3llO trpAEl(X) (2), both hosts in which Atrp cannot replicate. The trpAE1 deletion mutation removes the whole trp operon. In order to demonstrate uniquely P,-promoted or Ptrp-promoted synthesis of trp mRNA, strain WDB558groP (which retains a tryptophan regulator (trpR) gene) or trpAEl(A), lysogenic for A and therefore possessing h repressor, was infected with htrpE-A in the presence or absence of Ltryptophan, respectively. In all the experiments, DNA of @Otrp phages carrying various trp gene segments was used as a DNA complement in DNA-RNA hybridization assays to determine trp mRNA, and l-strand DNA of himm2’ phage was employed to assay A mRNA from the b2-J region, which is located downstream from the translocated trp operon in the AtrpE-A. The genetic maps of these phages are shown in Fig. 1. Production of the oop RNA (II) was negligible as compared to that of the b2-J mRNA in the present system: The hybridization value of [3HlRNA from trpAE1 infected with AtrpE-A in the presence of L-tryptophan with 1 -strand DNAs of himm2’, hatt80immR0 (15) and 480 were 0.49, 0.54 and 0.06% of total input, respectively, and the difference of the last two values was 0.48%. Thus, the hybrid value of the oop mRNA (0.490.48%) was very small compared with that of b2-J mRNA. In Fig. 2a it is shown that, after infection of WDB558groP with AtrpE-A , the P,.promoted transcription proceeded sequentially in the order of trpED, trpC-A and the b2J regions. The synthesis of mRNA reached a maximum during the initial period after infection and then declined until it reached a steady state (16’). The decline in the rate of synthesis seen after several minutes of infection is thought to be caused by the function of the tof gene whose product acts at the 0, operator of the N gene and substantially reduces transcription of the l-strand of the early region of A soon after the initiation of phage development (17). The reduction in the rate
_.-_-_--_--_- -_..... :. __.__-_--_- __.__ __._
__ __- __ ______._. _____6aot,,c-A
FIG. 1. Simplified molecular maps of relevant coliphage. The relative size of trp genes carried by each 48Otrp phage is estimated from the molecular weight of the corresponding polypeptides (23). The location of termini of trp operon segment carried by sites has each @Otrp relative to known mutation been described previously (241. The genetic map of AtrpE-A is based on the data of Nishimune (25) and Fiandt et al. (26). Location of the right endpoint of the bacterial substitution in the phage and that of the left endpoint of the imm” substitution in himm” (261 are represented aligned on a scale relative to A chromosome length (27). Solid, broken, zigzag and double lines indicate the region of A, 480, 21 and bacterial chromosome segments, respectively. The length of dual transcription of the translocated trp operon of AtrpE-A is represented by two open arrows, according to the conclusions inferred in this paper.
of synthesis of trpED, trpC-A and b2J mRNA was also sequential in that order as was the appearance of those mRNAs, though the decline of b2-J mRNA was rather slower than those of the other two trp mRNA species probably because of a gradual magnifying of heterogeneity in the bacterial population of the rate of transcription seen on the region far from the initiation site. These results strongly support the notion that the transcription of the b2-J region is controlled by the P, promoter and the O1. operator. (For those cells, any transcription of the trp operon from the host bacterial chromosome was repressed by the trp repressor.) An argument that the effect observed might be due to some physiological feature related to phage growth is exluded by using a host bearing the groP mutation (14, 18). An
SHORT COMMUNICATIONS
FIG. 2. Comparison of the time course of transcription originated at the P, (a) and Ptrp (b) promoters. Strains WDB558groP (14) (a), kindly supplied by Dr. T. Ogawa, or W3110 trpAE1 (h) (b) grown at 30” were infected with htrpE-A and incubated in the presence or absence of L-tryptophan (50 pg/ml), respectively. pulse-labeling was carried out with 100 PCi of 13HJuridine (19 Ci/mmole) for successive 1-min periods after infection. Other conditions and procedures for bacterial growth, pulse-labeling, preparation of RNA, and assay of trp mRNA by hybridization with 480, (bSodrpE0 and #KkrpC-A DNAs were described previously (I). Hybridization of pulse-labeled RNA (a, 6690 pg; b, 22-26 pg) with l-strand DNA of himm” (0.25 pg) was carried out in 50 ~1 of 2x SSC solution for 4 hr at 66”. Hybrids were collected on a Millipore filter by filtration and washed with 100 ml of 2x SSC. From each hybrid value, background values (a, 0.024%; b, 0.080%) of 13HlRNA from uninfected cells with the I-strand DNA of himm” were subtracted, respectively. Relatively high background values seen with the RNA from trpAEl(h) may be attributed to constitutive but weak expression of the int gene on the prophage (28). Between 105 and 143 gg of 13HlRNA (sp act, 4.9 x 1V-4.7 x 103 cpm/pg) (a) or 6.1 and 7.2 pg of VHIRNA (sp act, 1.2 x lff-1.8 x 104 cpm/gg) (b) were used for each of trp mRNA assay. Data are plotted in the middle of each period of pulse-labeling. (O), trpED mRNA; CO),trpC-A mRNA; (O), b2J mRNA.
essentially similar effect was also observed in trpAE1 infected with htrpE-A in the presence of tryptophan, despite the fact that replication of phage DNA could occur (data not shown). In contrast, during transcription initiated at the Ptrp promoter, no significant increase in the rate of transcription of the b2J region was observed (Fig. 2b). In the host employed, trpAE1 (A), replication of the phage DNA was repressed by the X
183
repressor to the same extent as in the WllB558g~0P host. The amount of trp mRNA hybridizable to @0t~pED DNA was approximately two times higher than that hybridizable to ~#&OtrpC-ADNA. This would be due to relatively high production of trpE mRNA (19, 20). Although this much inequality of the mRNA synthesized at opposite ends of the trp operon has been consistently observed under the conditions used, the amount of trpD, trpCB and trpA mRNAs produced correlates with the relative length of those segments of the trp operon (19,20). These results indicate that the transcription initiated at the P, promoter by Escherichia coli RNA polymerase is so modified that the signal for terminating transcription at the end of the trp operon is ignored. Thus, the transcription of the trp operon as a result of read-through from the P,-promoter shows at least three major differences from Ptrp-promoted transcription: (i) The transcription fails to terminate at a normal stop signal; (ii) the translational termination at nonsense mutation sites of the P,-promoted transcript fails to produce polarity (1,9,10>; and (iii) the trp mRNA as a part of P,-promoted transcript is chemically stable (16). The phenomenon of the relief of polarity of nonsense mutations has also been found in studies of the trp (8,9) orgu2 (10) operon translocated into phages $80 or A. Franklin (9) and Adhya et al. (10) have suggested that transcription initiated at the P, promoter by E. coli RNA polymerase is modified by interaction with the N protein in such a way that normal transcription stop signals in fused E. coli genes are ignored; however, the nature of the tx termination signal (9) and of a transcriptional termination signal at the end of the bioA gene (10) are still obscure (9, II 1. These disparate changes are difficult to rationalize with a single model. At least the first phenomenon must presumably occur at the level of RNA polymerase. It might arise through the interaction of a phage product with RNA polymerase, and Richardson et al. (21) have suggested that a change in polymerase that makes it insensitive to the terminating effect of rho
184
SHORT
COMMUNICATIONS
factor might account for the failure of both termination and polarity (effects 1 and 2). However, such a mechanism has several drawbacks: (i) It requires the act hoc assumption that the phage-modified polymerase binds only at some promoters, since Ptrp-promoted transcripts are normal; (ii) it is not clear how a modified polymerase could affect mRNA decay; and (iii) it is not clear why expression and functional inactivation of P,-promoted mRNA remain normal. It seems reasonable that, as an alternative, there is a more general mechanism which couples transcription, polarity, and chemical degradation but not functional inactivation, to the translational machinery (cf. 22). A phage-specific protein could then affect the coupling mechanism, perhaps through an interaction with the ribosome. ACKNOWLEDGMENTS We wish to express appreciation to Dr. D. Schlessinger for his critical reading of the manuscript. REFERENCES
1. SEGAWA, T., and IMAMOTO, F., J. Mol. Biol. 87, 741-745 (1974). 2. IIKAMOTO, F., and TANI, S., Nature New Biol. 240, 172175 (1972). 3. IMAMOTO, F., and KANO, Y., Nature New Biol. 232, 169-173 (1971). 4. SCHLESSINGER, D., In “Stadler Symposia, University of Missouri, Columbia,” Vol. 3, pp. I12. 1971. 5. CRAIG, E., Genetics, 70, 331-336 (1972). 6. IMAMOTO, F., J. Mol. Biol. 74, 113-136 (1973). 7. STENT, G. S., Science, 144, 816820 (1964). 8. ZALKIN, H., YANOFSKY, C., and SQUIRES, C. L., J. Biol. Chem. 249, 465-475 (1974). 9. FRANKLIN, N. C., J. Mol. Biol. 89, 33-48 (1974). 10. ADHYA, S., G~TTESMAN, M., and DE CROM-
BRUGGHE, B., Proc. Nat. Acad. Sci. USA 71, 2534-2538 (1974). HAYES, S., and SZYBALSKI, W., Mol. Gen. Genet. ll. 126, 275-290 (1973). S., and IMAMOTO, F., J. Mol. Biol. 92,30512. TANI, 309 (1975). 13. ROBERTS, J. W., Cold Spring Harbor Symp. Quant. Biol. 35, 121-126 (1970). 14, OGAWA, T., J. Mol. Biol. 91, in the press (1975). 15. FRANKLIN, N. C., DOVE, W. F., and YANOFSKY, C., B&hem. Biophys. Res. Commun. 18, 91@ 923 (1965). 16. YAMAMOTO, T., and IMAMOTO, F., J. Mol. Biol. 92, 289-304 (1975). 17. SZYBALSKI, W., BQVRE, K., FIANDT, M., HAYES, S., HRADECNA, Z., KUMAR, S., LOZERON, H. A., NIJKAMP, H. J. J., and STEVENS, W. F.,
Cold Spring Harbor Symp. Quant. Biol. 35, 341-353 (1970).
18. GEORGOPOIJL~~, C.
P., and HERSKOWITZ, I., In “The Bacteriophage Lambda” (A. D. Hershey, ed.), pp. 553-564. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y., 1971. 19. IMAMOTO, F., J. Mol. Biol. 43, 51-69 (1969). 20. IMAMOTO, F., Mol. Gen. Genet. 106, 123-138 (1970). 21. RICHARDSON, J. P., GRIMLEY, C., and LOWERY,
C., Proc. Nat. Acad. Sci. USA 72, 17251728 (1975). 22. IMAMOTO, F., and SCHLESSINGER, D., Mol. Gen. Genet. 135, 29-38 (19’74). 23. IMAMOTO, F., and YANOFSKY, C., J. Mol. Biol. 28, l-23 (1967). 24. IMAMOTO, F., KANO, Y., and TANI, S., Cold
Spring Harbor Symp. Quant. Biol. 35,471-490 (1970). 25. NISHIMUNE, Y., Virology, 53, 236246 (1973). 26. FIANDT, M., SZYBALSKI, W., and IMAMOTO, F., Virology, 61, 312-314 (1974). 27. DAVIDSON, N., and SZYBALSKI, W., In “The Bacteriophage Lambda” (A. D. Hershey. ed.), pp. 45-82. Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. 1971. 28. SHIMADA, K., and CAMPBELL, A., Proc. Nat. Acad. Sci. USA 71, 237-241 (1974).