Vol. 16, No. 6 Printed in U.SA.
JOURNAL OF VIROLOGY, Dec. 1975, p. 1683-1687 Copyright C) 1975 American Society for Microbiology
Chemical Stability of Bacteriophage T7 Early mRNA YOSHIHIKO YAMADA, PATRICIA A. WHITAKER, AND DAI NAKADA* Department of Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261
Received for publication 17 June 1975
T7 early mRNA produced by a gene 1 amber mutant phage, T7 am27, is chemically stable in terms of acid insolubility and T7 DNA hybridizability. However, the messenger activity of individual T7 early mRNA species, transcripts of gene 1, gene 0.7, and gene 1.3, decay with a half-life of about 6.5 min at 30 C. An extensive secondary structure is present in all T7 early mRNA species and is probably responsible for the chemical stability of the RNAs after the loss of functional activity. It is unlikely that ribosomes protect T7 early mRNA from nucleolytic degradation. We have previously shown that bacteriophage T7 early mRNA is functionally unstable and decays rather rapidly, although the same RNA is chemically stable in terms of acid insolubility and T7 DNA hybridizability (9, 10). This functional instability seems sufficient to explain why late in T7 infection only late proteins are produced in the presence of chemically stable early mRNA together with late mRNA, since discriminatory translational control against early mRNA in favor of late mRNA (1, 5) has not yet been found in T7-infected host Escherichia coli F- cells (Y. Yamada and D. Nakada, J. Mol. Biol., in press). Here we report our finding of the presence of an extensive secondary structure in T7 early mRNA which is probably responsible for the chemical stability of the RNA. Radioactive T7 early mRNA samples were prepared from UV light-irradiated E. coli D10 F- cells infected with T7 am27 (a gene 1 amber mutant which does not make T7-specific RNA polymerase and, consequently, late mRNA) and labeled with [3H]uridine. Rifampin was added 8 min after infection. Soon after the addition of rifampin, the synthesis of radioactive T7 early mRNA, in terms of acid insolubility and T7 DNA hybridizability, slowed and stopped but the accumulated RNA remained stable, as shown previously (9). The messenger activity of the accumulated total T7 early mRNA extracted from the cells showed a rapid decay with a half-life about 6.5 min at 30 C when tested in directing in vitro protein synthesis as previously reported (9, 10). Figure 1 shows the electrophoretic profiles of T7 early mRNAs in acrylamide-agarose-SDS (sodium dodecyl sulfate) gels (A, B, and C) and in nonaqueous formamide-acrylamide gels which denature RNA molecules (D, E, and F).
In acrylamide-agarose-SDS gels, gene 1 mRNA, gene 0.7 mRNA, gene 1.3 mRNA, and a mixture of gene 0.3 and gene 1.1 mRNAs were well separated (Fig. 1, A, B, and C) and they were designated as peaks I, II, III, and IV (3, 4, 8). Formamide-acrylamide gel electrophoresis was carried out by the method of Staynov, Pinder, and Gratzer (7). With RNAs of known length, we have confirmed that under the conditions used the electrophoretic mobility of RNA molecules is proportional to the molecular weight. From the gel profiles in Fig. 1, we conclude that T7 early mRNAs have an extensive secondary structure similar in extent to that of E. coli ribosomal RNAs. This conclusion was reached on the basis of the mobilities of the early mRNAs relative to ribosomal RNAs in nondenaturing and denaturing conditions (compare Fig. 1A and D). E. coli ribosomal RNAs are known to have an extensive secondary structure involving 60 to 70% of the total bases in the RNA (2). At 20 min after T7 infection (12 min after rifampin addition), at which time total T7 early mRNA has lost messenger activity, individual mRNA species still remain as large size RNAs (Fig. 1C) even under denaturing conditions in formamide-acrylamide gel (Fig. iF). However, these RNA species were found to be functionally inactive when tested individually in directing codon-specific f-Met-tRNAf binding to ribosomes. Functional decay of individual T7 early mRNA species is shown in Fig. 2. For this experiment, T7 early mRNA samples, prepared at different times after the addition of rifampin at 8 min of T7 infection, were electrophoresed in' acrylamide-agarose-SDS gels. Individual mRNA species were eluted from the gel slices at respective peak positions as shown in Fig.
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FRACTION NUMBER FIG. 1. Electrophoretic patterns of T7 early mRNA in acrylamide-agarose-SDS gels (A, B, and C) and in formamide-acrylamide gels (D, E, and F). UV-irradiated E. coli D10 F- cells were infected with T7 am27 phage at a multiplicity of 10 at 30 C as described previously (9, 10) and labeled with [3H]uridine (20 pACi per 1 pg/ml). Rifampin was added 8 min after infection and RNA was prepared from the cells withdrawn 8, 10, and 20 min after infection. RNA samples (about 5 x 105 counts/min per sample) were mixed with [14C]uracillabeled total E. coli RNA and subjected to electrophoresis in 2.5% acrylamide-05% agarose-0.2% SDS gels (A, B, and C) for 165 min at 5 mA per gel (4) and in formamide-3.6% acrylamide gels for 280 min at20 V per cm (7). Gels were sliced and radioactivity of each slice was counted. The direction of electrophoresis was from left to right in the figure: A and D, T7 am27 RNA 8 min after infection; B and E, 10 min; C and F, 20 min. Symbols: *-*, [3H]uridine-labeled T7 am27 RNA; -----, [14C]uracil-labeled E. coli RNA, 23S and 16S, from left to right.
amide-agarose-SDS gels as described in Fig. 1, and gel slices comprising each peak (peaks I through IV) were pooled. RNA was eluted from the pooled gel 0 80 slices and used as messenger to direct f-Met-tRNA m binding to ribosomes. The assay mixture, in a total 60 0 I iI0 volume of 0.1 ml, contained: 30 pg of 1 M NH4Cl1.0~~~~~ washed ribosomes; 8 pg of initiation factors; 5 pmol 40 of Tris-hydrochloride, pH 72; 0.5 ,mol of magnesium acetate; 5 pwol of NH4Cl; 0.05 pmol of guano0 sine 5'-triphosphate; 05 ,u.mol of mercaptoethanol; 0 1.4 x 105 counts/min of [35S] labeled f-Met-tRNA (6 x 20 104 counts/min per pmol). Incubation was at 35 C for Wi L W W 15 min and membrane filter-retainable radioactivity It)In 5 5 5 (Millipore Corp.) was counted. The RNA added to each reaction was adjusted to contain the same [3H] TIME (min) AFTER RIFAMPICIN ADDITION radioactivity as the RNA of time 0 sample. (A) Gene 1 FIG. 2. Functional decay of individual T7 early mRNA (peak I in acrylamide-agarose-SDS gel). The mRNA. UV-irradiated cells were infected with T7 100% value was 6,325 counts/min. (B) Gene 0.7 am27 and labeled with [3H]uridine as in Fig. 1, and mRNA (peak II), 1(I)% value, 7,184 counts/min. (C) rifampin was added 8 min after infection (time 0 in Gene 13 mRNA (peak III), 100% value, 8,431 the figure). At indicated times thereafter, aliquots of counts/min. (D) A mixture of gene 03 mRNA and the culture were withdrawn and RNA was prepared gene 1.1 mRNA (peak IV), 100% value, 8,612 from each sample. RNA was electrophoresed in acryl- counts/min. A.
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VOL. 16, 1975
1A, B, and C, and used as messenger. Messenactivity of gene 1 mRNA, gene 0.7 mRNA, and gene 1.3 mRNA (Fig. 2A, B, and C, respectively) in directing initiation factor-dependent f-[35S]Met-tRNA binding to ribosomes decayed with a half-life of about 6.5 min at 30 C which is similar to the half-life of unfractionated total T7 early mRNA. A mixture of gene 0.3 mRNA
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and gene 1.1 mRNA showed a slightly slower decay (Fig. 2D), but the reason for this observation is not known. Gene 1 mRNA was eluted from acrylamideagarose-SDS gel after electrophoresis of total T7 early mRNA and was subjected to re-electrophoresis in acrylamide-agarose-SDS gel and in formamide-acrylamide gel. Figure 3 shows the
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FRACTION NUMBER FIG. 3. Electrophoretic pattern offunctionally active and inactive 77 gene 1 mRNA in acrylamide-agaroseSDS gels and formamide-acrylamide gels. [3Hluridine-labeled T7 early mRNA was prepared from UVirradiated, T7am27-infected cells treated with rifampin at8 min after infection. RNA samples, prepared from the cells 8 and 20 min after infection, were subjected to electrophoresis in acrylamide-agarose-SDS gels as shown in Fig. 1. [3H]uridine-labeled gene 1 mRNA was eluted from the peak I region of gel slices and mixed with [14C]uracil-labeled marker E. coli RNA. The RNA mixture was subjected to electrophoresis in acrylamide-agarose-SDS gels (A and B) and in formamide-acrylamide gels (C and D) as described in Fig. I. The direction of electrophoresis was from left to right. The 8-min gene 1 mRNA represents the functionally active form (Fig. 2A, time 0) and the 20-min gene 1 RNA represents the inactive form (Fig. 2A, 12 min after rifampin addition). (A) 8-min gene 1 RNA in acrylamide-agarose-SDS gel. (B) 20-min gene RNA in acrylamideagarose-SDS gel. (C) 8-min gene 1 RNA in formamide-acrylamide gel. (D) 20-min gene 1 RNA in formamide-acrylamide gel. Symbols: 0-0, [3Hluridine-labeled gene RNA; -----, [14C]uracil-labeled E. coli RNA marker, 23S and 16S RNA from left to right.
1686
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FRACTION NUMBER FIG. 4. Polysome patterns of T7 am27-infected cells before and after the addition of rifampin. Cells were grown in the presence of [14C]uracil (0.1 pCi per 0.17 jg/ml) for several generations at 30 C and then infected with T7 am27 at a multiplicity ofinfection of10. After infection (4 min) the cells were labeled with [3H]uridine (20 pCi per 05 pg/mi), and 5 min after infection rifampin was added to a final concentration of 400 pg/ml. Cells were harvested at 5 min (A) and 17 min (B) after infection and lysed by the method ofSchwartz, Craig and Kennell (6) using Brij 58, deoxycholate and DNase. The lysate was layered on a 14 to 28% sucrose gradient containing 0.005 M Tris-hydrochloride, pH 72, 0.006 M MgS04, and 0.06 MKC1, and centrifuged at 30,000 rpm for2 h in an SW41 rotor. Radioactivity ofeach fraction was counted using an 0.025-ml portion. The rest of each fraction was used to prepare RNA for T7 DNA-hybridization after pooling fractions into three groups, as shown in the figure as gradient regions I, II, and III. Arrows indicate the position of 70S ribosomes. The direction ofcentrifugation was from right to left. (A) Polysomes 5 min after infection ([3H]uridine labeling fori min). (B)Polysomes 17 min after infection ([3H]uridine labeling forl min, then rifampin treatment for 12 min). Symbols: [3H]uridine-labeled RNA after infection. -----, [14C]uracil-labeled RNA before infection. 0,
electrophoretic profiles of gene 1 mRNA isolated at 8 and 20 min after T7 infection (rifampin was added at 8 min). In nondenaturing conditions, both RNA- samples comigrated with
23S ribosomal RNA (Fig. 3A and B). However, in denaturing conditions, the 20-min RNA moved slightly but significantly faster than the 23S RNA while the 8-min RNA comigrated
VOL. 16, 1975
with the 23S RNA (Fig. 3C and D). The results indicate that the 20-min RNA which has lost messenger activity (Fig. 2A, 12 min after rifampin) is slightly smaller in size than the 8-min RNA which is functionally active (Fig. 2A, 0 min) and that the inactive 20-min RNA still contains an extensive secondary structure. Because messenger activity in directing fMet-tRNA binding depends on the presence of the initiation codon at or near the 5'-end of messenger RNA, it may be suggested that the 20-min RNA has lost a small nucleotide sequence at or near the 5'-end of the RNA molecule. The removal of a small sequence from the 5'-end region of RNA may be a common primary inactivation step involved in the functional decay of all T7 early mRNA species. The secondary structure in all individual T7 early mRNA species reported here is probably responsible for the chemical stability of the RNAs after the loss of functional activity. Finally, we briefly describe an experiment in which polysome patterns of T7-infected cells after the addition of rifampin to halt new RNA synthesis were analyzed. T7 early mRNA-specific polysomes decayed rapidly at a similar rate as polysomes of uninfected cells after the addition of rifampin. When a 1-min pulse of [3H]uridine was given after 4 min of T7 infection, about 60% of T7 DNA-hybridizable RNA was associated with polysomes (Fig. 4A, gradient region I). After the addition of rifampin at 5 min and an additional incubation for 12 min (17 min after infection), polysomes had disappeared and only 6% of total T7 DNA-hybridizable RNA was found in the polysome region (Fig. 4B. gradient region I). On the other hand, about 70% of T7 DNA-hybridizable RNA was recovered in the gradient region III (Fig. 4B) containing materials sedimenting slower than
NOTES
1687
the 70S ribosomes. Therefore, we would like to eliminate the possibility that ribosomal protection is the reason for the chemical stability of T7 early mRNA. This work was supported by Public Health Service research grants GM 18185 and GM 21504 from the National Institutes of General Medical Sciences and from the National Science Foundation (GB 33809) to D. N. P. A. W. was supported by a Public Health Service training grant. We thank E. G. Minkley and S. Phillips for their advice on electrophoresis and F. W. Studier for T7 phages.
LITERATURE CITED 1. Blumberg, D. D., and M. H. Malamy. 1974. Evidence for the presence of nontranslated T7 late mRNA in infected F' (PIF+) episome-containing cells. J. Virol. 13:378-385. 2. Fellner, P. 1974. Structure of the 16s and 23s ribosomal RNAs, p. 169-191. In M. Nomura, A. Tisieres, and P. Lengyel (ed.), Ribosomes. Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 3. Minkley, E. G., Jr. 1974. Transcription of the early region of bacteriophage T7: characterization of the in vivo transcripts. J. Mol. Biol. 83:289-304. 4. Minkley, E. G., and D. Pribnow. 1973. Transcription of the early region of bacteriophage T7: selective initiation with dinucleotides. J. Mol. Biol. 77:255-277. 5. Morrison, T. G., and M. H. Malamy. 1971. T7 translational control mechanisms and their inhibition by F factors. Nature (London) New Biol. 231:3741. 6. Schwartz, T., E. Craig, and D. Kennell. 1970. Inactivation and degradation of messenger ribonucleic acid from the lactose operon of Escherichia coli. J. Mol. Biol. 54:299-311. 7. Staynov, D. Z., J. C. Pinder, and W. B. Gratzer. 1972.
Molecular weight determination by gel electrophoresis in non-aqueous solution. Nature (London) New Biol. 235:108-110. 8. Studier, F. W. 1973. Analysis of bacteriophage T7 early RNAs and proteins on slab gels. J. Mol. Biol. 79:237248. 9. Yamada, Y., P. A. Whitaker, and D. Nakada. 1974. Functional instability of T7 early mRNA. Nature (London) 248:335-338. 10. Yamada, Y., P. A. Whitaker, and D. Nakada. 1974. Early to late switch in bacteriophage T7 development: functional decay of T7 early messenger RNA. J. Mol. Biol. 89:293-303.
JOURNAL OF VIROLOGY, Dec. 1975, p. 1683-1687 Copyright C) 1975 American Society for Microbiology
Chemical Stability of Bacteriophage T7 Early mRNA YOSHIHIKO YAMADA, PATRICIA A. WHITAKER, AND DAI NAKADA* Department of Biochemistry, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania 15261
Received for publication 17 June 1975
T7 early mRNA produced by a gene 1 amber mutant phage, T7 am27, is chemically stable in terms of acid insolubility and T7 DNA hybridizability. However, the messenger activity of individual T7 early mRNA species, transcripts of gene 1, gene 0.7, and gene 1.3, decay with a half-life of about 6.5 min at 30 C. An extensive secondary structure is present in all T7 early mRNA species and is probably responsible for the chemical stability of the RNAs after the loss of functional activity. It is unlikely that ribosomes protect T7 early mRNA from nucleolytic degradation. We have previously shown that bacteriophage T7 early mRNA is functionally unstable and decays rather rapidly, although the same RNA is chemically stable in terms of acid insolubility and T7 DNA hybridizability (9, 10). This functional instability seems sufficient to explain why late in T7 infection only late proteins are produced in the presence of chemically stable early mRNA together with late mRNA, since discriminatory translational control against early mRNA in favor of late mRNA (1, 5) has not yet been found in T7-infected host Escherichia coli F- cells (Y. Yamada and D. Nakada, J. Mol. Biol., in press). Here we report our finding of the presence of an extensive secondary structure in T7 early mRNA which is probably responsible for the chemical stability of the RNA. Radioactive T7 early mRNA samples were prepared from UV light-irradiated E. coli D10 F- cells infected with T7 am27 (a gene 1 amber mutant which does not make T7-specific RNA polymerase and, consequently, late mRNA) and labeled with [3H]uridine. Rifampin was added 8 min after infection. Soon after the addition of rifampin, the synthesis of radioactive T7 early mRNA, in terms of acid insolubility and T7 DNA hybridizability, slowed and stopped but the accumulated RNA remained stable, as shown previously (9). The messenger activity of the accumulated total T7 early mRNA extracted from the cells showed a rapid decay with a half-life about 6.5 min at 30 C when tested in directing in vitro protein synthesis as previously reported (9, 10). Figure 1 shows the electrophoretic profiles of T7 early mRNAs in acrylamide-agarose-SDS (sodium dodecyl sulfate) gels (A, B, and C) and in nonaqueous formamide-acrylamide gels which denature RNA molecules (D, E, and F).
In acrylamide-agarose-SDS gels, gene 1 mRNA, gene 0.7 mRNA, gene 1.3 mRNA, and a mixture of gene 0.3 and gene 1.1 mRNAs were well separated (Fig. 1, A, B, and C) and they were designated as peaks I, II, III, and IV (3, 4, 8). Formamide-acrylamide gel electrophoresis was carried out by the method of Staynov, Pinder, and Gratzer (7). With RNAs of known length, we have confirmed that under the conditions used the electrophoretic mobility of RNA molecules is proportional to the molecular weight. From the gel profiles in Fig. 1, we conclude that T7 early mRNAs have an extensive secondary structure similar in extent to that of E. coli ribosomal RNAs. This conclusion was reached on the basis of the mobilities of the early mRNAs relative to ribosomal RNAs in nondenaturing and denaturing conditions (compare Fig. 1A and D). E. coli ribosomal RNAs are known to have an extensive secondary structure involving 60 to 70% of the total bases in the RNA (2). At 20 min after T7 infection (12 min after rifampin addition), at which time total T7 early mRNA has lost messenger activity, individual mRNA species still remain as large size RNAs (Fig. 1C) even under denaturing conditions in formamide-acrylamide gel (Fig. iF). However, these RNA species were found to be functionally inactive when tested individually in directing codon-specific f-Met-tRNAf binding to ribosomes. Functional decay of individual T7 early mRNA species is shown in Fig. 2. For this experiment, T7 early mRNA samples, prepared at different times after the addition of rifampin at 8 min of T7 infection, were electrophoresed in' acrylamide-agarose-SDS gels. Individual mRNA species were eluted from the gel slices at respective peak positions as shown in Fig.
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FRACTION NUMBER FIG. 1. Electrophoretic patterns of T7 early mRNA in acrylamide-agarose-SDS gels (A, B, and C) and in formamide-acrylamide gels (D, E, and F). UV-irradiated E. coli D10 F- cells were infected with T7 am27 phage at a multiplicity of 10 at 30 C as described previously (9, 10) and labeled with [3H]uridine (20 pACi per 1 pg/ml). Rifampin was added 8 min after infection and RNA was prepared from the cells withdrawn 8, 10, and 20 min after infection. RNA samples (about 5 x 105 counts/min per sample) were mixed with [14C]uracillabeled total E. coli RNA and subjected to electrophoresis in 2.5% acrylamide-05% agarose-0.2% SDS gels (A, B, and C) for 165 min at 5 mA per gel (4) and in formamide-3.6% acrylamide gels for 280 min at20 V per cm (7). Gels were sliced and radioactivity of each slice was counted. The direction of electrophoresis was from left to right in the figure: A and D, T7 am27 RNA 8 min after infection; B and E, 10 min; C and F, 20 min. Symbols: *-*, [3H]uridine-labeled T7 am27 RNA; -----, [14C]uracil-labeled E. coli RNA, 23S and 16S, from left to right.
amide-agarose-SDS gels as described in Fig. 1, and gel slices comprising each peak (peaks I through IV) were pooled. RNA was eluted from the pooled gel 0 80 slices and used as messenger to direct f-Met-tRNA m binding to ribosomes. The assay mixture, in a total 60 0 I iI0 volume of 0.1 ml, contained: 30 pg of 1 M NH4Cl1.0~~~~~ washed ribosomes; 8 pg of initiation factors; 5 pmol 40 of Tris-hydrochloride, pH 72; 0.5 ,mol of magnesium acetate; 5 pwol of NH4Cl; 0.05 pmol of guano0 sine 5'-triphosphate; 05 ,u.mol of mercaptoethanol; 0 1.4 x 105 counts/min of [35S] labeled f-Met-tRNA (6 x 20 104 counts/min per pmol). Incubation was at 35 C for Wi L W W 15 min and membrane filter-retainable radioactivity It)In 5 5 5 (Millipore Corp.) was counted. The RNA added to each reaction was adjusted to contain the same [3H] TIME (min) AFTER RIFAMPICIN ADDITION radioactivity as the RNA of time 0 sample. (A) Gene 1 FIG. 2. Functional decay of individual T7 early mRNA (peak I in acrylamide-agarose-SDS gel). The mRNA. UV-irradiated cells were infected with T7 100% value was 6,325 counts/min. (B) Gene 0.7 am27 and labeled with [3H]uridine as in Fig. 1, and mRNA (peak II), 1(I)% value, 7,184 counts/min. (C) rifampin was added 8 min after infection (time 0 in Gene 13 mRNA (peak III), 100% value, 8,431 the figure). At indicated times thereafter, aliquots of counts/min. (D) A mixture of gene 03 mRNA and the culture were withdrawn and RNA was prepared gene 1.1 mRNA (peak IV), 100% value, 8,612 from each sample. RNA was electrophoresed in acryl- counts/min. A.
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VOL. 16, 1975
1A, B, and C, and used as messenger. Messenactivity of gene 1 mRNA, gene 0.7 mRNA, and gene 1.3 mRNA (Fig. 2A, B, and C, respectively) in directing initiation factor-dependent f-[35S]Met-tRNA binding to ribosomes decayed with a half-life of about 6.5 min at 30 C which is similar to the half-life of unfractionated total T7 early mRNA. A mixture of gene 0.3 mRNA
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and gene 1.1 mRNA showed a slightly slower decay (Fig. 2D), but the reason for this observation is not known. Gene 1 mRNA was eluted from acrylamideagarose-SDS gel after electrophoresis of total T7 early mRNA and was subjected to re-electrophoresis in acrylamide-agarose-SDS gel and in formamide-acrylamide gel. Figure 3 shows the
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FRACTION NUMBER FIG. 3. Electrophoretic pattern offunctionally active and inactive 77 gene 1 mRNA in acrylamide-agaroseSDS gels and formamide-acrylamide gels. [3Hluridine-labeled T7 early mRNA was prepared from UVirradiated, T7am27-infected cells treated with rifampin at8 min after infection. RNA samples, prepared from the cells 8 and 20 min after infection, were subjected to electrophoresis in acrylamide-agarose-SDS gels as shown in Fig. 1. [3H]uridine-labeled gene 1 mRNA was eluted from the peak I region of gel slices and mixed with [14C]uracil-labeled marker E. coli RNA. The RNA mixture was subjected to electrophoresis in acrylamide-agarose-SDS gels (A and B) and in formamide-acrylamide gels (C and D) as described in Fig. I. The direction of electrophoresis was from left to right. The 8-min gene 1 mRNA represents the functionally active form (Fig. 2A, time 0) and the 20-min gene 1 RNA represents the inactive form (Fig. 2A, 12 min after rifampin addition). (A) 8-min gene 1 RNA in acrylamide-agarose-SDS gel. (B) 20-min gene RNA in acrylamideagarose-SDS gel. (C) 8-min gene 1 RNA in formamide-acrylamide gel. (D) 20-min gene 1 RNA in formamide-acrylamide gel. Symbols: 0-0, [3Hluridine-labeled gene RNA; -----, [14C]uracil-labeled E. coli RNA marker, 23S and 16S RNA from left to right.
1686
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FRACTION NUMBER FIG. 4. Polysome patterns of T7 am27-infected cells before and after the addition of rifampin. Cells were grown in the presence of [14C]uracil (0.1 pCi per 0.17 jg/ml) for several generations at 30 C and then infected with T7 am27 at a multiplicity ofinfection of10. After infection (4 min) the cells were labeled with [3H]uridine (20 pCi per 05 pg/mi), and 5 min after infection rifampin was added to a final concentration of 400 pg/ml. Cells were harvested at 5 min (A) and 17 min (B) after infection and lysed by the method ofSchwartz, Craig and Kennell (6) using Brij 58, deoxycholate and DNase. The lysate was layered on a 14 to 28% sucrose gradient containing 0.005 M Tris-hydrochloride, pH 72, 0.006 M MgS04, and 0.06 MKC1, and centrifuged at 30,000 rpm for2 h in an SW41 rotor. Radioactivity ofeach fraction was counted using an 0.025-ml portion. The rest of each fraction was used to prepare RNA for T7 DNA-hybridization after pooling fractions into three groups, as shown in the figure as gradient regions I, II, and III. Arrows indicate the position of 70S ribosomes. The direction ofcentrifugation was from right to left. (A) Polysomes 5 min after infection ([3H]uridine labeling fori min). (B)Polysomes 17 min after infection ([3H]uridine labeling forl min, then rifampin treatment for 12 min). Symbols: [3H]uridine-labeled RNA after infection. -----, [14C]uracil-labeled RNA before infection. 0,
electrophoretic profiles of gene 1 mRNA isolated at 8 and 20 min after T7 infection (rifampin was added at 8 min). In nondenaturing conditions, both RNA- samples comigrated with
23S ribosomal RNA (Fig. 3A and B). However, in denaturing conditions, the 20-min RNA moved slightly but significantly faster than the 23S RNA while the 8-min RNA comigrated
VOL. 16, 1975
with the 23S RNA (Fig. 3C and D). The results indicate that the 20-min RNA which has lost messenger activity (Fig. 2A, 12 min after rifampin) is slightly smaller in size than the 8-min RNA which is functionally active (Fig. 2A, 0 min) and that the inactive 20-min RNA still contains an extensive secondary structure. Because messenger activity in directing fMet-tRNA binding depends on the presence of the initiation codon at or near the 5'-end of messenger RNA, it may be suggested that the 20-min RNA has lost a small nucleotide sequence at or near the 5'-end of the RNA molecule. The removal of a small sequence from the 5'-end region of RNA may be a common primary inactivation step involved in the functional decay of all T7 early mRNA species. The secondary structure in all individual T7 early mRNA species reported here is probably responsible for the chemical stability of the RNAs after the loss of functional activity. Finally, we briefly describe an experiment in which polysome patterns of T7-infected cells after the addition of rifampin to halt new RNA synthesis were analyzed. T7 early mRNA-specific polysomes decayed rapidly at a similar rate as polysomes of uninfected cells after the addition of rifampin. When a 1-min pulse of [3H]uridine was given after 4 min of T7 infection, about 60% of T7 DNA-hybridizable RNA was associated with polysomes (Fig. 4A, gradient region I). After the addition of rifampin at 5 min and an additional incubation for 12 min (17 min after infection), polysomes had disappeared and only 6% of total T7 DNA-hybridizable RNA was found in the polysome region (Fig. 4B. gradient region I). On the other hand, about 70% of T7 DNA-hybridizable RNA was recovered in the gradient region III (Fig. 4B) containing materials sedimenting slower than
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the 70S ribosomes. Therefore, we would like to eliminate the possibility that ribosomal protection is the reason for the chemical stability of T7 early mRNA. This work was supported by Public Health Service research grants GM 18185 and GM 21504 from the National Institutes of General Medical Sciences and from the National Science Foundation (GB 33809) to D. N. P. A. W. was supported by a Public Health Service training grant. We thank E. G. Minkley and S. Phillips for their advice on electrophoresis and F. W. Studier for T7 phages.
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