Vol. 127, No. 3 Printed in U.S.A.

JOURNAL OF BACTCRIOOGY, Sept. 1976, p. 1292-1297 Copyright C) 1976 American Society for Microbiology

Temperature-Sensitive Ribonucleic Acid Polymerase Mutant of Salmonella typhimurium with a Defect in the ,3' Subunit BARBARA S. YOUNG,* SONIA K. GUTERMAN,

AND

ANDREW WRIGHT

Department of Molecular Biology and Microbiology, Tufts University School of Medicine, Boston, Massachusetts 02111 Received for publication 24 May 1976

Localized mutagenesis of Salmonella typhimurium followed by a [3H]uridine enrichment procedure yielded a temperature-sensitive strain with a mutation in the rpo region of the chromosome. Ribonucleic acid (RNA) polymerase (EC 2.7.7.6; nucleoside triphosphate: RNA nucleotidyltransferase) purified from this mutant was considerably less active at the nonpermissive temperature than wild-type enzyme. Furthermore, the enzyme from this mutant, unlike RNA polymerase of previously isolated temperature-sensitive mutants, was as thermostable as wild-type enzyme when preincubated at 500C. Subunit reconstitution experiments have shown that the temperature sensitivity is caused by an alteration in the /3' subunit of the enzyme.

The deoxyribonucleic acid (DNA)-dependent ribonucleic acid (RNA) polymerase of Escherichia coli (EC 2.7.7.6; nucleoside triphosphate: RNA nucleotidyltransferase) consists of at least four polypeptide subunits: a, /3, /3', and a- (4, 23). The genes for the two large subunits, /8 and l3', are located adjacent to one another at min 89 on the E. coli chromosome and are coordinately expressed (2, 8, 14, 15, 16); the gene for the a subunit is located at min 72 (12). The location of the gene for the o- subunit is unknown. The role that the individual subunits play in the transcription process is not well understood. A promising approach to the problem of determining subunit functions is the study of mutationally altered RNA polymerase. Resistance to the drug rifampin is conferred by mutations located in the structural gene for the /3 subunit. Only one temperature-sensitive mutant that produces conditionally defective RNA polymerase has been characterized, with the defect being in the /3' subunit (12, 18). These mutants have yielded information on the /8 and ,3' subunits, but the precise functions of these subunits have yet to be defined. Mutants with temperature-sensitive RNA polymerase activity have been difficult to isolate because of the lack of a powerful selection for them. We have attempted to isolate such mutants in Salmonella typhimurium using the method of localized mutagenesis described by Hong and Ames (11). Although S. typhimurium RNA polymerase had not been characterized, we have found no difference between this enzyme and the E. coli RNA polymerase. When the localized mutagenesis procedure was fol-

lowed by a [3H]uridine suicide enrichment, a large number of mutants temperature sensitive specifically in uridine incorporation were isolated. In this paper we describe one strain in which temperature sensitivity is caused by a mutation in the /3' subunit of RNA polymerase. MATERIALS AND METHODS Bacterial and phage strains. Mutants were derived from S. typhimurium LT-2 strain DB53 Cys-His- (designated as wild type in the figures and text; obtained from D. Botstein). Strain PMR4 carrying spontaneous rifampin resistance mutation rpoB4 was isolated from DB53 by P. Morris. In E. coli the genes for the RNA polymerase subunits a, ,8, and /3' have been designated rpoA, -B, and -C, respectively (9). We have used this nomenclature for S. typhimurium RNA polymerase genes. BY324 is strain DB53 rpoC32 and was constructed by transduction. The genotypes of the bacterial strains are summarized in Table 1. All of these strains were derived from strain DB53 Cys-His-. Transductions were carried our as described by Ebel-Tsipis and Botstein (7) using recipient strains lysogenic for P22 amNll sie- (21) to increase the yield of transductants when necessary. In some cases strains were subsequently cured of this prophage. Localized mutagenesis and [3Hluridine killing. The high-frequency transducing phage P22 HT/4 (19) grown on PMR4 (rpoB4) was mutagenized with hydroxylamine-hydrochloride to 5 to 10% survival (11). The mutagenized phage were mixed with DB53 lysogenic for P22 amNll sie- cells at a multiplicity of 8 to 10 particles per cell (2 x 108 to 3 x 108 cells per ml) at 370C for 10 min, diluted 50-fold in LB broth (7) containing P22 antiserum (K = 4), incubated for 3 h at 300C, concentrated 50-fold, and spread on OMglucose plates [1.1% K2HPO4-0.45% KH2PO4-0.05%

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ts RNA POLYMERASE MUTANT OF S. TYPHIMURIUM

TABLE 1. Bacterial strains used Strains Relevant genotype PMR4 rpoB4 BYL Lysogenic for P22 amNll sieBY32 rpoB4 rpoC32; Lysogenic for P22 amNII sieBY324 rpoC32

MgSO4-0.1% (NH4)2 S04-0.047% sodium citrate1.5% agar-0.2% glucose] (17) supplemented with 20

,ug of cysteine per ml, 20 ,ug of histidine per ml, and 50 ,ug of rifampin per ml. After 16 to 24 h at 300C the confluent lawns of Rifr transductants were trans-

1293

procedure have an increased frequency of mutations in the rpo (formerly called rif) region of the chromosome (11). We incubated a population of Rifr transductants with [3H]uridine at 42°C for various times, washed the cells, and stored them at 4°C to enrich for mutants that make reduced amounts of RNA at the nonpermissive temperature (42C) (1, 6). Figure 1 shows the rate of tritium killing after 10, 20, 30 and 60 min of incorporation of [3Hluridine. One percent of the survivors of the [3H]uridine selection gave rise to temperature-sensitive clones that grew at 30°C but not at 42°0, compared to 0.01% of Rifr transductants screened directly. We measured [3H]uridine incorporation in cultures of mutants after a shift from 300C to

ferred to M9 medium (0.022 M KH2PO4-0.042 M Na2HPO4-0.018 M NH4Cl-1 mM MgSO4-8.5 mM NaCl) (20) containing 1.5% Casamino Acids and 0.2% glucose (M9 CAA) and grown at 300C to an optical density at 600 nm of 0.2. Cultures were shifted to 429C for 10 min, at which time [3H]uridine (100 ,&Ci/ml; 1.0 ,ig/ml) was added. After 10, 20, 30, and 60 min, samples were filtered, washed with M9 CAA medium and then with water, suspended in M9 CAA medium (6), stored at 4°C, and assayed for viable cell count. RNA polymerase purification. RNA polymerase was purified from log-phase cells through the highsalt glycerol gradient step as described by Burgess (4). RNA synthesis in vitro. The standard reaction mixture contained 0.04 M tris(hydroxymethyl)aminomethane (Tris), pH 7.9, 0.01 M Mg9l2, 0.2 mM dithiothreitol, 0.15 M KCl, 0.1 mM ethylenediaminetetraacetate (EDTA), 0.2 mM K2HPO4, 0.2 mM KH2PO4, 15 to 25 ,ug of polydeoxyadenosine-deoxythymidine (dA-dT) (Miles Laboratories) per ml [2,83H]adenosine 5'-triphosphate (2 ,uCi/ml) (New England Nuclear), and purified RNA polymerase in a final volume of 0.25 ml. The reactions were stopped by the addition of 2 ml of 5% trichloroacetic acid containing 0.01 M sodium pyrophosphate. Precipitates were collected on Whatman GF/A filters, and the radioactivity was determined in a liquid scintillation counter. Separation and reconstitution of RNA polymerase subunits. Subunits of purified RNA polymerase were separated by electrophoresis on cellulose acetate (10) with the buffer system described by Boyd et al. (3). Core enzymes were reconstituted by dialysis of subunit mixtures against 0.05 M Tris (pH 8)-0.5 M KCl-0.01 M MgCl2-1 mM dithiothreitol-20% glycerol overnight at 4°C, followed by 3 h at room temperature with no change of dialysis fluid (22). The enzyme was reconstituted at a protein concentration of 100 jAg/ml.

0. Co

RESULTS Isolation and in vivo characterization of temperature-sensitive RNA synthesis mutants. The generalized transducing phage P22 grown on a rifampin-resistant (Rifr) donor was mutagenized with hydroxylamine (11) and used to transduce a rifampin-sensitive (Rifs) recipient to Rifr. Rifr transductants isolated by this

STORAGE AT 40 (days) FIG. 1. Kinetics of tritium killing after [3H]uridine incorporation. Cultures ofRift transductants were grown in the presence of [3H]uridine for 10, 20, 30 and 60 min at 42°C, filtered, and stored as described in Materials and Methods. Viable cell counts were determined for samples from each time point during storage at 4°C. Symbols: *, control; A, 10 min; *, 20 min; A, 30 min; 0, 60 min.

_

c

108

z

10 CO.)

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YOUNG, GUTERMAN, AND WRIGHT

J. BACTERIOL.

42°C. When a culture of the temperature-sensitive strain BY13 was shifted from 30°C to 42°C, the rate of uridine incorporation decreased dramatically within 5 min and then sharply increased between 5 and 20 min after the shift (Fig. 2A). In contrast, the rate of uridine incorporation in a culture of strain BY32 decreased sharply by 5 min and remained at the low level for the duration of the experiment (Fig. 2B). Only strain BY32 displayed this second pattern of uridine incorporation, whereas the patterns of 18 independent temperature-sensitive mutants resembled that of strain BY13. In the wild-type control the rate of uridine incorporation increased after the shift to 42°C (Fig. 2C). DNA synthesis in mutant and wild-type strains was unaffected by the temperature shift. We purified RNA polymerase from several mutants, including strain BY32, to determine whether any produced a temperature-sensitive enzyme. RNA polymerase purified from strain BY32 was temperature sensitive in vitro (see below), whereas enzymes from other mutants were not. In vitro temperature sensitivity of BY324 RNA polymerase. The rate of RNA synthesis

by wild-type RNA polymerase using a poly(dAdT) template was twofold greater at 45°C than at 30°C (Fig. 3). In contrast, the highly purified enzyme from strain BY324 (rpoC32) was less active at 450C than at 300C. After 60 min of incubation (Fig. 3), the 45°C to 30°C activity ratio was 0.5 for mutant enzyme and greater than 2 for wild-type enzyme. The temperature sensitivity of the purified enzyme from strain BY324 suggests that the temperature-sensitive mutation is located in a gene for one ofthe RNA polymerase subunits. To determine whether the temperature sensitivity of the mutant RNA polymerase was due to thermolability of the enzyme protein as found for the temperature-sensitive rpoC mutant T16 in E. coli (18), we measured the effect of preincubation of wild-type and mutant enzymes at 450C and 500C (Fig. 4). The enzymes were incubated at these temperatures in assay buffer without DNA template or nucleoside triphosphates. The residual enzyme activity was measured at 300C at the times indicated. Mutant and wild-type enzymes lost activity at the same rates, indicating that the temperature sensitivity of the BY324 RNA polymerase was

-A.

B.

10.0

a. 0

to 0

TIME (min) 2. FIG. RNA and DNA synthesis by BY13 (A), BY32 (B), and wild-type (C) cells at 30°C and 42°C. The rates of RNA synthesis in exponentially growing cultures in M9 CAA at 30°C and after a shift to 42°C (arrow) were measured by incubating 1 ml of cells for 2 min with [3H]uridine (2 ,uCi; 5 4g) at 30'C or 42°C at indicated times. Cells were precipitated with 1 ml of 10% trichloroacetic acid, and the precipitates were collected on Whatman GFIA filters and counted in a liquid scintillation counter. DNA synthesis was measured in parallel cultures growing in the presence of deoxyadenosine (300 pglml) and [3H]thymidine (2 ,uCi; 1 lAg/ml). Samples were removed, and acid-insoluble radioactivity was measured as above. Symbols: 0, RNA synthesis at 30°C; 0, RNA synthesis at 42°C; A, DNA synthesis at 30°C; A, DNA synthesis at 42°C.

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ts RNA POLYMERASE MUTANT OF S. TYPHIMURIUM

overall low recovery ofenzymatic activity was a result of subunit separation, since reconstitution of enzymes without prior subunit separation gave greater than 50% recovery of activity (data not shown). In this experiment the ts+ enzyme was rifampin resistant (purified from PMR4). The 1' subunit from BY324 was identified as the altered subunit in another experiment using ts+ rifampin-sensitive enzyme (data not shown). In E. coli the 13 subunit determines the response to rifampin (3, 10). To find out which subunit confers resistance to this drug in S.

5.00

10

20 40 30 ASSAY TIME (min)

1295

SO

60

FIG. 3. RNA synthesis in vitro at 30°C and 450C by purified wild-type and mutant RNA polymerase. Reaction mixtures containinig about 2 pg of purified RNA polymerase were incubated at 30°C or 42°C. The reactions were stopped at the times indicated, and incorporation of radioactivity into acid-insoluble counts was determined as described in Materials and Methods. Symbols: *, wild type at 30°C; O wild type at 42°C; A, BY324 at 30°C; A, BY324 at 42°C.

not due to an increased instability of the mutant protein. We are currently exploiting this property of the enzyme to determine the step at which transcription is blocked at the nonper-

50s

loU. 4

missive temperature. Identifi'cation of the defective subunit. S. typhimurium RNA polymerase consists of four maj]or polypeptide subunits that have electrophoretic properties identical to those of the four 4 10~ ~ 5101 major subunits of E. coli RiNA polymerase (unpublished data). We have thus used the E. coli nomenclature for the S. typhimurium subunits. Subunits a, ft, and ,8' from ts+ (PNMR4) and temperature-sensitive (BY324) enzymes were separated, and used in the .combinations shown in Table 2 to reconstitute RNA polymerase core enzyme (10, 22). All of the reconstituted enzymes that contained the BY324 ,8' PREINCUBATION TIME (min) subunit (lines 2, 3, 4 and 5) were temperature FIG. 4. Inactivation of wild-type and BY324 ensensitive, as indicated by a low 50°C to 30°C zyme at 45°C and 50°C. Wild-tpe and BY324 enactivity ratio (column 4). zymes were added to preheated airsay buffer (0.04 M The presence of the mutant ,B' subunit re- Tris [pH 7.91-0.01 M MgC1,=015 M KCL-0.1 mM sulted not only in temperature sensitivity but EDTA-0.2 mM dithiothreitol-02 mM KJIPOr0.2 also in a decrease in the recovery of enzymatic mM KH2PO.) to give a final protein concentration of 1 0 pg/ml. At the times indicated portions containing activity. Reconstitution of ts+ subunits (column 2.5 pg of enzyme were added to nucleoside tripho-s2, lineo 1) gave 9.8% recovery of specific enzy- phates, poly(dA-dT), and [5-3H]uridine 5'-triphosmatic activity, but reconstitution of BY324 sub- phate and incubated at 30°C for 20 min. Incorporaunits gave only 2.0% recovery (colujmn 2, line tion of radioactivity into acid-insoluble counts was 2). All enzymes reconstituted with the mutant determined and recorded as the percentage of the ,8' subunit (column 2, lines 2, 3, 4, and 5) were original activity. Symbols: *0, wild type at 45°C; O, less active than those with the wild-type 8' wild type at 50°C; A, BY324 at 45°C; A, BY324 at subunit (column 2, lines 1, 6, 7, and 8). The 50°C.

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TABLE 2. Identification of the temperature-sensitive polypeptide by reconstitution of RNA polymerase from isolated subunits Subunit RNA (cpm) 50°/30°C ac- +rifb RNA (cpm) +rif/-rif actionP 30°C 50°C tivity ratio (300C) tivity ratiof a /3 /3' 1.17 16,400 19,150 7,330 0.45 300 0.31 40 0.04 1,000 (E) t)

GO O a

(E)

O,

(p

a

8 (g)

O0)(!) /3'

0/X3 ,l3' a

) /3'

a

/3 /3'

/3

la'

a

(i) (E)

(n) @)

(Do

15,

2,020

190

0.09

40

2,300

150

0.07

470

0.20

1,560 12,610

300 24,850

0.20

340

0.22

1.97

90

0.01

15,860 8,250

15,220

3,290

0.21

22,080

0.96 2.68

0.02

90 260 70 60 100 50

120 510 90 60 70 50

120 60 100 50 50 40 50

0.02

(i) PMR4 161,530 1.43 230,840 0.91 147,720 0.48 BY324 49,400 23,720 0.02 1,050 a Core enzymes consisting of combinations of BY324 (rpoC32) and PMR4 (rpoB4) subunits were reconstituted as described in Materials and Methods. Approximately 2.4 p.g of each mixture was assayed in the standard reaction mixture at 30°C or 50°C for 40 min with [5_3H]uridine 5'-triphosphate as the labeled nucleotide. Subunits from the BY324 enzyme are indicated by the circled symbols. A control mixture (no enzyme) gave a value of 40 cpm, which was subtracted from each of the above numbers. b Samples of enzymes were incubated with rifampin (2 pg/ml) for 30 s prior to the addition of the substrates and poly(dA-dT) template. ' Ratios were calculated from the data in column 2 (-rif) and column 5 (+rif).

typhimurium we used a Rifr ts+ RNA polymerase (purified from PMR4) in the experiment described in Table 2 and assayed the reconstituted enzymes in the absence and presence of rifampin (columns 2 and 5). The ratios of these activities (column 6) indicate that the PMR4 18 subunit confers resistance to rifampin (lines 1, 4, 5, and 7). Genetic characterization of rpoC32. The mutations obtained by the localized mutagenesis technique described above were co-transducible with the rpoB4 (/3) marker. The rpoB4 (13) and rpoC32 (,3') mutations were co-transduced by phage P22 at a frequency of 68%, and the map order is argF-rpoB4 (/3)-rpoC32 (13') (unpublished data). We have found that the orientation of these genes is the same as that in E. coli (8). Although we were unable to detect spontaneous wild-type revertants of BY32, revertants were obtained at a frequency of 10-8 to 10-9 after treatment with the mutagen 2-aminopurine. RNA polymerase extracted from one such revertant was indistinguishable from wild-type

RNA polymerase with respect to its temperature optimum, and the temperature independence of this strain was co-transducible with rpoB4. From these results we assume that the temperature-sensitive phenotype of BY32 is caused by a point mutation in the 13' gene. DISCUSSION The investigation of temperature-sensitive RNA polymerase mutants is desirable for studies of subunit function, the control of subunit synthesis, and the overall control of transcription within the cell. We have found that localized mutagenesis followed by a [3Hluridine enrichment provides a powerful selection for such mutants. When 20 temperature-sensitive mutants isolated by this procedure were examined in vivo for temperature sensitivity of [3H]uridine incorporation, one (BY32) displayed a dramatic reduction of [3H]uridine incorporation at 42°0. The RNA polymerase purified from BY32 was found to have temperature-sensitive activity in vitro. The technique used to isolate this mutant has the potential to generate 18

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ts RNA POLYMERASE MUTANT OF S. TYPHIMURIUM

mutants as well as additional 3' mutants. By localized mutagenesis of the spc region of the chromosome it could be used to generate a mutants as well. The temperature sensitivity of RNA polymerase purified from BY32 and its derivatives is caused by a mutation in the gene for the ,3' subunit (rpoC). The order ofthe rpoB and rpoC genes in S. typhimurium was found to be identical to that in E. coli, and no electrophoretic differences between the RNA polymerases of the two organisms were detected. The mutant BY32 should be more useful for studying transcription both in vivo and in vitro than previously described strains with temperature-sensitive mutations in rpo genes. Transcription by RNA polymerase containing the rpoC32 mutation is temperature sensitive, but the enzyme displays wild-type stability during preincubation at high temperatures and throughout purification and storage. We intend to exploit these properties to determine which steps of the transcription reaction are altered with the mutant enzyme. Several steps in the transcription process, binding, initiation, elongation, and termination, have been defined using in vitro assays (reviewed in reference 5). One or more of these steps may be affected by the rpoC32 mutation. Measurements of the effect of high temperatures on each step of transcription by BY324 RNA polymerase will hopefully provide information on the role of the (3' subunit in the process of RNA synthesis. LITERATURE CITED 1. Babinet, C. 1970. A mutation which affects the resistance of E. coli to rifampicin, p. 37-45. In L. Silvestri (ed.), Lepetit colloquium: RNA polymerase and tran2.

3. 4.

5.

6.

7.

scription. North Holland Publishing Co., Amsterdam. Bachman, B. J., K. B. Low, and A. L. Taylor. 1976. Recalibrated linkage map of Escherichia coli K-12. Bacteriol. Rev. 40:116-167. Boyd, D. H., W. Zillig, and F. J. G. Scaife. 1974. Reference mutations for the ,B subunit of RNA polymerase. Mol. Gen. Genet. 130:315-320. Burgess, R. R. 1969. A new method for the large scale purification of Escherichia coli deoxyribonucleic aciddependent ribonucleic acid polymerase. J. Biol. Chem. 244:6160-6167. Chamberlin, M. J. 1974. The selectivity of transcription. Annu. Rev. Biochem. 43:721-775. Cronan, J. E., T. K. Ray, and P. R. Vagelos. 1970. Selection and characterization of an E. coli mutant defective in membrane lipid biosynthesis. Proc. Natl. Acad. Sci. U.S.A. 65:737-744. Ebel-Tsipis, J., and D. Botstein. 1971. Superinfection exclusion by P22 prophage in lysogens of Salmonella typhimurium. Virology 45:629-637.

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8. Erington, L., R. E. Glass, R. S. Hayward, and J. G. Scaife. 1974. Structure and orientation of an RNA polymerase operon in Escherichia coli. Nature (London) 249:519-522. 9. Hayward, R. S., and J. G. Scaife. 1976. Systematic nomenclature for the RNA polymerase genes of prokaryotes. Nature (London) 260:646-648. 10. Heil, A., and W. Zillig. 1970. Reconstitution of bacterial DNA-dependent RNA-polymerase from isolated subunits as a tool for the elucidation of the role of the subunits in transcription. FEBS Lett. 11:165-168. 11. Hong, J.-S., and B. N. Ames. 1971. Localized mutagenesis of any specific small region of the bacterial chromosome. Proc. Natl. Acad. Sci. U.S.A. 68:3158-3162. 12. Jaskunas, S. R., R. R. Burgess, and M. Nomura. 1975. Identification of a gene for the a-subunit of RNA polymerase at the str-spc region of the Escherichia coli chromosome. Proc. Natl. Acad. Sci. U.S.A. 72:5036-5040. 13. Khesin, R. B., Zh. M. Gorlenko, M. F. Shemyakin, S. L. Stvolinsky, S. Z. Mindlin, and T. S. Ilyina. 1969. Studies on the functions of the RNA polymerase components by means of mutations. Mol. Gen. Genet. 105:243-261. 14. Kirschbaum, J. B., I. V. Claeys, S. Nasi, B. Molholt, and J. H. Miller. 1975. Temperature-sensitive RNA polymerase mutants with altered subunit synthesis and degradation. Proc. Natl. Acad. Sci. U.S.A.

72:2375-2379. 15. Matzura, H., S. Molin, and 0. Maaloe. 1971. Sequential biosynthesis of the 3 and ,B' subunits of the DNAdependent RNA polymerase from Escherichia coli. J. Mol. Biol. 59:17-25. 16. Oeschger, M. P., and M. K. B. Berlyn. 1975. Regulation of RNA polymerase synthesis in Escherichia coli: a mutant unable to synthesize enzyme at 43°. Proc. Natl. Acad. Sci. U.S.A. 72:911-915. 17. Ozeki, H. 1959. Chromosome fragments participating in transduction in Salmonella typhimurium. Genetics

44:457-470. 18. Panny, S. R., A. Heil, B. Mazus, P. Palm, W. Zillig, S. Z. Mindlin, T. S. Ilyina, and R. B. Khesin. 1974. A temperature sensitive mutation of the ,8'-subunit of DNA-dependent RNA polymerase from E. coli T16. FEBS Lett. 48:241-245. 19. Schmieger, H. 1971. A method for detection of phage mutants with altered transducing abilit. Mol. Gen. Genet. 110:378-381. 20. Smith, H. O., and M. Levine. 1964. Two sequential repressions of DNA synthesis in the establishment of lysogeny by phage P22 and its mutants. Proc. Natl. Acad. Sci. U.S.A. 52:356-363. 21. Suskind, M. M., D. Botstein, and A. Wright. 1974. Superinfection exclusion by P22 prophage in lysogens of Salmonella typhimurium. III. Failure of superinfecting phage DNA to enter sie A+ lysogens. Virology

62:350-366.

22. Yarbrough, L. R., and J. Hurwitz. 1974. The reversible denaturation of deoxyribonucleic acid-dependent ribonucleic acid polymerase ofEscherichia coli. J. Biol. Chem. 249:5394-5399. 23. Zillig, W., E. Fuchs, P. Palm, D. Rabussay, and K. Zechel. 1970. On the different subunits of DNA-dependent RNA-polymerase from E. coli and their role in the complex function of the enzyme, p. 151-157. In L. Silvestri (ed.), Lepetit coloquium: RNA polymerase and transcription. North Holland Publishing Co.,

Amsterdam.