Proc. Natl. Acad. Sci. USA Vol. 74, No. 5, pp. 1831-1835, May 1977
Biochemistry
Chromosomal location of a structural gene for the RNA polymerase oa factor in Escherichia coli* (F' plasmid/gene dosage effect/Salmonella a factor/immunoprecipitation/dnaG region)
YOSHIKAZU NAKAMURA, TOSHIO OSAWA, AND TAKASHI YURA Institute for Virus Research, Kyoto University, Kyoto, Japan
Communicated by Norton D. Zinder, January 31, 1977
streaking on appropriate selective medium, and the resulting hybrid strains of S. typhimurium harboring an E. coli F' plasmid were tested for a factor synthesis. Media and Chemicals. Minimal medium used in most experiments was medium E (19) supplemented with 0.5% glucose and amino acids, purines, or pyrimidines at 50 ,ug/ml, and vitamins at 1 ,g/ml as required. F' strains were grown in appropriate selective media either lacking certain nutrients required by F- segregants, or containing sugar not utilizable by F- segregants, to minimize growth of F- cells. Chemicals used are generally those described previously (16). Radioactive Labeling of Cells. To determine the rate of synthesis of RNA polymerase including a, a logarithmic phase culture at about 5 X 108 cells per ml was labeled at 37° with 10 gCi/ml of a synthetic mixture of 15 3H-labeled L-amino acids (New England Nuclear Corp., NET-250) for 2 min and chased with 1% casamino acids (Difco) for 3 min, unless otherwise indicated. Sodium azide (20 mM) was added in an ice bath, and cells were collected by centrifugation and washed in Tris-HCI (pH 7.8) containing EDTA and NaCl (20). Preparation of Crude Extract. Cells were lysed by treatment with lysozyme/EDTA and Brij 58, and then by sonication as described previously (20). Determination of the Polymerase Proteins. The polymerase proteins were selectively precipitated from crude extract by treatment with antiserum against E. coli RNA polymerase holoenzyme, and treated with sodium dodecyl sulfate (NaDodSO4) as described previously (20). In most experiments presented below, logarithmic-phase cells of an F- strain (KY1400) labeled for 1 hr with ['4C]leucine (330 mCi/mmol, 1 ,uCi/ml) were used as internal reference; cells containing 7 X 105 cpm of trichloroacetic acid-insoluble radioactivity were added to each sample of 3H-labeled cells before crude extract was prepared. Polyacrylamide gel electrophoresis with NaDodSO4 and estimation of differential rate of synthesis of polymerase proteins was carried out by a modification of the method described previously (21). The 3H/14C ratio for each protein band was divided by that for bulk protein to estimate relative differential synthesis rates.
A set of F' strains of Escherichia coli K-12 ABSTRACT partially diploid for various chromosomal segments has been examined for possible gene dosage effects in the synthesis of a factor of the DNA-dependent RNA polymerase (RNA nucleotidyltransferase; nucleoside-triphosphate:RNA nucleotidyltransferase, EC 2.7.7.6). It was found that all F' strains diploid for the dnaG region synthesize a at rates two to three times higher than other F' or F- strains. Moreover, strains of Salmonella typhimurium harboring these F' plasmids produce E. coli a in addition to Salmonella a. This has been shown on the basis of the finding that Salmonella a can be precipitated with antiserum against E. coli RNA polymerase but is distinguishable from E. coli a in its mobility in sodium dodecyl sulfate/polyacrylamide gel electrophoresis. E. coli a polypeptides thus produced seem to be stable in cells of S. typhimurium. These results indicate that a structural gene for a (rpoD) is located at the metC-argG region, probably near the dnaG locus (66 min on the current genetic map of E. coli).
RNA polymerase (RNA nucleotidyltransferase; nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) of Escherichia coli is composed of at least four different proteins, a, (',and a (1). The problem of organization and expression of genes determining the structure of these polymerase proteins is basic to any understanding of gene regulation at the level of transcription. The structural gene for f (rpoB) has been identified by mutations conferring on cells resistance to several antibiotics including rifampicin (2-6), and located at 88.5 min on the current genetic map (7). The gene for ,B' (rpoC) is closely linked to that for 1 (8-10), and appears to form an operon with the ( gene (11, 12). The a gene (rpoA) has recently been identified and mapped near the cluster of ribosomal protein genes at 72 min (13-15). We now report that a structural gene for the a factor, rpoD, is located at the metC-argG region, probably near the dnaG locus (around 66 min) on the chromosome of E. coli. It also became apparent during this study that the a gene of E. coli present on F' plasmids can be expressed efficiently in cells of Salmonella typhimurium. Moreover, E. coli a polypeptides thus produced appear to be metabolically stable in S. typhimurium.
MATERIALS AND METHODS Bacterial Strains. Strains of E. coli K-12 and of S. typhimurium used are listed in Table 1. A number of F' plasmids carrying different segments of the E. coli chromosome (17) were obtained from B. Bachmann (see Fig. 1). Salmonella strains were kindly provided by T. Iino and K. E. Sanderson; they were mated with each of the E. coli F' strains by cross-
RESULTS Gene Dosage Effects in a Factor Synthesis in E. coli Merodiploids. A set of 19 F' strains partially diploid for different portions of the E. coli chromosome was first examined for the synthesis of a and other RNA polymerase proteins. Because the F' plasmids used (Fig. 1) cover most parts of the entire chromosome with partial overlaps, some of the F' strains may be expected to be diploid for a structural gene for a (rpoD) and
Abbreviations: Gene symbols for E. coli and S. typhimurium are those described in refs. 7 and 18, respectively. NaDodSO4, sodium dodecyl sulfate. * Presented at the 48th Annual Meeting of the Genetics Society of Japan (October 1976, Osaka).
might therefore produce significantly higher levels of a than F- or other F' strains, due to gene dosage effect. Table 2 shows some of the typical results obtained in such an experiment. In
1831
1832
Proc. Natl. Acad. Sci. USA 74 (1977)
Biochemistry: Nakamura et al. Table 1. Bacterial strains Strain
Sex
Chromosomal markers
Source or ref.
Escherichia coli K-12 F141/JC1553
B. J. Bachmann F' argG metBhis leu (CGSC 4248) recA mitl xyl malA
gal lacY str tonA tsx supE F126/KL181 F' his trp pyrD thi recA mitl xyl malA galK str F116/KL110 F' The same as in F141/JC1553 except thyA F152/KL253 F' his trp pyrD thi tyrA recA mtl xyl malA galK str F- his ilv argH KY1400 trpE9829 tyr lacZ tonA tsx bfe sup- 126 F- leu thyA dnaG tonB PC3 str Salmonella typhimurium F- thy argE [correST4 sponds to argG of E. coli (18)] F' The same as in ST4 F116/ST4 F' The same as in ST4 F122/ST4 F- pro leu ara gal xyl SW1292 rha str F134/SW1292 F' The same as in SW1292
B. J. Bachmann (CGSC 4253) B. J. Bachmann
(CGSC 4254) B. J. Bachmann
(CGSC 4287) Ref. 16 1t116
129
122
Y. Hirota
K. E. Sanderson This paper This paper T. Iino
This paper
this and all subsequent experiments, differential synthesis rates of polymerase subunits among total protein synthesized have been normalized to those for the E. coli F- strain continuously labeled with [14C]leucine. It is evident that strains harboring F141 or F 116 synthesize at rates two to three times higher than those for other F' strains, whereas they produce other polymerase proteins essentially at normal rates. F- segregants derived from these F' strains produce a at normal rates, indicating that the higher rates of synthesis is correlated with the presence of an F' plasmid. The differential synthesis rate of for F- haploid strains under these conditions is approximately 0.1% of total protein synthesized, and represents about one-third of that for core polymerase subunits on molar basis (20, 22, 23). The relative differential synthesis rates of a for all F' strains tested are presented in Fig. 2 as a function of the map location of the chromosomal segment carried by each of the F' plasmids used. It can be seen that the strains partially diploid for the metC-argG region, corresponding approximately to 64-68 min on the genetic map (7), invariably exhibit two to three times higher rates of a synthesis than other strains. Again, F- segregants derived from these strains show normal levels of synthesis (data not shown). All the remaining strains with one or two possible exceptions (those harboring F134 or F101) produce normal levels of These results provided evidence, though indirect, that a structural gene for is located at the metC-argG region. S. typliimurium Strains Harboring E. coil F' Plasmids. A more direct test for the location of a structural gene for on the E. coli chromosome was carried out by using strains of S. tya
a
a
a
a.
a
a
FIG. 1. Genetic map of E. coli K-12 showing approximate locations of chromosomal segments carried by the F' plasmids used. Each F' is represented by an arc with a number assigned by K. B. Low (17); the "map numbers" rather than original numbers are used throughout this paper for convenience in presenting the data. The presence of F' in each merodiploid strain has been checked by its sensitivity to male-specific phages, appearance of F- segregants, and by its ability to transfer F' to other F- recA strains. F' plasmids drawn with heavy lines are those that were shown to carry a structural gene for af (see text).
phimurium harboring an E. coli F' plasmid, because it was recently found that a protein of S. typhimurium can be precipitated by antiserum to F. coli RNA polymerase and migrates slightly behind that of E. coli in NaDodSO4/polyacrylamide gel electrophoresis (24). Thus, several Salmonella strains harboring an F' plasmid suspected of carrying a af gene were first examined for their differential rates of a synthesis. It was expected that these hybrid F' strains might produce E. coli af protein as well as Salmonella a, giving rise to apparently higher levels of total cr synthesis, provided that the E. coli af gene can be expressed in S. typhimurium. That this is indeed the case is shown in Table 3; both the strain harboring Fl16 and the strain harboring F122 produced af at rates significantly higher than those for the parental F- strain (ST4). In constrast, the level of af synthesis for the strain harboring F134 did not appreciably exceed that for the parental haploid strain. It should be noted that F' plasmid F134, when present in cells of E. coli, brought about significantly higher levels of af production under similar conditions (see Fig. 2). The rates of synthesis of (3 and (3' subunits in the Salmonella strains were not affected appreciably by introduction of any of the E. coli F' plasmids tested. These data are therefore consistent with, and lend further support to, the suggestion that a ar gene is located at the metC-argG region of the E. coli chromosome, although af polypeptides of F. coli and of S. typhimurium were not resolved in these experiments. Identification' of E. coili a Synthesized in Salmonella Hybrid F' Strains. We then attempted to demonstrate the synthesis of F. coli a in Salmonella strains harboring an E. coli F' plasmid, F116 or F122. In the first experiment, 314-labeled proteins extracted from such strains were precipitated by antiserum and were subjected to prolonged electrophoresis on longer columns. When a mixture of differentially labeled polymerase proteins was analyzed under these conditions, a polypeptides of E. coli and of S. typhimurium were clearly
Proc. Natl. Acad. Sci. USA 74 (1977)
Biochemistry: Nakamura et al. Table 2. Synthesis of RNA polymnerase proteins in E. coli merodiploids
1833
Table 3. Synthesis of RNA polymerase proteins in S. typhimurium strains harboring an E. coli F' plasmid
Relative differential synthesis rates
Relative differential synthesis rates
Strain
,B
,s
a
a
Strain
a
,B
,B
F141/JC1553 F- segregant of above*
0.87 0.76 0.81 0.77 0.86 0.71
0.59 0.74 0.80 0.72 0.74 0.55
2.37 1.12 3.17 0.88 1.17 1.11
0.55 0.60 0.60 0.55 0.51 0.55
ST4 (F-) F116/ST4 F122/ST4 SW1292 (F-) F134/SW1292
1.03 3.87 3.38 1.15 1.49
1.20 1.50 1.34 0.69 0.72
1.21 1.58 1.44 0.75 1.00
F116/KL110 F- segregant of above* F126/KL181 F152/KL253
General procedures and conditions are as described in Materials and Methods. Cells were grown in selective media and pulse-labeled at 370 with a mixture of 3H-labeled amino acids (10 MCi/ml). F- cells (KY1400) labeled with [14Clleucine for 1 hr were added to each sample of 3H-labeled cells containing I to 2 X 106 cpm of acid-insoluble radioactivity. Crude extracts were prepared, treated with antiserum to precipitate RNA polymerase proteins, and analyzed by NaDodSO4 gel electrophoresis. The 3H/14C ratio for each polymerase protein was divided by that for bulk protein to obtain relative differential synthesis rates: (3H/14C)subunit/(3H/'4C)bulk protein. Apparent deviation of the values for f', f, and a from 1.0 is presumably due to the different isotopic compounds and other conditions used for preparing 3H- arid 14C-labeled cells, and to differences in amino acid composition among polymerase subunits. * These segregants were obtained by spontaneous loss of an F' plasmid.
distinguished, as seen in Fig. 3A. In the same experiment, polymerase proteins obtained from the Salmonella strain harboring F1 16 gave two distinct peaks that approximately correspond to Salmonella and E. coli a proteins (Fig. 3B). At least twice as much radioactivity was associated with E. coli a as with Salmonella a, presumably reflecting the relative rate of synthesis of the two species of a proteins. The amount of Salmonella a synthesized is also comparable to that in the parental F- strain (ST4). Similar results were obtained with another Salmonella strain harboring F122 (data not shown). In contrast, a strain of S. typhimurium harboring F134 gave rise
General procedures and conditions are as described in Table 2 and Materials and Methods, except that the pulse labeling was carried out for 5 min followed by a chase for 2 min, and gel electrophoresis was run for a longer period (16 hr). The values for a subunit are not included, because a proteins ran off the gel in this experiment. Each sample of 3H-labeled cells contained 2 to 4 X 106 cpm of acid-insoluble radioactivity. Note that the values represent differential synthesis rates relative to those for E. coli F- cells.
to a single peak corresponding to Salmonella a under the same conditions (data not shown). In the second experiment, the antibody precipitates obtained
from these cells labeled with [4C]ileucine were analyzed by slab gel electrophoresis (Fig. 4). It can be seen that E. coli and Salmonella a proteins are unequivocally identified by this procedure. The results of autoradiography (Fig. 4B) clearly indicate that the Salmonella F' strains harboring either F1 16 or F122 (samples 3 and 2) can synthesize protein with an electrophoretic mobility very similar to that of E. coli a, beside Salmonella a. Furthermore, the protein patterns of stained gels from the same experiment (Fig. 4A) also revealed two bands that correspond to E. coli and Salmonella a proteins. The strain harboring F134 exhibited a single band coinciding with Salmonella a (data not shown). These results taken together with those already presented strongly suggest that E. coli a proteins can be synthesized in these hybrid F' strains. Because the capacity to produce E. coli a in Salmonella is associated with an F'plasmid (F116 or F122)
122
3
116
-
a
6
102
.cn
140-
c o
cnz 0
~0 10 .o
10 101,-
11
143
254
1
.>
.-a
134 101
=
x
114
1 26
104128
150
133
_1l29
15 2
'a
_ 104.
111-
0,
IV
5
-1
o
o
5
209
th r
lac
gal
trp
0
his ~~I
metC rgG
ilIv
Int
rpoB
I
I -,A&
38
0
10
20
30
40
50
60
70
80
90
100
Map location, min
FIG. 2. Relative differential synthesis rates of in E. coli F' strains diploid for different segments of the chromosome. Experimental procedures and conditions are as described in Table 2 and Materials and Methods. The abscissa indicates the map location in a 100 min scale (7) with some marker genes. Bars indicate chromosomal segments carried by each F' plasmid. F' numbers are in Fig. 1. The ordinate indicates the rate of a synthesis relative to the F- control strain. The complete data of these experiments and those on other polymerase subunits will be presented elsewhere (Y. Nakamura, a
unpublished).
o
L
W-
-
39 37 Distance from origin, cm
38
FIG. 3. Prolonged NaDodSO4/polyacrylamide gel electrophoresis of proteins synthesized by the strain of S. typhimurium harboring an E. coli F' plasmid, F116. General procedures and conditions are as described in Table 3 and Materials and Methods, except that electrophoresis was continued for 70 hr on longer columns (50 X 0.5 cm), and gels were stained and cut into 1 mm thick pieces to obtain best resolution. Only portions of the gels corresponding to a proteins are shown. Samples of 3H-labeled cells contained 2.4 X 106 cpm and 2.9 X 106 cpm of acid-insoluble radioactivity for A and B, respectively. (A) ST4 (F-); (B) F116/ST4; 0-0, 3H radioactivity; 0- -, 14C radioactivity (E. coli KY1400). a
Biochemistry: Nakamura et al.
1834
2
3
5
4
6
Proc. Natl. Acad. Sci. USA 74 (1977) 7
A
Table 4. Tests for markers carried by F' plasmids Marker (map position, min)
F'
plasmid AOrWs
VAW*
Salmonellai E.
3
,'
4
5
col/I,
6
-_-
I
1)
Salmonella E. coli
FIG. 4. NaDodSO4/polyacrylamide gel electrophoresis of RNA polymerase proteins synthesized by strains harboring F116 or F122. Cells were labeled at 370 with [14C]leucine (330 mCi/mmol, 0.4 ,uCi/ml) for 1 hr, and crude extracts obtained were treated with antiserum as described in Materials and Methods. Electrophoresis was carried out on 15 cm long, 1 mm thick slab gels (15 X 14 X 0.1 cm; 5% stacking gels and 10% separation gels) using a discontinuous buffer system essentially as described by Laemmli (25). Migration is from top to bottom as shown here. After running for 6 hr at 20 mA, gels were stained with Coomassie brilliant blue and dried. Autoradiographs were taken by exposing Fuji x-ray films to gel slabs for 5 days. (A) Stained gels; (B) Autoradiographs. Samples 1 and 7, partially purified E. coli RNA polymerase holoenzyme, not treated with antiserum; sample 2, F122/ST4; sample 3, F116/ST4; sample 4, a mixture (1:1, wt/wt) of S. typhimurium (ST4) and E. coli (KY1400); sample 5, E. coli (KY1400); sample 6, S. typhimurium (ST4).
that also enables E. coli cells to produce higher amounts of a, it may be concluded that the chromosomal segment containing the metC-argG region carries a gene determining the structure and synthesis of the protein. In addition, the data of Fig. 4A suggest that E. coli a polypeptides produced in cells of S. tya
phimurium are metabolically stable during steady-state growth. Close Linkage between the Genes rpoD and dnaG. In view of the results so far presented, it was important to determine or reconfirm the end points of chromosomal segment carried by each of the F' plasmids that was shown to contain the rpoD gene. Thus, these plasmids were examined to find whether they carry each of several known markers at the metC-argG region. As shown in Table 4, all these F' plasmids were found to contain dnaG but lack either argG or metC with one exception (F122). These, results are in complete agreement with those already published (17). The present findings therefore suggest strongly that rpoD is located within a small segment of the chromosome containing dnaG. DISCUSSION Our contention that a structural gene for factor is located in the metC-argG region of the E. coli chromosome is based on the following observations. (i) The gene dosage effect on the synthesis of (but not other polymerase subunits) was consistently found with all E. coli F' strains tested that are diploid for the metC-argG region; (ii) S. typhimurium strains harboring F116 or F122 (each carrying the metC-argG region of the E. coli chromosome) synthesize proteins (including both Salmonella and E. coli a) at the rates higher than the parental Fa
a
a
F102 F116 F122 F140 F141
thyA
metC dnaG* argG
(60.5) (64) +
+
(66) + +
argRt
(68) (69.5) rpoDt +
+
-
-
+ +
+
+
+
+
-
+
-
-
+ +
+ +
+ +
+ +
Cells of F' strains were cross-streaked on appropriate media against cells of F- recA strains that are mutant for the marker being tested. Colonies that appeared at the intersection were purified and further examined for partial diploidy. + and - indicate presence and absence, respectively, of each marker. * Checked by ability to convert an F- dnaG (temperature-sensitive growth) strain (PC3) to temperature-independent phenotype. t Data taken from ref. 17. * Deduced from the present data.
strain; and (iii) these hybrid F' strains produce a protein that can react with antiserum to RNA polymerase and is indistinguishable from E. coli a in mobility in high-resolution NaDodSO4/gel electrophoresis. An alternative possibility, although unlikely for many reasons, cannot be excluded entirely at present. It might be argued that degradation or modification of j3', fl, or a proteins of Salmonella RNA polymerase, provoked by the functioning of an E. coli gene located at the metC-argG region, could lead to the formation of protein indistinguishable from E. coli a in electrophoretic mobility. However, this would not explain the gene dosage effect in a synthesis observed with E. coli merodiploids or Salmonella F' hybrids. The results of further experiments to rule out these possibilities more rigorously (Y. Nakamura, unpublished data) will be reported subsequently. At least two genes that might be related to a have been mapped at the metC-argG region on the E. coli chromosome; dnaG controlling the initiation of synthesis of nascent (Okazaki) fragments of DNA (26) is located between metC and argG (7), whereas nusA involved in the functioning of N protein in X-phage-infected bacteria has been mapped near argG (27). A a gene (rpoD) as identified here may be closely linked and possibly coregulated with, if not identical with, one of these genes. In this connection, genes for at least two ribosomal proteins (S15 and L21) were recently reported to be near argG in E. coli (28). In view of the frequent occurrence of clusters containing genes for both RNA polymerase subunit(s) and ribosomal proteins (13, 29), it would be interesting to see whether rpoD and several ribosomal protein genes form another cluster at the metC-argG region. The rate of a factor synthesis has previously been shown to be relatively constant, unlike that of core subunits of RNA polymerase (20, 22). However, recent observations suggest that a specific regulatory mechanism is operative in the control of a gene expression in E. coli. Thus, a synthesis can be enhanced by structural alteration of core polymerase (16, 21), the N gene product of phage X (30), or alteration of a transcription termination factor p (30). Some of the F' plasmids used in this study (F134 and possibly F101) carrying a chromosomal segment other than the metC-argG region brought about enhanced synthesis of a during steady-state growth (Fig. 2). Such an effect was not observed when F134 was present in cells of S. typhimurium (Table 3). Although the mechanism for enhanced a synthesis in this case is not known, it is conceivable that F134
Biochemistry:
Nakamura et al.
carries a gene somehow involved in specific regulation of a gene expression. Although the specific role of a factor in transcription initiation has been well established in intro, little is known about the role of ai in transcription in vivo, and more specifically, in promoter selection, which is of great importance from the point of view of transcriptional control. The results reported here should facilitate isolation of a mutants that would be most useful
for future studies along this line. Note Added in Proof. After this paper was submitted, J. Harris and R. Calendar kindly informed us that they have confirmed our conclusion on the location of the a gene in an independent work (personal communication). On the other hand, Friesen et al. (31) reported evidence suggesting that a a factor gene might lie on Xdpo!CdapD-9.
We are grateful to Drs. B. J. Bachmann, Y. Hirota, T. Iino, H. Ozeki, and K. E. Sanderson for bacterial strains used. Our sincere thanks are also due to Dr. K. Ito for his interest and valuable suggestions throughout this study, and to Dr. S. Hiraga for helpful discussion. This work was supported by a grant from The Ministry of Education, Science and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges from funds made available to support the research which is the subject of the article. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 1. Burgess, R. (1971) Annu. Rev. Biochem. 40, 711-740. 2. Tocchini-Valentini, G. P., Marino, P. & Colvill, A. J. (1968)
Nature 220, 275-276. 3. Yura, T. & Igarashi, K. (1968) Proc. Natl. Acad. Sci. USA 61, 1313-1319. 4. Schleif, R. (1969) Nature 223, 1068-1069. 5. Heil, A. & Zillig, W. (1970) FEBS Lett. 11, 165-168. 6. Iwakura, Y., Ishihama, A. & Yura, T. (1973) Mo!. Gen. Genet. 121, 181-196. 7. Bachmann, B. J., Low, K. B. & Taylor, A. L. (1976) Bacteriol. Rev. 40, 116-167.
Proc. Natl. Acad. Sci. USA 74 (1977)
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8. Matzura, H., Molin, S. & Maalte, 0. (1971) J. Mol. Biol. 59, 17-25. 9. Nakamura, Y. & Yura, T. (1973) Biochem. Biophys. Res. Commun. 53, 645-652. 10. Austin, S. (1974) Nature 252,596-597. 11. Errington, L., Glass, R. E., Hayward, R. S. & Scaife, J. G. (1974) Nature 249, 519-522. 12. Kirschbaum, J. B. & Scaife, J. (1974) Mol. Gen. Genet. 132, 193-201. 13. Jaskunas, S. R., Burgess, R. R. & Nomura, M. (1975) Proc. Natl. Acad. Sci. USA 72,5036-5040. 14. Sunshine, M. G. & Sauer, B. (1975) Proc. Natl. Acad. Sci. USA
72,2770-2774. 15. Fujiki, H., Palm, P., Zillig, W., Calendar, R. & Sunshine, M. (1976) Mol. Gen. Genet. 145, 19-22. 16. Nakamura, Y. & Yura, T. (1975) Mol. Gen. Genet. 141, 97111. 17. Low, K. B. (1972) Bacteriol. Rev. 36,587-607. 18. Sanderson, K. E. (1972) Bacteriol. Rev. 36,558-586. 19. Vogel, H. J. & Bonner, D. M. (1956) J. Biol. Chem. 218, 97106. 20. Nakamura, Y. & Yura, T. (1975) J. Mol. Biol. 97,621-642. 21. Nakamura, Y. & Yura, T. (1976) Mol. Gen. Genet. 145, 227237. 22. Iwakura, Y., Ito, K. & Ishihama, A. (1974) Mol. Gen. Genet. 133, 1-23. 23. Engbaek, F., Gross, C. & Burgess, R. R. (1976) Mol. Gen. Genet. 143,291-295. 24. Fukuda, R., Taketo, M., Saitoh, T. & Ishihama, A. (1976) Seikagaku 48,758. 25. Laemmli, U. K. (1970) Nature 227,680-685. 26. Lark, K. G. (1972) Nature New Biol. 240, 237-240. 27. Friedman, D. I. & Baron, L. S. (1974) Virology 58, 141-148. 28. Takata, R. & Kobata, K. (1976) Mol. Gen. Genet. 149, 159165. 29. Lindahl, L., Jaskunas, S. R., Dennis, P. P. & Nomura, M. (1975) Proc. Natl. Acad. Sci. USA 72, 2743-2747. 30. Nakamura, Y. & Yura, T. (1976) Proc. Natl. Acad. Sci. USA 73, 4405-4409. 31. Friesen, J. D., Parker, J., Watson, R. J., Bendiak, D., Reeh, S. V., Pedersen, S. & Fiil, N. P. (1976) Mol. Gen. Genet. 148,93-98.
Biochemistry
Chromosomal location of a structural gene for the RNA polymerase oa factor in Escherichia coli* (F' plasmid/gene dosage effect/Salmonella a factor/immunoprecipitation/dnaG region)
YOSHIKAZU NAKAMURA, TOSHIO OSAWA, AND TAKASHI YURA Institute for Virus Research, Kyoto University, Kyoto, Japan
Communicated by Norton D. Zinder, January 31, 1977
streaking on appropriate selective medium, and the resulting hybrid strains of S. typhimurium harboring an E. coli F' plasmid were tested for a factor synthesis. Media and Chemicals. Minimal medium used in most experiments was medium E (19) supplemented with 0.5% glucose and amino acids, purines, or pyrimidines at 50 ,ug/ml, and vitamins at 1 ,g/ml as required. F' strains were grown in appropriate selective media either lacking certain nutrients required by F- segregants, or containing sugar not utilizable by F- segregants, to minimize growth of F- cells. Chemicals used are generally those described previously (16). Radioactive Labeling of Cells. To determine the rate of synthesis of RNA polymerase including a, a logarithmic phase culture at about 5 X 108 cells per ml was labeled at 37° with 10 gCi/ml of a synthetic mixture of 15 3H-labeled L-amino acids (New England Nuclear Corp., NET-250) for 2 min and chased with 1% casamino acids (Difco) for 3 min, unless otherwise indicated. Sodium azide (20 mM) was added in an ice bath, and cells were collected by centrifugation and washed in Tris-HCI (pH 7.8) containing EDTA and NaCl (20). Preparation of Crude Extract. Cells were lysed by treatment with lysozyme/EDTA and Brij 58, and then by sonication as described previously (20). Determination of the Polymerase Proteins. The polymerase proteins were selectively precipitated from crude extract by treatment with antiserum against E. coli RNA polymerase holoenzyme, and treated with sodium dodecyl sulfate (NaDodSO4) as described previously (20). In most experiments presented below, logarithmic-phase cells of an F- strain (KY1400) labeled for 1 hr with ['4C]leucine (330 mCi/mmol, 1 ,uCi/ml) were used as internal reference; cells containing 7 X 105 cpm of trichloroacetic acid-insoluble radioactivity were added to each sample of 3H-labeled cells before crude extract was prepared. Polyacrylamide gel electrophoresis with NaDodSO4 and estimation of differential rate of synthesis of polymerase proteins was carried out by a modification of the method described previously (21). The 3H/14C ratio for each protein band was divided by that for bulk protein to estimate relative differential synthesis rates.
A set of F' strains of Escherichia coli K-12 ABSTRACT partially diploid for various chromosomal segments has been examined for possible gene dosage effects in the synthesis of a factor of the DNA-dependent RNA polymerase (RNA nucleotidyltransferase; nucleoside-triphosphate:RNA nucleotidyltransferase, EC 2.7.7.6). It was found that all F' strains diploid for the dnaG region synthesize a at rates two to three times higher than other F' or F- strains. Moreover, strains of Salmonella typhimurium harboring these F' plasmids produce E. coli a in addition to Salmonella a. This has been shown on the basis of the finding that Salmonella a can be precipitated with antiserum against E. coli RNA polymerase but is distinguishable from E. coli a in its mobility in sodium dodecyl sulfate/polyacrylamide gel electrophoresis. E. coli a polypeptides thus produced seem to be stable in cells of S. typhimurium. These results indicate that a structural gene for a (rpoD) is located at the metC-argG region, probably near the dnaG locus (66 min on the current genetic map of E. coli).
RNA polymerase (RNA nucleotidyltransferase; nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) of Escherichia coli is composed of at least four different proteins, a, (',and a (1). The problem of organization and expression of genes determining the structure of these polymerase proteins is basic to any understanding of gene regulation at the level of transcription. The structural gene for f (rpoB) has been identified by mutations conferring on cells resistance to several antibiotics including rifampicin (2-6), and located at 88.5 min on the current genetic map (7). The gene for ,B' (rpoC) is closely linked to that for 1 (8-10), and appears to form an operon with the ( gene (11, 12). The a gene (rpoA) has recently been identified and mapped near the cluster of ribosomal protein genes at 72 min (13-15). We now report that a structural gene for the a factor, rpoD, is located at the metC-argG region, probably near the dnaG locus (around 66 min) on the chromosome of E. coli. It also became apparent during this study that the a gene of E. coli present on F' plasmids can be expressed efficiently in cells of Salmonella typhimurium. Moreover, E. coli a polypeptides thus produced appear to be metabolically stable in S. typhimurium.
MATERIALS AND METHODS Bacterial Strains. Strains of E. coli K-12 and of S. typhimurium used are listed in Table 1. A number of F' plasmids carrying different segments of the E. coli chromosome (17) were obtained from B. Bachmann (see Fig. 1). Salmonella strains were kindly provided by T. Iino and K. E. Sanderson; they were mated with each of the E. coli F' strains by cross-
RESULTS Gene Dosage Effects in a Factor Synthesis in E. coli Merodiploids. A set of 19 F' strains partially diploid for different portions of the E. coli chromosome was first examined for the synthesis of a and other RNA polymerase proteins. Because the F' plasmids used (Fig. 1) cover most parts of the entire chromosome with partial overlaps, some of the F' strains may be expected to be diploid for a structural gene for a (rpoD) and
Abbreviations: Gene symbols for E. coli and S. typhimurium are those described in refs. 7 and 18, respectively. NaDodSO4, sodium dodecyl sulfate. * Presented at the 48th Annual Meeting of the Genetics Society of Japan (October 1976, Osaka).
might therefore produce significantly higher levels of a than F- or other F' strains, due to gene dosage effect. Table 2 shows some of the typical results obtained in such an experiment. In
1831
1832
Proc. Natl. Acad. Sci. USA 74 (1977)
Biochemistry: Nakamura et al. Table 1. Bacterial strains Strain
Sex
Chromosomal markers
Source or ref.
Escherichia coli K-12 F141/JC1553
B. J. Bachmann F' argG metBhis leu (CGSC 4248) recA mitl xyl malA
gal lacY str tonA tsx supE F126/KL181 F' his trp pyrD thi recA mitl xyl malA galK str F116/KL110 F' The same as in F141/JC1553 except thyA F152/KL253 F' his trp pyrD thi tyrA recA mtl xyl malA galK str F- his ilv argH KY1400 trpE9829 tyr lacZ tonA tsx bfe sup- 126 F- leu thyA dnaG tonB PC3 str Salmonella typhimurium F- thy argE [correST4 sponds to argG of E. coli (18)] F' The same as in ST4 F116/ST4 F' The same as in ST4 F122/ST4 F- pro leu ara gal xyl SW1292 rha str F134/SW1292 F' The same as in SW1292
B. J. Bachmann (CGSC 4253) B. J. Bachmann
(CGSC 4254) B. J. Bachmann
(CGSC 4287) Ref. 16 1t116
129
122
Y. Hirota
K. E. Sanderson This paper This paper T. Iino
This paper
this and all subsequent experiments, differential synthesis rates of polymerase subunits among total protein synthesized have been normalized to those for the E. coli F- strain continuously labeled with [14C]leucine. It is evident that strains harboring F141 or F 116 synthesize at rates two to three times higher than those for other F' strains, whereas they produce other polymerase proteins essentially at normal rates. F- segregants derived from these F' strains produce a at normal rates, indicating that the higher rates of synthesis is correlated with the presence of an F' plasmid. The differential synthesis rate of for F- haploid strains under these conditions is approximately 0.1% of total protein synthesized, and represents about one-third of that for core polymerase subunits on molar basis (20, 22, 23). The relative differential synthesis rates of a for all F' strains tested are presented in Fig. 2 as a function of the map location of the chromosomal segment carried by each of the F' plasmids used. It can be seen that the strains partially diploid for the metC-argG region, corresponding approximately to 64-68 min on the genetic map (7), invariably exhibit two to three times higher rates of a synthesis than other strains. Again, F- segregants derived from these strains show normal levels of synthesis (data not shown). All the remaining strains with one or two possible exceptions (those harboring F134 or F101) produce normal levels of These results provided evidence, though indirect, that a structural gene for is located at the metC-argG region. S. typliimurium Strains Harboring E. coil F' Plasmids. A more direct test for the location of a structural gene for on the E. coli chromosome was carried out by using strains of S. tya
a
a
a
a.
a
a
FIG. 1. Genetic map of E. coli K-12 showing approximate locations of chromosomal segments carried by the F' plasmids used. Each F' is represented by an arc with a number assigned by K. B. Low (17); the "map numbers" rather than original numbers are used throughout this paper for convenience in presenting the data. The presence of F' in each merodiploid strain has been checked by its sensitivity to male-specific phages, appearance of F- segregants, and by its ability to transfer F' to other F- recA strains. F' plasmids drawn with heavy lines are those that were shown to carry a structural gene for af (see text).
phimurium harboring an E. coli F' plasmid, because it was recently found that a protein of S. typhimurium can be precipitated by antiserum to F. coli RNA polymerase and migrates slightly behind that of E. coli in NaDodSO4/polyacrylamide gel electrophoresis (24). Thus, several Salmonella strains harboring an F' plasmid suspected of carrying a af gene were first examined for their differential rates of a synthesis. It was expected that these hybrid F' strains might produce E. coli af protein as well as Salmonella a, giving rise to apparently higher levels of total cr synthesis, provided that the E. coli af gene can be expressed in S. typhimurium. That this is indeed the case is shown in Table 3; both the strain harboring Fl16 and the strain harboring F122 produced af at rates significantly higher than those for the parental F- strain (ST4). In constrast, the level of af synthesis for the strain harboring F134 did not appreciably exceed that for the parental haploid strain. It should be noted that F' plasmid F134, when present in cells of E. coli, brought about significantly higher levels of af production under similar conditions (see Fig. 2). The rates of synthesis of (3 and (3' subunits in the Salmonella strains were not affected appreciably by introduction of any of the E. coli F' plasmids tested. These data are therefore consistent with, and lend further support to, the suggestion that a ar gene is located at the metC-argG region of the E. coli chromosome, although af polypeptides of F. coli and of S. typhimurium were not resolved in these experiments. Identification' of E. coili a Synthesized in Salmonella Hybrid F' Strains. We then attempted to demonstrate the synthesis of F. coli a in Salmonella strains harboring an E. coli F' plasmid, F116 or F122. In the first experiment, 314-labeled proteins extracted from such strains were precipitated by antiserum and were subjected to prolonged electrophoresis on longer columns. When a mixture of differentially labeled polymerase proteins was analyzed under these conditions, a polypeptides of E. coli and of S. typhimurium were clearly
Proc. Natl. Acad. Sci. USA 74 (1977)
Biochemistry: Nakamura et al. Table 2. Synthesis of RNA polymnerase proteins in E. coli merodiploids
1833
Table 3. Synthesis of RNA polymerase proteins in S. typhimurium strains harboring an E. coli F' plasmid
Relative differential synthesis rates
Relative differential synthesis rates
Strain
,B
,s
a
a
Strain
a
,B
,B
F141/JC1553 F- segregant of above*
0.87 0.76 0.81 0.77 0.86 0.71
0.59 0.74 0.80 0.72 0.74 0.55
2.37 1.12 3.17 0.88 1.17 1.11
0.55 0.60 0.60 0.55 0.51 0.55
ST4 (F-) F116/ST4 F122/ST4 SW1292 (F-) F134/SW1292
1.03 3.87 3.38 1.15 1.49
1.20 1.50 1.34 0.69 0.72
1.21 1.58 1.44 0.75 1.00
F116/KL110 F- segregant of above* F126/KL181 F152/KL253
General procedures and conditions are as described in Materials and Methods. Cells were grown in selective media and pulse-labeled at 370 with a mixture of 3H-labeled amino acids (10 MCi/ml). F- cells (KY1400) labeled with [14Clleucine for 1 hr were added to each sample of 3H-labeled cells containing I to 2 X 106 cpm of acid-insoluble radioactivity. Crude extracts were prepared, treated with antiserum to precipitate RNA polymerase proteins, and analyzed by NaDodSO4 gel electrophoresis. The 3H/14C ratio for each polymerase protein was divided by that for bulk protein to obtain relative differential synthesis rates: (3H/14C)subunit/(3H/'4C)bulk protein. Apparent deviation of the values for f', f, and a from 1.0 is presumably due to the different isotopic compounds and other conditions used for preparing 3H- arid 14C-labeled cells, and to differences in amino acid composition among polymerase subunits. * These segregants were obtained by spontaneous loss of an F' plasmid.
distinguished, as seen in Fig. 3A. In the same experiment, polymerase proteins obtained from the Salmonella strain harboring F1 16 gave two distinct peaks that approximately correspond to Salmonella and E. coli a proteins (Fig. 3B). At least twice as much radioactivity was associated with E. coli a as with Salmonella a, presumably reflecting the relative rate of synthesis of the two species of a proteins. The amount of Salmonella a synthesized is also comparable to that in the parental F- strain (ST4). Similar results were obtained with another Salmonella strain harboring F122 (data not shown). In contrast, a strain of S. typhimurium harboring F134 gave rise
General procedures and conditions are as described in Table 2 and Materials and Methods, except that the pulse labeling was carried out for 5 min followed by a chase for 2 min, and gel electrophoresis was run for a longer period (16 hr). The values for a subunit are not included, because a proteins ran off the gel in this experiment. Each sample of 3H-labeled cells contained 2 to 4 X 106 cpm of acid-insoluble radioactivity. Note that the values represent differential synthesis rates relative to those for E. coli F- cells.
to a single peak corresponding to Salmonella a under the same conditions (data not shown). In the second experiment, the antibody precipitates obtained
from these cells labeled with [4C]ileucine were analyzed by slab gel electrophoresis (Fig. 4). It can be seen that E. coli and Salmonella a proteins are unequivocally identified by this procedure. The results of autoradiography (Fig. 4B) clearly indicate that the Salmonella F' strains harboring either F1 16 or F122 (samples 3 and 2) can synthesize protein with an electrophoretic mobility very similar to that of E. coli a, beside Salmonella a. Furthermore, the protein patterns of stained gels from the same experiment (Fig. 4A) also revealed two bands that correspond to E. coli and Salmonella a proteins. The strain harboring F134 exhibited a single band coinciding with Salmonella a (data not shown). These results taken together with those already presented strongly suggest that E. coli a proteins can be synthesized in these hybrid F' strains. Because the capacity to produce E. coli a in Salmonella is associated with an F'plasmid (F116 or F122)
122
3
116
-
a
6
102
.cn
140-
c o
cnz 0
~0 10 .o
10 101,-
11
143
254
1
.>
.-a
134 101
=
x
114
1 26
104128
150
133
_1l29
15 2
'a
_ 104.
111-
0,
IV
5
-1
o
o
5
209
th r
lac
gal
trp
0
his ~~I
metC rgG
ilIv
Int
rpoB
I
I -,A&
38
0
10
20
30
40
50
60
70
80
90
100
Map location, min
FIG. 2. Relative differential synthesis rates of in E. coli F' strains diploid for different segments of the chromosome. Experimental procedures and conditions are as described in Table 2 and Materials and Methods. The abscissa indicates the map location in a 100 min scale (7) with some marker genes. Bars indicate chromosomal segments carried by each F' plasmid. F' numbers are in Fig. 1. The ordinate indicates the rate of a synthesis relative to the F- control strain. The complete data of these experiments and those on other polymerase subunits will be presented elsewhere (Y. Nakamura, a
unpublished).
o
L
W-
-
39 37 Distance from origin, cm
38
FIG. 3. Prolonged NaDodSO4/polyacrylamide gel electrophoresis of proteins synthesized by the strain of S. typhimurium harboring an E. coli F' plasmid, F116. General procedures and conditions are as described in Table 3 and Materials and Methods, except that electrophoresis was continued for 70 hr on longer columns (50 X 0.5 cm), and gels were stained and cut into 1 mm thick pieces to obtain best resolution. Only portions of the gels corresponding to a proteins are shown. Samples of 3H-labeled cells contained 2.4 X 106 cpm and 2.9 X 106 cpm of acid-insoluble radioactivity for A and B, respectively. (A) ST4 (F-); (B) F116/ST4; 0-0, 3H radioactivity; 0- -, 14C radioactivity (E. coli KY1400). a
Biochemistry: Nakamura et al.
1834
2
3
5
4
6
Proc. Natl. Acad. Sci. USA 74 (1977) 7
A
Table 4. Tests for markers carried by F' plasmids Marker (map position, min)
F'
plasmid AOrWs
VAW*
Salmonellai E.
3
,'
4
5
col/I,
6
-_-
I
1)
Salmonella E. coli
FIG. 4. NaDodSO4/polyacrylamide gel electrophoresis of RNA polymerase proteins synthesized by strains harboring F116 or F122. Cells were labeled at 370 with [14C]leucine (330 mCi/mmol, 0.4 ,uCi/ml) for 1 hr, and crude extracts obtained were treated with antiserum as described in Materials and Methods. Electrophoresis was carried out on 15 cm long, 1 mm thick slab gels (15 X 14 X 0.1 cm; 5% stacking gels and 10% separation gels) using a discontinuous buffer system essentially as described by Laemmli (25). Migration is from top to bottom as shown here. After running for 6 hr at 20 mA, gels were stained with Coomassie brilliant blue and dried. Autoradiographs were taken by exposing Fuji x-ray films to gel slabs for 5 days. (A) Stained gels; (B) Autoradiographs. Samples 1 and 7, partially purified E. coli RNA polymerase holoenzyme, not treated with antiserum; sample 2, F122/ST4; sample 3, F116/ST4; sample 4, a mixture (1:1, wt/wt) of S. typhimurium (ST4) and E. coli (KY1400); sample 5, E. coli (KY1400); sample 6, S. typhimurium (ST4).
that also enables E. coli cells to produce higher amounts of a, it may be concluded that the chromosomal segment containing the metC-argG region carries a gene determining the structure and synthesis of the protein. In addition, the data of Fig. 4A suggest that E. coli a polypeptides produced in cells of S. tya
phimurium are metabolically stable during steady-state growth. Close Linkage between the Genes rpoD and dnaG. In view of the results so far presented, it was important to determine or reconfirm the end points of chromosomal segment carried by each of the F' plasmids that was shown to contain the rpoD gene. Thus, these plasmids were examined to find whether they carry each of several known markers at the metC-argG region. As shown in Table 4, all these F' plasmids were found to contain dnaG but lack either argG or metC with one exception (F122). These, results are in complete agreement with those already published (17). The present findings therefore suggest strongly that rpoD is located within a small segment of the chromosome containing dnaG. DISCUSSION Our contention that a structural gene for factor is located in the metC-argG region of the E. coli chromosome is based on the following observations. (i) The gene dosage effect on the synthesis of (but not other polymerase subunits) was consistently found with all E. coli F' strains tested that are diploid for the metC-argG region; (ii) S. typhimurium strains harboring F116 or F122 (each carrying the metC-argG region of the E. coli chromosome) synthesize proteins (including both Salmonella and E. coli a) at the rates higher than the parental Fa
a
a
F102 F116 F122 F140 F141
thyA
metC dnaG* argG
(60.5) (64) +
+
(66) + +
argRt
(68) (69.5) rpoDt +
+
-
-
+ +
+
+
+
+
-
+
-
-
+ +
+ +
+ +
+ +
Cells of F' strains were cross-streaked on appropriate media against cells of F- recA strains that are mutant for the marker being tested. Colonies that appeared at the intersection were purified and further examined for partial diploidy. + and - indicate presence and absence, respectively, of each marker. * Checked by ability to convert an F- dnaG (temperature-sensitive growth) strain (PC3) to temperature-independent phenotype. t Data taken from ref. 17. * Deduced from the present data.
strain; and (iii) these hybrid F' strains produce a protein that can react with antiserum to RNA polymerase and is indistinguishable from E. coli a in mobility in high-resolution NaDodSO4/gel electrophoresis. An alternative possibility, although unlikely for many reasons, cannot be excluded entirely at present. It might be argued that degradation or modification of j3', fl, or a proteins of Salmonella RNA polymerase, provoked by the functioning of an E. coli gene located at the metC-argG region, could lead to the formation of protein indistinguishable from E. coli a in electrophoretic mobility. However, this would not explain the gene dosage effect in a synthesis observed with E. coli merodiploids or Salmonella F' hybrids. The results of further experiments to rule out these possibilities more rigorously (Y. Nakamura, unpublished data) will be reported subsequently. At least two genes that might be related to a have been mapped at the metC-argG region on the E. coli chromosome; dnaG controlling the initiation of synthesis of nascent (Okazaki) fragments of DNA (26) is located between metC and argG (7), whereas nusA involved in the functioning of N protein in X-phage-infected bacteria has been mapped near argG (27). A a gene (rpoD) as identified here may be closely linked and possibly coregulated with, if not identical with, one of these genes. In this connection, genes for at least two ribosomal proteins (S15 and L21) were recently reported to be near argG in E. coli (28). In view of the frequent occurrence of clusters containing genes for both RNA polymerase subunit(s) and ribosomal proteins (13, 29), it would be interesting to see whether rpoD and several ribosomal protein genes form another cluster at the metC-argG region. The rate of a factor synthesis has previously been shown to be relatively constant, unlike that of core subunits of RNA polymerase (20, 22). However, recent observations suggest that a specific regulatory mechanism is operative in the control of a gene expression in E. coli. Thus, a synthesis can be enhanced by structural alteration of core polymerase (16, 21), the N gene product of phage X (30), or alteration of a transcription termination factor p (30). Some of the F' plasmids used in this study (F134 and possibly F101) carrying a chromosomal segment other than the metC-argG region brought about enhanced synthesis of a during steady-state growth (Fig. 2). Such an effect was not observed when F134 was present in cells of S. typhimurium (Table 3). Although the mechanism for enhanced a synthesis in this case is not known, it is conceivable that F134
Biochemistry:
Nakamura et al.
carries a gene somehow involved in specific regulation of a gene expression. Although the specific role of a factor in transcription initiation has been well established in intro, little is known about the role of ai in transcription in vivo, and more specifically, in promoter selection, which is of great importance from the point of view of transcriptional control. The results reported here should facilitate isolation of a mutants that would be most useful
for future studies along this line. Note Added in Proof. After this paper was submitted, J. Harris and R. Calendar kindly informed us that they have confirmed our conclusion on the location of the a gene in an independent work (personal communication). On the other hand, Friesen et al. (31) reported evidence suggesting that a a factor gene might lie on Xdpo!CdapD-9.
We are grateful to Drs. B. J. Bachmann, Y. Hirota, T. Iino, H. Ozeki, and K. E. Sanderson for bacterial strains used. Our sincere thanks are also due to Dr. K. Ito for his interest and valuable suggestions throughout this study, and to Dr. S. Hiraga for helpful discussion. This work was supported by a grant from The Ministry of Education, Science and Culture of Japan. The costs of publication of this article were defrayed in part by the payment of page charges from funds made available to support the research which is the subject of the article. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 1. Burgess, R. (1971) Annu. Rev. Biochem. 40, 711-740. 2. Tocchini-Valentini, G. P., Marino, P. & Colvill, A. J. (1968)
Nature 220, 275-276. 3. Yura, T. & Igarashi, K. (1968) Proc. Natl. Acad. Sci. USA 61, 1313-1319. 4. Schleif, R. (1969) Nature 223, 1068-1069. 5. Heil, A. & Zillig, W. (1970) FEBS Lett. 11, 165-168. 6. Iwakura, Y., Ishihama, A. & Yura, T. (1973) Mo!. Gen. Genet. 121, 181-196. 7. Bachmann, B. J., Low, K. B. & Taylor, A. L. (1976) Bacteriol. Rev. 40, 116-167.
Proc. Natl. Acad. Sci. USA 74 (1977)
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8. Matzura, H., Molin, S. & Maalte, 0. (1971) J. Mol. Biol. 59, 17-25. 9. Nakamura, Y. & Yura, T. (1973) Biochem. Biophys. Res. Commun. 53, 645-652. 10. Austin, S. (1974) Nature 252,596-597. 11. Errington, L., Glass, R. E., Hayward, R. S. & Scaife, J. G. (1974) Nature 249, 519-522. 12. Kirschbaum, J. B. & Scaife, J. (1974) Mol. Gen. Genet. 132, 193-201. 13. Jaskunas, S. R., Burgess, R. R. & Nomura, M. (1975) Proc. Natl. Acad. Sci. USA 72,5036-5040. 14. Sunshine, M. G. & Sauer, B. (1975) Proc. Natl. Acad. Sci. USA
72,2770-2774. 15. Fujiki, H., Palm, P., Zillig, W., Calendar, R. & Sunshine, M. (1976) Mol. Gen. Genet. 145, 19-22. 16. Nakamura, Y. & Yura, T. (1975) Mol. Gen. Genet. 141, 97111. 17. Low, K. B. (1972) Bacteriol. Rev. 36,587-607. 18. Sanderson, K. E. (1972) Bacteriol. Rev. 36,558-586. 19. Vogel, H. J. & Bonner, D. M. (1956) J. Biol. Chem. 218, 97106. 20. Nakamura, Y. & Yura, T. (1975) J. Mol. Biol. 97,621-642. 21. Nakamura, Y. & Yura, T. (1976) Mol. Gen. Genet. 145, 227237. 22. Iwakura, Y., Ito, K. & Ishihama, A. (1974) Mol. Gen. Genet. 133, 1-23. 23. Engbaek, F., Gross, C. & Burgess, R. R. (1976) Mol. Gen. Genet. 143,291-295. 24. Fukuda, R., Taketo, M., Saitoh, T. & Ishihama, A. (1976) Seikagaku 48,758. 25. Laemmli, U. K. (1970) Nature 227,680-685. 26. Lark, K. G. (1972) Nature New Biol. 240, 237-240. 27. Friedman, D. I. & Baron, L. S. (1974) Virology 58, 141-148. 28. Takata, R. & Kobata, K. (1976) Mol. Gen. Genet. 149, 159165. 29. Lindahl, L., Jaskunas, S. R., Dennis, P. P. & Nomura, M. (1975) Proc. Natl. Acad. Sci. USA 72, 2743-2747. 30. Nakamura, Y. & Yura, T. (1976) Proc. Natl. Acad. Sci. USA 73, 4405-4409. 31. Friesen, J. D., Parker, J., Watson, R. J., Bendiak, D., Reeh, S. V., Pedersen, S. & Fiil, N. P. (1976) Mol. Gen. Genet. 148,93-98.
