Cell, Vol . 15, 231-236, September 1978, Copyright © 1978 by MIT
Isolation and Characterization of a Promoter Mutant in the str Ribosomal Protein Operon in E . coli Leonard E . Post, Ann E . Arfsten and Masayasu Nomura* Departments of Genetics and Biochemistry Institute for Enzyme Research University of Wisconsin Madison, Wisconsin 53706 S . Richard Jaskunas Department of Chemistry Indiana University Bloomington, Indiana 47401
Summary The afus3 transducing phage carries several operons for ribosomal proteins of E . call, including the str operon . A mutant transducing phage with a promoter mutation in this operon has been isolated . This mutant shows reduced stimulation of synthesis of proteins encoded by the operon, S12, S7, and elongation factors G and Tu, in ultraviolet-irradiated cells . This mutation also abolishes in vitro transcription from the str promoter . The DNA sequence of the mutant promoter shows that it is a point mutation 6 bases upstream from the in vitro transcription start site, changing the "Pribnow box" sequence from TAAAATT to TAAAACT . These results indicate that the site altered by the mutation, which is in the region just preceding the transcription start site, is important for the expression of the str operon . Introduction Isolation of mutants having alterations in promoter regions has been useful in analyzing the mechanisms involved in the expression and regulation of several bacterial operons such as the /ac operon (for reviews, see Gilbert, 1976 ; Reznikoff and Abelson, 1978) . Isolation of such promoter mutants in operons essential for growth, such as operons for ribosomal components, has been difficult in the past, and no promoter mutants in essential operons have been reported . To study the mechanism of regulation of ribosome biosynthesis, we have initiated isolation of promoter mutants in ribosomal protein operons . This paper reports isolation and characterization of the first promoter mutant which decreases the expression of all the genes in the str operon . We have demonstrated that the mutation involves a single nucleotide substitution at a site preceding the in vitro transcription start point and defines a site essential for the initiation of the transcription of the str operon . * To whom requests for reprints should be addressed .
Results and Discussion Isolation of Xfus3-P105 We have previously described isolation of two mutants of xfus3 transducing phages, Xfus3-4101 and kfus3-1103, which greatly decreased the activity of all the genes in the str operon . These mutants are a small deletion (101) and an insertion (1103), and were used to define the operon . Mutants were isolated using the same method as described for A101 and 1103 (Jaskunas et al ., 1975b) . Briefly, we started from a lysogen (NO1380) carrying Xfus3 and with chromosomal markers recA - , trkA - , spcr, strr and fusr . Xfus3 carries spc`, strs and fuss alleles . It is known that the three drugsensitive alleles are dominant over corresponding drug-resistant alleles and, therefore, the lysogen is sensitive to all three antibiotics-that is, Spc-S, Str-S and Fus-S . Spontaneous Str-R mutants were selected, and mutants which were Fus-R but Spc-S were investigated further . Transducing phages were isolated from these mutant bacteria, and phages carrying the mutations were characterized with respect to their density, the restriction enzyme digestion patterns of their DNA and their ability to stimulate the synthesis of proteins coded for by the str operon . Afus3-P105, which was used in this study, did not show any detectable density difference, but the Hind II digestion pattern of its DNA indicated a slight but definite alteration in the Hind II 314 base pair (bp) fragment compared to the parent Xfus3 (Figure 1) . As is described below, the P105 mutation involves a single nucleotide substitution in the 314 by fragment, and the observed mobility change reflects the nucleotide change rather than small deletions (or additions) . Nucleotide Sequence of the str Promoter Region of the afus3-P105 Mutant The Hind II 314 by fragment carrying the str promoter was cloned from the mutant Xfus3-P105 DNA in the same way described in the accompanying paper (Post et al ., 1978) for the cloning of the wildtype 314 by fragment . The hybrid plasmid thus obtained (pNO2011) was used to sequence the cloned mutant Hind II 314 by fragment . DNA sequence gels were carried out according to the method of Maxam and Gilbert (1977) ; Figure 2 shows portions of the gels which are pertinent to the sequence difference between the two promoters . We have found that the P105 mutation involves a single AT to GC transition at position -6-that is, within the Pribnow box discussed in the accompanying paper (Figure 3) . By sequencing from both the Hind II cut and the Hae III cut (see the accompanying paper), both strands of the DNA around the mutation were sequenced . No other sequence difference was observed .
Cell 232
Figure DNAs
1. Hind II Digestion
Products
from
Afus3
DNA and Mutant
The following DNAs were digested with Hind II restriction enzyme: pN02006 DNA (lane I), AfusS DNA (lane 2), Mus3-P107 DNA (lane 3). hfus3-PI05 DNA (lane 4) and hfus3-A104 DNA (lane 5). Digests were electrophoresed on 4% polyactylamide gels in 50 mM Trisborate (pH 6.3), 1 mM EDTA at 10 V/cm for 5 hr. hfus3-P107 is a spontaneous point mutant which has reduced the activities of all the genes in the str operon (our unpublished experiments). (pN02006 is a plasmid consisting of mini-Co1 El DNA with two Hpa I fragments from AfusS inserted into the Hind II site. This plasmid DNA was used as a reference; the smallest Hind II fragment is the 314 bp fragment which includes thestr promoter. Its position is indicated by an arrow.) It can be seen that the Hind II 314 bp fragment with the str promoter is overlapped with another DNA fragment of the same mobility in Afusd digests, but it moved slightly faster in Afus3-PI07 and Afus3-P105, and was absent in Afus3-A104. This photograph is a portion of the gel showing fragments of sizes approximately 200-900 bp.
Effects of the P105 Mutation on the Expression of the str Operon in Vivo The expression of genes in the str operon carried by the hfus3-P105 mutant was examined in ultraviolet-irradiated E. coli cells and compared with that of the corresponding genes carried by the parent, Afus3. The irradiated cells were infected with purified transducing phages; proteins synthesized after phage infection were labeled with 35S-methionine and analyzed by SDS-polyacrylamide gel electrophoresis followed by autoradiography (Figure 4; see also Table 1). In other experiments, cells were prelabeled with 14C-leucine, irradiated and infected with phages; the proteins synthesized after phage infection were labeled with 3H-leucine. The ratio of 3H/14C for most of the r proteins was
determined after separation of the r proteins by the standard two-dimensional polyacrylamide gel electrophoresis. The data were normalized to the expression of the S4 gene which is carried by the transducing phages but is in a different operon [Jaskunas, Fallon and Nomura, 1977; see Figure 1 of the accompanying paper by Post et al. (1978)]. The results are given in Table 1. It is clear that the PI05 mutation greatly (70-90%) reduced the expression of all the genes in the str operon-that is, genes for S12, S7, EF-G and EF-Tu. Synthesis of none of the other r proteins analyzed was affected. We conclude that the site altered by the P105 mutation, which is in the region just preceding the in vitro transcription start, is indeed important for the expression of the str operon in vivo. In the same experiments described above, three other mutant phages were also analyzed-hfus3AlOl, hfus3-1103 and hfus3-A104. The first two mutations were previously described (Jaskunas et al., 1975b). Al01 is a deletion removing about 1.6 kilobases (kb) DNA covering the right-hand coli-h DNA junction, the str promoter region and a part or all of thestr structural gene (Jaskunaset al., 1975b; L. Post and M. Nomura, unpublished experiments). 1103 is an insertion with the size of IS2 (about 1.3 kb) which is located within the Hind II 314 bp fragment [Jaskunas et al., 1975b; L. Post and M. Nomura, unpublished experiments; see also the accompanying paper by Post et al. (1978)]. The data shown in Figure 4 and Table 1 confirm the previous conclusion that these two mutations cause strong polar effects on the expression of distal genes in the same operon. The third mutation, A104, has not been described previously. This is a small deletion removing a DNA segment of about 500 bp, which covers the 314/2300 Hind IIsensitive site and the 530/1180 Hae Ill-sensitive site, but not the 500/314 Hind II-sensitive site (S. R. Jaskunas, L. Post and M. Nomura, unpublished experiments; for the respective restriction enzymesensitive sites, see Figure 1 of the accompanying paper). The exact position of the left end of the Al04 deletion in the promoter region has not been determined. From the results shown in Figure 4 and Table 1, one can see that the polar effect caused by the P105 mutation is as strong as the effects caused by the two deletions and stronger than the effect caused by the 1103 insertion. Since at least one of the two deletions, AlOl, removes the entire promoter region, the results underscore the importance of the base pair affected by the P105 mutation in the promoter function. Effects of the P105 Mutation on in Vitro Transcription of the str Operon The accompanying paper (Post et al., 1978) describes transcription experiments using the Hind II
Promoter 233
Mutant
in Ribosomal
Protein
Operon
a
Figure
2. DNA-Sequencing
Gels
Showing
b
the Mutation
in P105
The Hind II ends of the 314 bp fragments from pN02001 (a) or pN02011 (b) were was purified and sequenced according to Maxam and Gilbert (1977). Shown are been electrophoresed for 35 hr at 1000 V. Note that the Hind II end is labeled, so messenger RNA-that is, the bottom strand in Figure 3. The sequence indicated mutation.
5’ 3”
Figure 3. Mutational Alteration Promoter in hfus3-P105
of the DNA
The DNA sequence around the Pribnow promoter is shown. The mutational change, the P105 mutation is indicated by an arrow.
Sequence
in the str
box of the wild-type TA to CG. caused by
314 bp fragment as template. We observed the synthesis of RNA transcripts about 80 nucleotides long which define the specific in vitro start site, Experiments were repeated using the corresponding Hind II 314 bp fragment obtained from the P105 mutant as template, and radioactive RNAs synthesized were compared with those obtained with the standard template by polyacrylamide gel electrophoresis followed by autoradiography. As can be seen in Figure 5, the 80 nucleotide long RNA (band 1 in Figure 5) was produced only
labeled and digested with Hae Ill, and the 149 bp fragment portions of 20% polyacrylamide gels in 7 M urea that had the sequence read is the strand complementary to thestr represents the Pribnow box and includes the A + G P105
with the template fragment with the wild-type promoter, and not with the template fragment with the mutant promoter. This conclusion was obtained under various experimental conditions (see the legend to Figure 5). Similarly, the smaller RNA (band 2 in Figure 5) representing the first part (about 24-31 nucleotides long) of the sfr mRNA was also synthesized only with the wild-type promoter and not with the mutant promoter. In contrast, both templates were active in the synthesis of RNAs with larger sizes, which presumably represent RNA (or its aggregates) initiated nonspecifically. This indicates that the failure of the synthesis of the 80 nucleotide long RNA is not due to some trivial reason, such as general “damage” of the mutant DNA template. In addition, the same results were obtained using the DNA templates with and without DEAE column chromatographic purification [see Experimental Procedures in the accompanying paper by Post et al. (1978)]. We conclude that the synthesis of the 80 nucleotide long RNA reflects the in vivo transcription initiation, and that the mutant promoter is inefficient both in vivo and in vitro.
Cell 234
-
EF-G
-a
EF-Tu
In conclusion, we have demonstrated that the P105 mutation is a point mutation, which involves an AT to GC bp substitution at a position (position -6) within the Pribnow box for the str operon, and that this mutational change causes a great reduction in the transcription of the operon both in vivo and in vitro. It is interesting to note that position -6, which was affected by the mutation, corresponds to the sixth of the seven Pribnow box nucleotides, 5’-TATPuATPu-S’, and that the nucleotide (T) at this position is the one most conserved in promoter sequences reported to date (for review, see Reznikoff and Abelson, 1978). Further isolation and characterization of promoter mutants in the present system will certainly be useful for the analysis of the structure and function of r protein promoters. In addition, these results show that analysis of transcription initiation in essential operons can be aided by isolation of mutants, and encourages attempts to isolate similar promoter mutants in other essential operons.
Synthesized in Ultraviolet-Irradiated Various Transducing Phages
1 Figure
Table
2
3
4
5
4. SDS-Polyacrylamide
1. Relative
Gel
Synthesis
of Proteins Transducing
Protein
of
in Ultraviolet-Irradiated
after
Infection
with
Ultraviolet-irradiated S159(h) cells were infected with (1) AC1657S7, (2) Afus3, (3) hfus3-AlOl, (4) Afus3-1103, (5) Afus3-A104 and (6) hfus3-P105. 35S-methionine was added to infected cells 30 min after infection, and incorporation was allowed for 10 min at 37°C. Samples were prepared and analyzed by electrophoresis on 6.75% polyactylamide-0.1% SDS gels followed by autoradiography. Experimental details are the same as described previously (Jaskunas et al., 1975b). An autoradiogram is shown.
6 Electrophoresis
Bacteria
Proteins
Bacteria
after
Infection
with
Mutants
of AfusS
Phages Afus3-I 103
Afus3-Al04
hfus3-P105
Afusa
Afus3-Al01
s12
1 .o
0.06
0.20
0.05
0.05
57
1 .o
0.39
0.46
0.23
0.29
EF-G
1 .o
0.05
0.29
0.23
0.17
EF-TU
1 .o
0.16
0.36
0.24
0.16
Others
1 .o
0.96
0.97
1.02
0.97
Strain S159(A) was irradiated with ultraviolet light and infected with purified transducing phages, and the proteins synthesized after infection were analyzed as previously described (Jaskunas et al., 1975b). Briefly, this was carried out as follows. The relative amounts of EF-G and EF-TU synthesized after infection were determined from autoradiograms similar to those shown in Figure 4. The autoradiograms were scanned with a Joyce-Loebl microdensitometer, and the amounts of EF-G and EF-TU synthesized relative to the amount of RNA polymerase subunit a synthesized were calculated from the area of the peaks. These values were then normalized to the amount synthesized after infection with the parent phage. hfusd. To determine the relative synthesis of S7, S12 and other r proteins, cells were prelabeled with “C-leucine, irradiated and infected with transducing phages. Proteins synthesized after infection were labeled with JHleucine, and r proteins were separated by two-dimensional polyacrylamide gel electrophoresis (Kaltschmidt and Wittmann. 1970). The ‘H/ “C ratios for all the r proteins except Sl, L20, L31, and L34 were determined. The 3H/‘4C ratios were first normalized to the value for S4. The normalized ratios were then divided by the corresponding values obtained with the parent phage, tius3. Data for S7 and S12 are given. The stimulation of the synthesis of other r proteins was not significantly affected by the mutations studied. The average of the normalized relative stimulation for these proteins is also given. It should be noted that the genes for S4 and a are in a separate transcription unit [see Figure 1 of the accompanying paper by Post et al. (1976)]. and the synthesis of these proteins does not appear to be affected by any of the mutations studied in this work.
Promoter 235
Table
Mutant
in Ribosomal
2. Bacterial
Strains
Protein
Operon
Used
Strain
Genotype
NO1345
kdpABC5,
NO1380
NO1345
Source
and Comments
spc’, SW,fus’,
trkA401, (Afus3,
recA
hcl857S765156519xis6)
Jaskunas
et al. (1975a)
Jaskunas
et al. (1977)
Jaskunas
et al. (1975b)
Jaskunas
et al. (1975b)
NO1682
NO1345
(hfus3-AlOl,
NO1684
NO1345
(hfus3-1103,
NO1686
NO1345
(Afus3-A104,
Acl657S7b5156519xis6)
This work
NO1667
NO1345
(Afus3-P105.
Acl657S76515b519xis6)
This work
S159(A)
Apapa
lysogen
Acl857S7b5156519xis6)
and References
Acl657S76515b519xis6)
Jaskunas
of S159
et al. (1977)
Jaskunas et al.. 1975b, 1977). Afus3-Al04 and Afus3-P105 were also isolated in a similar way, starting from NO1380 (see the text and Table 2). Lysogens carrying these mutant phages as well as other bacterial strains used in the present work are listed in Table 2. Transducing phages were grown by thermal induction of lysogens carrying these phages and separated from helper phages according to published procedures (Miller, 1972).
llstr mRNA)
DNA SALTS
12
34
56
78
910
11 12
WM -I---It
WM
WM
WM
WM
WM
1
Ii
1
H
1
TIME
10’
90*
MF
+
Figure 5. RNA Transcribed the Wild-Type sfr Promoter the Mutant PI05 Promoter
from Hind II 314 bp Fragments with (W; lanes 1, 3, 5, 7. 9 and 11) or with (M; lanes 2, 4, 6, 6, 10 and 12)
RNA was synthesized in the presence of a-32P-UTP using Hind II 314 bp fragments as template. DNA, RNA polymerase, ATP and GTP were preincubated at 37°C for 10 min. A mixture of CTP and &*P-UTP (Rif, -; lanes l-8) or a mixture of CTP, r+?P-UTP and rifampicin (final concentration 100 pg/ml; Rif, +; lanesg-12) was added, and transcription was allowed at 37°C for 10 min (lanes l4) or 90 set (lanes 5-12). Both preincubation and transcription were carried out either in high salt conditions (salts, H; lanes 1, 2. 5, 6, 9 and 10) or in low salt conditions (salts, L; lanes 3. 4, 7, 6. 11 and 12). RNA was prepared and analyzed by polyacrylamide gel electrophoresis as described in the accompanying paper (Post et al., 1978). Autoradiograms after exposure of 24 hr (lanes l-4) or 48 hr (lanes 5-12) are shown.
Experimental Bacterial Isolation described
Procedures and Phage of mutants previously
Strains of Afus3, (Jaskunas.
Afus3.Al01 Lindahl
and hfus3-1103 was and Nomura, 1975a;
Transcription of DNA Templates The standard conditions are described in the accompanying paper (Post et al., 1978). The salt concentrations described are called “high salt conditions” in this paper. “Low salt conditions” are also used; the conditions are the same as the high salt conditions, except that concentrations of some components were decreased both in preincubation and in transcription reaction as follows: 50 mM Tris-HCI (pH 7.8) (instead of 100 mM). 5 mM MgCI, (instead of 10 mM), 40 mM KCI (instead of 80 mM). After preincubation of DNA with RNA polymerase, ATP and GTP, as described in the accompanying paper, the transcription was started by the addition of CTP and u-~~P-UTP (or CTP, &2P-UTP and rifampicin). Reaction was stopped after either 90 set or 10 min of incubation, and RNA was analyzed as described in the accompanying paper (Post et al., 1978). Other Procedures Cloning of the Hind II 314 bp fragment from Afus3-P105 DNA was carried out using mini-Co1 El plasmid (pVH51; Hershfield et al., 1976) as a vector, as described in the accompanying paper regarding cloning of the corresponding fragment from Afus3 DNA (Post et al., 1978). Isolation of phage DNA, digestion of DNA with restriction enzymes, methods used for DNA sequencing and preparation of template DNA fragments are described in the accompanying paper (Post et al., 1978). Infection of ultravioletirradiated S159(A) cells with various transducing phages and analysis of proteins synthesized after phage infection were performed according to the procedures described in previous papers (Jaskunas et al., 1975a. 1975b, 1977). These are outlined in the legends to Figure 4 and Table 1. The experiments were carried out according to the NIH Guidelines which recommend Pl physical and EKl biological containment. Acknowledgments This work was supported in part by the College of Agriculture and Life Sciences, University of Wisconsin, and by grants from the NIH (administered by M.N.), the NSF (administered by M.N.) and the NIH (administered by S.R.J.). L.E.P. was supported by an NSF predoctoral fellowship. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received
May 15.1978
Cell 236
References Gilbert, W . (1976) . Starting and stopping sequences for the RNA polymerase . In RNA Polymerase, R . Losick and M . Chamberlin, eds . (New York : Cold Spring Harbor Laboratory), pp . 193-205 . Hershfield, V ., Boyer, H . W ., Chow, L . and Helinski, D . R . (1976) . Characterization of a mini-CoIE1 plasmid . J . Bacteriol . 126, 447453 . Jaskunas, S . R ., Lindahl, L . and Nomura, M . (1975a) . Specialized transducing phages for ribosomal protein genes of Escherichia coli . Proc . Nat . Acad . Sci . USA 72, 6-10 . Jaskunas, S . R ., Lindahl, L ., Nomura, M . and Burgess, R . R . (1975b) . Identification of two copies of the gene for the elongation factor EF-Tu in E . coli . Nature 257, 458-462 . Jaskunas, S . R ., Fallon, A . M . and Nomura, M . (1977) . Identification and organization of ribosomal protein genes of Escherichia coli carried by \fus2 transducing phage . J . Biol . Chem . 252, 7323-7336 . Kaltschmidt, E . and Wittmann, H . G . (1970) . Ribosomal proteins . VII . Two-dimensional polyacrylamide gel electrophoresis for fingerprinting of ribosomal proteins . Anal . Biochem . 36, 401-412 . Maxam, A . M . and Gilbert, W . (1977) . A new method for sequencing DNA . Proc . Nat . Acad . Sci . USA 74, 560-564 . Miller, J . H . (1972) . Experiments in Molecular Genetics (New York : Cold Spring Harbor Laboratory), p. 321 . Post, L . E ., Arfsten, A . E ., Reusser, F . and Nomura, M . (1978) . DNA sequences of promoter regions for the str and spc ribosomal protein operons in E . coli . Cell 15, 215-229 . Reznikoff, W . S . and Abelson, J . N . (1978) . The lac promoter . In The Operon, J . H . Miller and W . S . Reznikoff, eds . (New York : Cold Spring Harbor Laboratory), in press .
Isolation and Characterization of a Promoter Mutant in the str Ribosomal Protein Operon in E . coli Leonard E . Post, Ann E . Arfsten and Masayasu Nomura* Departments of Genetics and Biochemistry Institute for Enzyme Research University of Wisconsin Madison, Wisconsin 53706 S . Richard Jaskunas Department of Chemistry Indiana University Bloomington, Indiana 47401
Summary The afus3 transducing phage carries several operons for ribosomal proteins of E . call, including the str operon . A mutant transducing phage with a promoter mutation in this operon has been isolated . This mutant shows reduced stimulation of synthesis of proteins encoded by the operon, S12, S7, and elongation factors G and Tu, in ultraviolet-irradiated cells . This mutation also abolishes in vitro transcription from the str promoter . The DNA sequence of the mutant promoter shows that it is a point mutation 6 bases upstream from the in vitro transcription start site, changing the "Pribnow box" sequence from TAAAATT to TAAAACT . These results indicate that the site altered by the mutation, which is in the region just preceding the transcription start site, is important for the expression of the str operon . Introduction Isolation of mutants having alterations in promoter regions has been useful in analyzing the mechanisms involved in the expression and regulation of several bacterial operons such as the /ac operon (for reviews, see Gilbert, 1976 ; Reznikoff and Abelson, 1978) . Isolation of such promoter mutants in operons essential for growth, such as operons for ribosomal components, has been difficult in the past, and no promoter mutants in essential operons have been reported . To study the mechanism of regulation of ribosome biosynthesis, we have initiated isolation of promoter mutants in ribosomal protein operons . This paper reports isolation and characterization of the first promoter mutant which decreases the expression of all the genes in the str operon . We have demonstrated that the mutation involves a single nucleotide substitution at a site preceding the in vitro transcription start point and defines a site essential for the initiation of the transcription of the str operon . * To whom requests for reprints should be addressed .
Results and Discussion Isolation of Xfus3-P105 We have previously described isolation of two mutants of xfus3 transducing phages, Xfus3-4101 and kfus3-1103, which greatly decreased the activity of all the genes in the str operon . These mutants are a small deletion (101) and an insertion (1103), and were used to define the operon . Mutants were isolated using the same method as described for A101 and 1103 (Jaskunas et al ., 1975b) . Briefly, we started from a lysogen (NO1380) carrying Xfus3 and with chromosomal markers recA - , trkA - , spcr, strr and fusr . Xfus3 carries spc`, strs and fuss alleles . It is known that the three drugsensitive alleles are dominant over corresponding drug-resistant alleles and, therefore, the lysogen is sensitive to all three antibiotics-that is, Spc-S, Str-S and Fus-S . Spontaneous Str-R mutants were selected, and mutants which were Fus-R but Spc-S were investigated further . Transducing phages were isolated from these mutant bacteria, and phages carrying the mutations were characterized with respect to their density, the restriction enzyme digestion patterns of their DNA and their ability to stimulate the synthesis of proteins coded for by the str operon . Afus3-P105, which was used in this study, did not show any detectable density difference, but the Hind II digestion pattern of its DNA indicated a slight but definite alteration in the Hind II 314 base pair (bp) fragment compared to the parent Xfus3 (Figure 1) . As is described below, the P105 mutation involves a single nucleotide substitution in the 314 by fragment, and the observed mobility change reflects the nucleotide change rather than small deletions (or additions) . Nucleotide Sequence of the str Promoter Region of the afus3-P105 Mutant The Hind II 314 by fragment carrying the str promoter was cloned from the mutant Xfus3-P105 DNA in the same way described in the accompanying paper (Post et al ., 1978) for the cloning of the wildtype 314 by fragment . The hybrid plasmid thus obtained (pNO2011) was used to sequence the cloned mutant Hind II 314 by fragment . DNA sequence gels were carried out according to the method of Maxam and Gilbert (1977) ; Figure 2 shows portions of the gels which are pertinent to the sequence difference between the two promoters . We have found that the P105 mutation involves a single AT to GC transition at position -6-that is, within the Pribnow box discussed in the accompanying paper (Figure 3) . By sequencing from both the Hind II cut and the Hae III cut (see the accompanying paper), both strands of the DNA around the mutation were sequenced . No other sequence difference was observed .
Cell 232
Figure DNAs
1. Hind II Digestion
Products
from
Afus3
DNA and Mutant
The following DNAs were digested with Hind II restriction enzyme: pN02006 DNA (lane I), AfusS DNA (lane 2), Mus3-P107 DNA (lane 3). hfus3-PI05 DNA (lane 4) and hfus3-A104 DNA (lane 5). Digests were electrophoresed on 4% polyactylamide gels in 50 mM Trisborate (pH 6.3), 1 mM EDTA at 10 V/cm for 5 hr. hfus3-P107 is a spontaneous point mutant which has reduced the activities of all the genes in the str operon (our unpublished experiments). (pN02006 is a plasmid consisting of mini-Co1 El DNA with two Hpa I fragments from AfusS inserted into the Hind II site. This plasmid DNA was used as a reference; the smallest Hind II fragment is the 314 bp fragment which includes thestr promoter. Its position is indicated by an arrow.) It can be seen that the Hind II 314 bp fragment with the str promoter is overlapped with another DNA fragment of the same mobility in Afusd digests, but it moved slightly faster in Afus3-PI07 and Afus3-P105, and was absent in Afus3-A104. This photograph is a portion of the gel showing fragments of sizes approximately 200-900 bp.
Effects of the P105 Mutation on the Expression of the str Operon in Vivo The expression of genes in the str operon carried by the hfus3-P105 mutant was examined in ultraviolet-irradiated E. coli cells and compared with that of the corresponding genes carried by the parent, Afus3. The irradiated cells were infected with purified transducing phages; proteins synthesized after phage infection were labeled with 35S-methionine and analyzed by SDS-polyacrylamide gel electrophoresis followed by autoradiography (Figure 4; see also Table 1). In other experiments, cells were prelabeled with 14C-leucine, irradiated and infected with phages; the proteins synthesized after phage infection were labeled with 3H-leucine. The ratio of 3H/14C for most of the r proteins was
determined after separation of the r proteins by the standard two-dimensional polyacrylamide gel electrophoresis. The data were normalized to the expression of the S4 gene which is carried by the transducing phages but is in a different operon [Jaskunas, Fallon and Nomura, 1977; see Figure 1 of the accompanying paper by Post et al. (1978)]. The results are given in Table 1. It is clear that the PI05 mutation greatly (70-90%) reduced the expression of all the genes in the str operon-that is, genes for S12, S7, EF-G and EF-Tu. Synthesis of none of the other r proteins analyzed was affected. We conclude that the site altered by the P105 mutation, which is in the region just preceding the in vitro transcription start, is indeed important for the expression of the str operon in vivo. In the same experiments described above, three other mutant phages were also analyzed-hfus3AlOl, hfus3-1103 and hfus3-A104. The first two mutations were previously described (Jaskunas et al., 1975b). Al01 is a deletion removing about 1.6 kilobases (kb) DNA covering the right-hand coli-h DNA junction, the str promoter region and a part or all of thestr structural gene (Jaskunaset al., 1975b; L. Post and M. Nomura, unpublished experiments). 1103 is an insertion with the size of IS2 (about 1.3 kb) which is located within the Hind II 314 bp fragment [Jaskunas et al., 1975b; L. Post and M. Nomura, unpublished experiments; see also the accompanying paper by Post et al. (1978)]. The data shown in Figure 4 and Table 1 confirm the previous conclusion that these two mutations cause strong polar effects on the expression of distal genes in the same operon. The third mutation, A104, has not been described previously. This is a small deletion removing a DNA segment of about 500 bp, which covers the 314/2300 Hind IIsensitive site and the 530/1180 Hae Ill-sensitive site, but not the 500/314 Hind II-sensitive site (S. R. Jaskunas, L. Post and M. Nomura, unpublished experiments; for the respective restriction enzymesensitive sites, see Figure 1 of the accompanying paper). The exact position of the left end of the Al04 deletion in the promoter region has not been determined. From the results shown in Figure 4 and Table 1, one can see that the polar effect caused by the P105 mutation is as strong as the effects caused by the two deletions and stronger than the effect caused by the 1103 insertion. Since at least one of the two deletions, AlOl, removes the entire promoter region, the results underscore the importance of the base pair affected by the P105 mutation in the promoter function. Effects of the P105 Mutation on in Vitro Transcription of the str Operon The accompanying paper (Post et al., 1978) describes transcription experiments using the Hind II
Promoter 233
Mutant
in Ribosomal
Protein
Operon
a
Figure
2. DNA-Sequencing
Gels
Showing
b
the Mutation
in P105
The Hind II ends of the 314 bp fragments from pN02001 (a) or pN02011 (b) were was purified and sequenced according to Maxam and Gilbert (1977). Shown are been electrophoresed for 35 hr at 1000 V. Note that the Hind II end is labeled, so messenger RNA-that is, the bottom strand in Figure 3. The sequence indicated mutation.
5’ 3”
Figure 3. Mutational Alteration Promoter in hfus3-P105
of the DNA
The DNA sequence around the Pribnow promoter is shown. The mutational change, the P105 mutation is indicated by an arrow.
Sequence
in the str
box of the wild-type TA to CG. caused by
314 bp fragment as template. We observed the synthesis of RNA transcripts about 80 nucleotides long which define the specific in vitro start site, Experiments were repeated using the corresponding Hind II 314 bp fragment obtained from the P105 mutant as template, and radioactive RNAs synthesized were compared with those obtained with the standard template by polyacrylamide gel electrophoresis followed by autoradiography. As can be seen in Figure 5, the 80 nucleotide long RNA (band 1 in Figure 5) was produced only
labeled and digested with Hae Ill, and the 149 bp fragment portions of 20% polyacrylamide gels in 7 M urea that had the sequence read is the strand complementary to thestr represents the Pribnow box and includes the A + G P105
with the template fragment with the wild-type promoter, and not with the template fragment with the mutant promoter. This conclusion was obtained under various experimental conditions (see the legend to Figure 5). Similarly, the smaller RNA (band 2 in Figure 5) representing the first part (about 24-31 nucleotides long) of the sfr mRNA was also synthesized only with the wild-type promoter and not with the mutant promoter. In contrast, both templates were active in the synthesis of RNAs with larger sizes, which presumably represent RNA (or its aggregates) initiated nonspecifically. This indicates that the failure of the synthesis of the 80 nucleotide long RNA is not due to some trivial reason, such as general “damage” of the mutant DNA template. In addition, the same results were obtained using the DNA templates with and without DEAE column chromatographic purification [see Experimental Procedures in the accompanying paper by Post et al. (1978)]. We conclude that the synthesis of the 80 nucleotide long RNA reflects the in vivo transcription initiation, and that the mutant promoter is inefficient both in vivo and in vitro.
Cell 234
-
EF-G
-a
EF-Tu
In conclusion, we have demonstrated that the P105 mutation is a point mutation, which involves an AT to GC bp substitution at a position (position -6) within the Pribnow box for the str operon, and that this mutational change causes a great reduction in the transcription of the operon both in vivo and in vitro. It is interesting to note that position -6, which was affected by the mutation, corresponds to the sixth of the seven Pribnow box nucleotides, 5’-TATPuATPu-S’, and that the nucleotide (T) at this position is the one most conserved in promoter sequences reported to date (for review, see Reznikoff and Abelson, 1978). Further isolation and characterization of promoter mutants in the present system will certainly be useful for the analysis of the structure and function of r protein promoters. In addition, these results show that analysis of transcription initiation in essential operons can be aided by isolation of mutants, and encourages attempts to isolate similar promoter mutants in other essential operons.
Synthesized in Ultraviolet-Irradiated Various Transducing Phages
1 Figure
Table
2
3
4
5
4. SDS-Polyacrylamide
1. Relative
Gel
Synthesis
of Proteins Transducing
Protein
of
in Ultraviolet-Irradiated
after
Infection
with
Ultraviolet-irradiated S159(h) cells were infected with (1) AC1657S7, (2) Afus3, (3) hfus3-AlOl, (4) Afus3-1103, (5) Afus3-A104 and (6) hfus3-P105. 35S-methionine was added to infected cells 30 min after infection, and incorporation was allowed for 10 min at 37°C. Samples were prepared and analyzed by electrophoresis on 6.75% polyactylamide-0.1% SDS gels followed by autoradiography. Experimental details are the same as described previously (Jaskunas et al., 1975b). An autoradiogram is shown.
6 Electrophoresis
Bacteria
Proteins
Bacteria
after
Infection
with
Mutants
of AfusS
Phages Afus3-I 103
Afus3-Al04
hfus3-P105
Afusa
Afus3-Al01
s12
1 .o
0.06
0.20
0.05
0.05
57
1 .o
0.39
0.46
0.23
0.29
EF-G
1 .o
0.05
0.29
0.23
0.17
EF-TU
1 .o
0.16
0.36
0.24
0.16
Others
1 .o
0.96
0.97
1.02
0.97
Strain S159(A) was irradiated with ultraviolet light and infected with purified transducing phages, and the proteins synthesized after infection were analyzed as previously described (Jaskunas et al., 1975b). Briefly, this was carried out as follows. The relative amounts of EF-G and EF-TU synthesized after infection were determined from autoradiograms similar to those shown in Figure 4. The autoradiograms were scanned with a Joyce-Loebl microdensitometer, and the amounts of EF-G and EF-TU synthesized relative to the amount of RNA polymerase subunit a synthesized were calculated from the area of the peaks. These values were then normalized to the amount synthesized after infection with the parent phage. hfusd. To determine the relative synthesis of S7, S12 and other r proteins, cells were prelabeled with “C-leucine, irradiated and infected with transducing phages. Proteins synthesized after infection were labeled with JHleucine, and r proteins were separated by two-dimensional polyacrylamide gel electrophoresis (Kaltschmidt and Wittmann. 1970). The ‘H/ “C ratios for all the r proteins except Sl, L20, L31, and L34 were determined. The 3H/‘4C ratios were first normalized to the value for S4. The normalized ratios were then divided by the corresponding values obtained with the parent phage, tius3. Data for S7 and S12 are given. The stimulation of the synthesis of other r proteins was not significantly affected by the mutations studied. The average of the normalized relative stimulation for these proteins is also given. It should be noted that the genes for S4 and a are in a separate transcription unit [see Figure 1 of the accompanying paper by Post et al. (1976)]. and the synthesis of these proteins does not appear to be affected by any of the mutations studied in this work.
Promoter 235
Table
Mutant
in Ribosomal
2. Bacterial
Strains
Protein
Operon
Used
Strain
Genotype
NO1345
kdpABC5,
NO1380
NO1345
Source
and Comments
spc’, SW,fus’,
trkA401, (Afus3,
recA
hcl857S765156519xis6)
Jaskunas
et al. (1975a)
Jaskunas
et al. (1977)
Jaskunas
et al. (1975b)
Jaskunas
et al. (1975b)
NO1682
NO1345
(hfus3-AlOl,
NO1684
NO1345
(hfus3-1103,
NO1686
NO1345
(Afus3-A104,
Acl657S7b5156519xis6)
This work
NO1667
NO1345
(Afus3-P105.
Acl657S76515b519xis6)
This work
S159(A)
Apapa
lysogen
Acl857S7b5156519xis6)
and References
Acl657S76515b519xis6)
Jaskunas
of S159
et al. (1977)
Jaskunas et al.. 1975b, 1977). Afus3-Al04 and Afus3-P105 were also isolated in a similar way, starting from NO1380 (see the text and Table 2). Lysogens carrying these mutant phages as well as other bacterial strains used in the present work are listed in Table 2. Transducing phages were grown by thermal induction of lysogens carrying these phages and separated from helper phages according to published procedures (Miller, 1972).
llstr mRNA)
DNA SALTS
12
34
56
78
910
11 12
WM -I---It
WM
WM
WM
WM
WM
1
Ii
1
H
1
TIME
10’
90*
MF
+
Figure 5. RNA Transcribed the Wild-Type sfr Promoter the Mutant PI05 Promoter
from Hind II 314 bp Fragments with (W; lanes 1, 3, 5, 7. 9 and 11) or with (M; lanes 2, 4, 6, 6, 10 and 12)
RNA was synthesized in the presence of a-32P-UTP using Hind II 314 bp fragments as template. DNA, RNA polymerase, ATP and GTP were preincubated at 37°C for 10 min. A mixture of CTP and &*P-UTP (Rif, -; lanes l-8) or a mixture of CTP, r+?P-UTP and rifampicin (final concentration 100 pg/ml; Rif, +; lanesg-12) was added, and transcription was allowed at 37°C for 10 min (lanes l4) or 90 set (lanes 5-12). Both preincubation and transcription were carried out either in high salt conditions (salts, H; lanes 1, 2. 5, 6, 9 and 10) or in low salt conditions (salts, L; lanes 3. 4, 7, 6. 11 and 12). RNA was prepared and analyzed by polyacrylamide gel electrophoresis as described in the accompanying paper (Post et al., 1978). Autoradiograms after exposure of 24 hr (lanes l-4) or 48 hr (lanes 5-12) are shown.
Experimental Bacterial Isolation described
Procedures and Phage of mutants previously
Strains of Afus3, (Jaskunas.
Afus3.Al01 Lindahl
and hfus3-1103 was and Nomura, 1975a;
Transcription of DNA Templates The standard conditions are described in the accompanying paper (Post et al., 1978). The salt concentrations described are called “high salt conditions” in this paper. “Low salt conditions” are also used; the conditions are the same as the high salt conditions, except that concentrations of some components were decreased both in preincubation and in transcription reaction as follows: 50 mM Tris-HCI (pH 7.8) (instead of 100 mM). 5 mM MgCI, (instead of 10 mM), 40 mM KCI (instead of 80 mM). After preincubation of DNA with RNA polymerase, ATP and GTP, as described in the accompanying paper, the transcription was started by the addition of CTP and u-~~P-UTP (or CTP, &2P-UTP and rifampicin). Reaction was stopped after either 90 set or 10 min of incubation, and RNA was analyzed as described in the accompanying paper (Post et al., 1978). Other Procedures Cloning of the Hind II 314 bp fragment from Afus3-P105 DNA was carried out using mini-Co1 El plasmid (pVH51; Hershfield et al., 1976) as a vector, as described in the accompanying paper regarding cloning of the corresponding fragment from Afus3 DNA (Post et al., 1978). Isolation of phage DNA, digestion of DNA with restriction enzymes, methods used for DNA sequencing and preparation of template DNA fragments are described in the accompanying paper (Post et al., 1978). Infection of ultravioletirradiated S159(A) cells with various transducing phages and analysis of proteins synthesized after phage infection were performed according to the procedures described in previous papers (Jaskunas et al., 1975a. 1975b, 1977). These are outlined in the legends to Figure 4 and Table 1. The experiments were carried out according to the NIH Guidelines which recommend Pl physical and EKl biological containment. Acknowledgments This work was supported in part by the College of Agriculture and Life Sciences, University of Wisconsin, and by grants from the NIH (administered by M.N.), the NSF (administered by M.N.) and the NIH (administered by S.R.J.). L.E.P. was supported by an NSF predoctoral fellowship. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Received
May 15.1978
Cell 236
References Gilbert, W . (1976) . Starting and stopping sequences for the RNA polymerase . In RNA Polymerase, R . Losick and M . Chamberlin, eds . (New York : Cold Spring Harbor Laboratory), pp . 193-205 . Hershfield, V ., Boyer, H . W ., Chow, L . and Helinski, D . R . (1976) . Characterization of a mini-CoIE1 plasmid . J . Bacteriol . 126, 447453 . Jaskunas, S . R ., Lindahl, L . and Nomura, M . (1975a) . Specialized transducing phages for ribosomal protein genes of Escherichia coli . Proc . Nat . Acad . Sci . USA 72, 6-10 . Jaskunas, S . R ., Lindahl, L ., Nomura, M . and Burgess, R . R . (1975b) . Identification of two copies of the gene for the elongation factor EF-Tu in E . coli . Nature 257, 458-462 . Jaskunas, S . R ., Fallon, A . M . and Nomura, M . (1977) . Identification and organization of ribosomal protein genes of Escherichia coli carried by \fus2 transducing phage . J . Biol . Chem . 252, 7323-7336 . Kaltschmidt, E . and Wittmann, H . G . (1970) . Ribosomal proteins . VII . Two-dimensional polyacrylamide gel electrophoresis for fingerprinting of ribosomal proteins . Anal . Biochem . 36, 401-412 . Maxam, A . M . and Gilbert, W . (1977) . A new method for sequencing DNA . Proc . Nat . Acad . Sci . USA 74, 560-564 . Miller, J . H . (1972) . Experiments in Molecular Genetics (New York : Cold Spring Harbor Laboratory), p. 321 . Post, L . E ., Arfsten, A . E ., Reusser, F . and Nomura, M . (1978) . DNA sequences of promoter regions for the str and spc ribosomal protein operons in E . coli . Cell 15, 215-229 . Reznikoff, W . S . and Abelson, J . N . (1978) . The lac promoter . In The Operon, J . H . Miller and W . S . Reznikoff, eds . (New York : Cold Spring Harbor Laboratory), in press .
