Ribosomal Protein S4 Acts in trans as a Translational Repressor to Regulate Expression of the a Operon in Escherichia colit
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JOURNAL OF BACTERIOLOGY, July 1982, p. 193-202 Vol. 151, No. 1
0021-9193/82/070193-10$02.00/0
Ribosomal Protein S4 Acts in trans as a Translational
Repressor to Regulate Expression of the a Operon in
Escherichia colit
SUE JINKS-ROBERTSON AND MASAYASU NOMURA*
Institute for Enzyme Research and the Departments of Genetics and Biochemistry, University of Wisconsin,
Madison, Wisconsin 53706
Received 24 November 1981/Accepted 4 March 1982
Ribosomal protein (r-protein) S4 is the translational repressor which regulates
the synthesis rates of r-proteins whose genes are in the a operon: r-proteins S13,
Sit, S4, and L17. In a strain having a mutation in the gene for r-protein S4
(rpsD), the mutant S4 fails to regulate expression of the a operon, resulting in
specific and significant overproduction of r-proteins S13, S11, and S4. This
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confirms and extends similar observations made with rpsD mutants
(M. 0. Olsson and L. A. Isaksson, Mol. Gen. Genet. 169:271-278, 1979) before
post-transcriptional regulation of r-protein synthesis was proposed and is consis-
tent with the established regulatory role of r-protein S4. The rpsD mutant has been
used to study the question of whether regulatory r-proteins function in trans or
strictly in cis as translational repressors. The mutant strain was lysogenized with
one or two specialized transducing phages carrying a wild-type S4 gene to obtain
strains which were diploid or triploid with respect to the a operon. The wild-type
and mutant forms of S4 were separated by two-dimensional polyacrylamide gel
electrophoresis, which allowed accurate measurement of the relative contribu-
tions of r-proteins from different a operons within a single cell. We found that
expression of r-proteins from the chromosomal a operon containing the rpsD
allele was reduced when the wild-type S4 was present, with the effect being
greater in the triploid strain than in the diploid strain. We conclude that the wild-
type S4 acts in trans as a translational repressor to regulate expression from the
chromosomal a operon.
The synthesis rates of most ribosomal pro- the r-proteins whose genes are in the same
teins (r-proteins) in exponentially growing Esch- transcription unit (operon) as the repressor r-
erichia coli are identical and respond coordi- protein (3, 8-10, 13, 37-39, 41).
nately to changes in environmental conditions The a operon, for example, contains the genes
(for reviews, see references 15 and 28). Based on for r-proteins S13, S11, S4, and L17 and RNA
gene dosage experiments in which we compared polymerase subunit a. It has been shown that S4
the synthesis rates of r-proteins with the synthe- specifically inhibits synthesis of itself, S13, and
sis rate of r-protein mRNA, we proposed a S11 in in vitro experiments utilizing a DNA-
model of post-transcriptional feedback regula- dependent protein synthesis system (37). In in
tion to explain the coordinate and stoichiometric vivo experiments in which the S4 gene (together
synthesis of r-proteins (12). We hypothesized with the S11 gene) has been fused to the lac
that r-protein synthesis and ribosome assembly promoter on a multicopy plasmid, induction of
are coupled so that when r-protein synthesis S4 synthesis causes a specific decrease in the
rates exceed those needed for ribosome assem- synthesis rates of S13, L17, and presumably a
bly, "free" r-proteins interact with their mRNA decrease in the synthesis rates of S4 and S11
and block further translation. This model has from the chromosomal a operon (8; D. Bedwell,
been confirmed and refined by both in vitro and D. Dean and M. Nomura, unpublished experi-
in vivo experiments. These experiments have ments).
identified specific r-proteins (S4, S7, S8, Li, L4, In our original gene dosage paper (12), we
and L1O) as "translational repressors" which considered available information on the domi-
selectively inhibit the synthesis of some or all of nance of wild-type Strs alleles over mutant Strr
alleles (5). Based on this information, we sug-
t Paper no. 2554 from the Department of Genetics, Univer- gested that the translational repressor r-proteins
sity of Wisconsin-Madison. would act mainly on mRNA at the site of their
193194 JINKS-ROBERTSON AND NOMURA J. BACTERIOL.
synthesis (cis regulation) and not on homologous ducing phage constructed in vitro by fusion of the lacZ
mRNA coming from a different genome in a gene with a 2,000-base-pair EcoRI-HindIII fragment
merodiploid strain (trans regulation). In this derived from A spcl (H. de Boer, this laboratory). The
paper, we describe the results of experiments 2,000-base-pair fragment contains the promoter for the
designed to study the question of whether this a operon (Pa) and the genes for r-proteins S13, S11,
and S4; it does not contain the genes for RNA poly-
cis model of regulation is correct. We isolated an merase subunit a or r-protein L17 (see A spc2 in Fig. 1
E. coli regulatory mutant which had an alter- for a map of this region). The left arm of A a-lacl was
ation in the gene for r-protein S4 (rpsD) and obtained by digesting XRS205-7 with HindIII. XRS205-
showed an increase in the relative synthesis 7 carries a fused trp-lac operon originally constructed
rates of r-proteins S13, Sit, and S4. This mutant by the W205 fusion (24) and was obtained from K.
is similar to those described previously by Ols- Bertrand and W. S. Reznikoff. The right arm of A a-
son and Isaksson (32, 33). The rpsD mutant lacl was obtained by digesting X459 with EcoRI. X459
strain was lysogenized with one or two special- was obtained from N. E. Murray and is a Asrll-2
ized transducing phages to obtain strains that phage having the normal att site and c1857 on the right
arm (27). Ligation of the right and left vector arms with
were diploid or triploid with respect to the a the 2,000-base-pair A spcl fragment produced a hybrid
operon. We present evidence that in such dip- phage with the lacZ gene fused to Pa in the correct
loid or triploid strains, the wild-type S4 func- orientation. X a-lacl was isolated as a plaque-forming
tions as a translational repressor to regulate the phage which produced Lac' lysogens after infection
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synthesis rate of the chromosomal mutant S4. of strain B2550. The structure of the phage was
We concluded that r-protein S4 (and presumably confirmed by analysis of the DNA with various restric-
all other repressor r-proteins) acts in trans to tion enzymes (Fig. 1).
regulate gene expression and not strictly in cis as A a-lacl was crossed with Apapa to replace the c1857
we originally suggested (12). In addition, we allele from X459 with the cI+ allele of Apapa so that
lysogens could be grown at 37°C (A. Miura, this
discuss the altered pattern of r-protein synthesis laboratory). A a-lacl cI+, the resulting recombinant
in our rpsD mutant and in those studied by phage, is used in the present study.
Olsson and Isaksson (33) in relation to the Construction of A h*, k spc2, and A a-lac cI+
regulatory role of S4 established previously. lysogens. Lysogens of N02514 and N02515 were
constructed by standard phage techniques. A trkA
MATERIALS AND METHODS lysogens contained kpapa as helper, and A spc2 lyso-
gens contained either Apapa or A a-lacl cI+ as helper.
Bacterial strains. A list of the E. coli K-12 strains The structures of X trkA and X spc2 are shown in Fig.
used in this study is given in Table 1. 1. X trkA and A spc2 lysogens were isolated by
Construction of A, a-lacl. A a-lacl is a hybrid trans- selecting for expression of the aroE+ allele carried on
TABLE 1. Bacterial strains
Designation Description Source, method of construction, or reference
B2550 A(lacOZ) lacYc A(trpEA)2 tna-2 Nalr K. Bertrand, W. S. Reznikoff
(= N02183)
N0816 F- aroE malA thi Laboratory collection
N02143 F- gal thi trkA401 kdpABC5 aroE lacY Laboratory collection
lacZ rpsL (Strr)
N02206 Same as B2550, but rpsL Spontaneous streptomycin-dependent mu-
tant of B2550
N02207 Same as B2550, but rpsD rpsL Spontaneous streptomycin-independent
"revertant" of N02206
N02246 Same as N0816, but aroE+ rpsD Transduction of N0816 by PI (NO2207),
selecting aroE+; screen for hypersensi-
tivity to streptomycin
N02514 Same as N02143, but trkA+ rpsL+ Transduction of N02143 by PI (NO2246),
selecting for trkA+; screen for aroE Strs
N02515 Same as N02143, but trkA+ rpsD rpsL+ Transduction of N02143 by Pl (NO2246),
selecting for trkA+; screen for aroE Strs,
temperature sensitivity
N02543 Apapa, A trkA double lysogen of N02514 This work
N02544 Apapa, A spc2 double lysogen of N02514 This work
N02545 A a-lacl cI+, A spc2 double lysogen of This work
N02514
N02546 Xpapa, A trkA double lysogen of N02515 This work
N02547 Apapa, A spc2 double lysogen of N02515 This work
N02548 A a-lacl cI+, A spc2 double lysogen of This work
N02515VOL. 151, 1982 TRANSLATIONAL REPRESSOR r-PROTEIN S4 195
locZ+ trpA+ trpB' W205 fusion
(a)
, 'c , , --
2 5 fso
205-7 "2L*" 459 *R"
X4a- lac I
S4 Sll S13 2000 bp frogment
PaT
(b)
--Pa -Pspc -PsI0
L17 a S4 SIl S13 L15L30S5U8 L6 S8 S14 L5 L24L14 SM29Lr6S322,1WL2L23L L3 Ssb
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[ X spc 2
Xtrk A
aroE trkA
Ikb
FIG. 1. Structures of specialized transducing phages. (a) Structure of A a-lacl. 205-7 "L" is the left arm of A
derived from digestion of XRS205-7 with Hindlll (h). 459 "R" is the right arm of A derived from digestion of X459
with EcoRI (+). Symbols: ga, phage DNA; o, bacterial DNA; U, deletion of phage DNA in X459. The 2,000-
base-pair EcoRI-HindlII fiagment is from A spcl and is identical to that shown for A spc2 in (b). (b) Structures of
X spc2 and A trkA. Symbols: y, phage DNA; o, bacterial DNA. EcoRI (+) and Hindll (+) sites for A spc2
bacterial DNA are shown (22). The bacterial DNA of A spc2 contains a single HindlIl site (+) between the S4 and
a genes.
these phages. A a-lacl cI+ lysogens were isolated by ABG and ABG-K2.5 minimal plates were used for
screening X-resistant cells for those producing 3-galac- selecting aroE+ and trkA+ transductants, respective-
tosidase. Since the recipient strains are lacY in addi- ly. ABG plates contained AB medium (6) supplement-
tion to lacZ, LacZ+ lysogens could not be directly ed with 0.2% glucose and 1 ,ug of thiamine per ml.
selected on standard indicator plates. Production of 0- ABG-K2.5 plates contained less potassium than ABG
galactosidase was shown by spraying colonies with plates, thus allowing growth of kdpABCS strains, but
toluene followed by the addition of 1 ml of 10 mM o- not kdpABCS trkA double mutants (11). ABG-K2.5
nitrophenylqf-D-galactopyranoside (23). LacZ+ cells medium was made the same as ABG, except that
turn bright yellow within several minutes, whereas sodium salts were substituted for potassium salts in
LacZ- cells remain white. AB medium and KCl was added to a final concentra-
The presence of A trkA or A spc2 and A a-lacl cI+ tion of 2.5 mM. A 10-ml amount of aro mix (5 mg of
prophage was confirmed by the production of aroE+ tyrosine, 5 mg of phenylalanine, 2 mg of shikimic acid,
and lacZ+ transducing phage, respectively, after in- 0.5 mg of p-aminobenzoic acid, and 0.5 mg of p-
duction of lysogens with 1 Fg of mitomycin C per ml. hydroxybenzoic acid per ml) was added per liter of
Growth cond_ions. LB medium contained 10 g of minimal ABG medium for growth of aroE strains.
tryptone, S g of yeast extract, 5 g of NaCl, and 1 ml of Details of the labeling conditions are given in footnote
1 N NaOH per liter. For growth and selection of a of Table 2.
streptomycin-dependent strains, LB was supplement- Preparation and analysis of r-proteins. To analyze
ed with 100 F.g of streptomycin per ml. Streptomycin unlabeled r-proteins from various strains, 1 liter of
sensitivity was used to distinguish rpsD rpsL double cells was grown in LB medium at 37°C to approximate-
mutants from rpsD rpsL+ single mutants in the trans- ly 5 x 108 cells per ml. Cells were harvested by
duction experiments; the former are more sensitive, centrifugation and washed with TMAI (10 mM Tris-
whereas the latter are less sensitive to streptomycin hydrochloride [pH 7.5], 30 mM NH4CI, 10 mM MgCl2,
than an rpsD+ rpsL+ strain (32). For determining the 6 mM P-mercaptoethanol). Washed cells were resus-
degree of streptomycin sensitivity, LB plates were pended in 7.5 ml of TMAI and lysed by sonication.
supplemented with 1, 3, 5, and 10 pLg of streptomycin Cellular debris was removed by centrifugation at
per ml. For growth of trkA mutants, the medium was 10,000 rpm for 1 h. 70S ribosomes were pelleted by
supplemented with 0.1 M KCI. centrifuging the supernatant for 4 h at 30,000 rpm in a1% JINKS-ROBERTSON AND NOMURA J. BACTERIOL.
TABLE 2. Relative synthesis rates of r-proteins in haploid, diploid, and triploid strains'
Relative synthesis rate of r-proteins
r-Proteinb N02514 control strains N02515 mutant strains
Haploid Diploid Triploid Haploid Diploid Triploid
Li 1.00 1.00 1.00 1.00 1.00 1.00
L2 0.82 0.97 0.87 0.92 1.00 1.01
L14 0.85 1.06 1.00 1.01 1.15 1.10
L17 0.95 0.95 0.93 1.19 1.37 1.11
S3 0.91 0.98 0.96 0.84 1.04 1.02
S4w 0.82 0.94 0.89 0.90 0.91
S4m 1.98 0.80 0.43
S7 0.95 0.97 1.00 0.98 0.99 1.01
Sit 0.93 1.09 1.07 1.89 1.74 1.32
S12 1.12 1.13 1.13 1.09 1.07 1.16
S13 0.93 1.14 1.10 1.87 1.62 1.32
a Haploid strains were Apapa, X trkA double lysogens; diploid strains were Xpapa, A spc2 double lysogens; and
triploid strains were k spc2, A a-lacl cI+ double lysogens. Cells were grown at 37°C in AB medium supplemented
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with 0.4% glucose and 1 ,ug of thiamine per ml (ABG medium). At a density of approximately 2 x 108 cells per ml,
cultures were pulse-labeled for 1 min with [3H]lysine (0.441 nmollml; 68 .Ci/nmol) followed by a 1-min chase
with excess nonradioactive lysine (500 ,ug/ml). At the end of the labeling period, cells were chilled rapidly on ice
and washed twice with 50 mM Tris-hydrochloride (pH 8.0). The growth rates of all strains under these conditions
were 1.3 doublings per h, except for that of the rpsD haploid strain N02515 (Xpapa, A trkA). The growth rate of
this strain was 0.5 doublings per h. Each 3H-labeled sample was divided; half was mixed with 14C-labeled rpsD
cells, and half was mixed with "4C-labeled wild-type cells. 14C-labeled cultures were prepared by growing cells in
ABG medium containing ["4C]lysine (5.85 nmol/ml; 342 mCi/mmol) for at least three generations, and these were
used as an internal standard for calculating the synthesis rates of individual r-proteins. Proteins were extracted
from the mixed cells and separated by two-dimensional gel electrophoresis. After staining and destaining of the
gels, individual r-protein spots were cut, dried, and oxidized in a Packard sample oxidizer to separate the 3H and
14C. The 3H/14C ratio was calculated for each r-protein, and these ratios were normalized to that for r-protein Li.
The normalized values are given and are the average value from four samples: 14C-labeled mutant cells were used
in two samples, and "'C-labeled wild-type cells were used in two samples. The same 3H/14C values were obtained
regardless of the type of 14C-labeled cells used, except in the case of L17 and S7. The values for these two r-
proteins are those obtained by using wild-type 14C-labeled cells as an internal standard. r-Protein S7 appears to
be turning over faster in the mutant strain than in the wild-type strain, so that the amount of 14C-labeled S7 is less
in the uniformly labeled mutant cells than in the wild type cells. As a result, all of the 3H/14C values obtained for
S7 by using the "4C-labeled mutant cells as a normalization standard are about 30o greater than the values
shown. L17 was overproduced slightly in the mutant strain relative to the wild-type strain, and the overproduced
protein was stable (similar to previous observations with Lii [21]). 3H/14C values for L17 with mutant cells as a
reference are thus slightly lower than those with wild-type cells. The values for r-proteins in the a operon which
are pertinent to the discussion in the text are in boldface type.
b
S4w, Wild-type S4; S4m, mutant S4.
Beckman 40 rotor. The 70S ribosome pellet was sus- described above. Labeled protein pellets were sus-
pended in 0.25 ml of TMAI and stored at -60°C. pended in 30 ,ul of sample buffer containing unlabeled
For two-dimensional gel analysis, r-proteins were r-proteins (5 absorbancy at 260 nm equivalents from
extracted from crude 70S ribosomes with 67% acetic N02514 and 5 absorbancy at 260 nm equivalents from
acid (18) and precipitated with 5 volumes of acetone N02515) and were separated by two-dimensional gel
(1). Samples for two-dimensional gels contained 5 to electrophoresis.
10 absorbancy at 260 nm equivalents of crude ribo-
somes and were suspended in a final volume of 30 ,ul of RESULTS
gel sample buffer (60 mM Tris, 75 mM boric acid, 6
mM dithiothreitol, 8 M urea). Two-dimensional poly- Isolation of an rpsD mutant. The rpsD mutant
acrylamide gel electrophoresis was done as described used in this study was isolated by the method of
by Howard and Traut (20). Olsson and Isaksson (32), which is based on the
To measure the synthesis rates of r-proteins, total following observations. An E. coli mutant hav-
cellular protein was extracted from labeled cells as ing a mutation in the gene for r-protein S12
follows. 3H-labeled and "4C-labeled cells were mixed, (rpsL) which renders it streptomycin dependent
washed with 50 mM Tris-hydrochloride (pH 8.0), and (2) will become phenotypically streptomycin in-
suspended in 50 p.1 of lysis solution (1.2 ml 50 mM Tris-
hydrochloride [pH 8.0], 150 ,ul of 0.1 M EDTA [pH dependent through acquisition of an additional
7.1], 60 ,ul of lysozyme [2 mg/ml]). Cells were lysed by mutation. Many of the secondary mutations are
freeze-thawing six times, and proteins were extracted in rpsD (ramA), the gene for r-protein S4 (19,
with acetic acid and precipitated with acetone as 32). Strains having such an rpsD mutation areVOL. 151, 1982 TRANSLATIONAL REPRESSOR r-PROTEIN S4 197
usually temperature sensitive, and this tempera- The rpsD mutation of N02207 was separated
ture sensitivity has been correlated with a failure from the rpsL mutation by P1 transduction and
to assemble 30S ribosomal subunits at the re- was subsequently introduced into a genetic
strictive temperature (30). In most cases, the background appropriate for the selection and
mutant protein has been found to be shorter than maintenance of diploid and triploid strains.
the wild-type protein and to have considerable N02514 and N02515 are the resulting isogenic
alteration of amino acid sequence at the car- aroE rpsD+ and aroE rpsD strains, respectively,
boxy-terminal end (14). which were used in the experiments described
A spontaneous streptomycin-dependent mu- below. It should be noted that our rpsD mutant
tant (NO2206) was isolated by plating cells of strains grew much better at 37 than at 30°C, and
strain B2550 on LB medium containing 100 pLg of that temperature sensitivity was more striking
streptomycin per ml. Spontaneous streptomy- when growth was compared at 37 and 42°C
cin-independent revertants of N02206 were iso- rather than at 30 and 42°C. All of the experi-
lated by plating cells in the absence of strepto- ments described in this paper were done at 37°C.
mycin at 30°C, and single colonies were Comparison of the synthesis rates of r-proteins
screened for a temperature-sensitive phenotype in rpsD and rpsD' haploid strains. The relative
at 42°C. Analysis of the r-proteins of one such synthesis rates of r-proteins in rpsD mutants has
temperature-sensitive revertant (NO2207) re- previously been examined by Olsson and Isaks-
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vealed the presence of an S4 spot with altered son (33). They found that the relative synthesis
electrophoretic mobility on a two-dimensional rates of r-proteins S4, S12, and S13 were elevat-
polyacrylamide gel (Fig. 2). The mutant S4 pro- ed in rpsD mutant strains, and they suggested
tein was approximately 15% shorter than the that S4 might be involved in regulating the
wild-type protein as judged from the mobility on synthesis rates of the promoter-proximal r-pro-
a sodium dodecyl sulfate-polyacrylamide gel, teins in both the str and a operons. Their work,
and peptide analysis has shown that the missing however, was done before the model of post-
15% corresponds to the carboxy-terminal end of transcriptional regulation of r-protein synthesis
the wild-type S4 (G. Craven, personal communi- was proposed, so it was important to confirm
cation). Although the exact nature of the rpsD and possibly extend their findings by using our
mutation has not been characterized, these ob- rpsD mutant. It appeared to us that rpsD mu-
servations suggest that the mutation causes pre- tants might be regulatory mutants which had lost
mature termination of translation of the S4 the translational repressor activity of the wild-
mRNA. type S4. We reasoned that if this were indeed the
a b
FIG. 2. Two-dimensional gel electrophoresis of 70S r-proteins. r-Proteins were extracted from crude 70S
ribosomes containing 10 absorbancy at 260 nm equivalents of (a) wild-type ribosomes from N02514 and (b)
mutant ribosomes from N02515. W, wild-type form of r-protein S4; M, mutant form of r-protein S4. Only the
basic sides of the gels are shown.198 JINKS-ROBERTSON AND NOMURA J. BACTERIOL.
case, then we should see overproduction only of 1). The haploid strain contains one mutant
those r-proteins encoded by the a operon (S13, (rpsD) a operon, the diploid strain contains one
Si1, S4, and L17). mutant and one wild-type (rpsD+) a operon, and
The synthesis rates of r-proteins in N02514 the triploid strain contains one mutant and two
(Xpapa, A trkA) and NO2515 (Xpapa, A trkA) wild-type a operons. The copy number of the a
were measured by pulse-labeling exponentially operon refers to the portion from the promoter
growing cultures for 1 min with [3H]lysine and through the S4 gene (see below). Since the
chasing for 1 min with an excess of nonradioac- mutant S4 can be clearly separated from the
tive lysine. 3H-labeled cells were mixed with wild-type S4 by two-dimensional gel electropho-
cells uniformly labeled with [14C]lysine, and the resis (Fig. 2), it is possible to examine the effect
ratio of 3H to 14C in individual r-proteins was of the wild-type S4 synthesized from the phage
determined (see footnote a of Table 2 for de- genomes on the "unregulated" synthesis of the
tails). The results are presented in Table 2 as 3H/ mutant S4 from the chromosome. Using the
14C values which have been normalized to the model of translational regulation, we made
value for r-protein LI. The values in Table 2 quantitative predictions as to what the synthesis
thus represent the synthesis rates of individual r- rates of a operon r-proteins should be if trans or
proteins relative to the synthesis rate of r-pro- cis regulation by the wild-type S4 were occur-
tein LI in each strain. Li was chosen as the ring in the diploid and triploid strains. These
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normalization standard because it separates well predictions are presented schematically in Fig. 3
from other r-proteins on two dimensional gels, and are based on the following assumptions. (i)
and because its gene is located in the rifregion at The S13, Sli, and S4 cistrons are sequentially
89 min on the E. coli chromosome rather than in translated (34, 40), and their relative synthesis
the str-spc region at 72 min (28). The addition of rates are identical. S4 acts at or near the begin-
identical 14C-labeled cells to different 3H-labeled ning of the S13 cistron mRNA and regulates Si1
samples allowed direct comparison of the syn- and S4 translation as a consequence of this
thesis rates of r-proteins in different strains as primary action (29, 40). (ii) The mutant S4 does
well as within a given strain. In the experiments not have any significant activity as a translation-
done here, all 3H-labeled samples were divided al repressor, and as a consequence the relative
so that r-proteins could be analyzed by using synthesis rates of S13, Sit, and S4 in the mutant
both mutant and wild-type 14C-labeled cells (see haploid strain increase by a factor of about two
below and footnote a of Table 2). under our experimental conditions (see above).
In agreement with the data of Olsson and (iii) In diploid and triploid strains containing
Isaksson, we found that the relative synthesis both forms of S4, only the wild-type S4 gets
rates of S4 and S13 were elevated about 90% in incorporated into ribosomes (33; our observa-
the rpsD mutant strain N02515 (Xpapa, A trkA). tions, not shown). The relative synthesis rate of
The synthesis rate of r-protein Sit has not been the wild-type S4 (coming from two phage ge-
previously examined in rpsD mutants, but, as nomes in the case of triploid) should always be
predicted, our experiments demonstrated an 1.0 so that its synthesis rate will be balanced
overproduction of this protein. It should be with the synthesis rates of other r-proteins that
noted that Sit was overproduced to the same are not regulated by S4. (iv) The ratio of the
extent as SI3 and S4. In contrast, we found that mutant S4 gene copy number to the wild-type S4
there was only a slight increase (about 20%o) in gene copy number is about 1:1 and 1:2 in the
the relative synthesis rate of L17 in the rpsD diploid and triploid strains, respectively. (We
strain. Olsson and Isaksson observed overpro- have not determined the sites of transducing
duction of S12 in addition to S4 and S13 in their phage integration in the strains used. Assuming
rpsD mutants, but our experiments failed to that the phages integrate at the normal X attach-
substantiate this finding. We conclude that the ment site, their copy numbers under our experi-
rpsD mutation affects the regulation of the syn- mental conditions would be about 20% lower
thesis rates of S13, Sli, and S4 (and possibly than if they integrated into the str-spc region [4,
L17; see below), but not that of r-protein S12. 7]. In our predictions, we have disregarded this
Diploid-triploid experiments with specialized small difference in copy number between the
transducing phages. Strain NO2515, which car- mutant S4 gene on the chromosome and the
ries the rpsD mutation, was used to study the wild-type S4 gene on the phages.)
question of whether regulatory r-proteins act in As shown in Fig. 3, one can use the assump-
trans or whether they function mainly in cis (see tions above to predict that if the wild-type S4
above). Strains diploid and triploid with respect acts only in cis, the relative synthesis rate of the
to the a operon were constructed by lysogeniz- mutant form of S4 should not be affected by the
ing this strain with specialized transducing presence of the wild-type protein and should
phages carrying the wild-type S4 gene (A spc2, remain at about 2 in both the diploid (rpsDI
or A spc2 and A a-lacl cI+; see below and Fig. rpsD+) and triploid (rpsDlrpsD+lrpsD+) strains.VOL. 151, 1982 TRANSLATIONAL REPRESSOR r-PROTEIN S4 199
S4 (M) Sll S13 p
Haploid
2 2 2
trans model cis model
I
S4 (M) Sl S13 P S4 (M) Sl S13 p I
1 1 1 ) 2 2 2
Diploid
1 1 S4 (W) Sl S13 P
1 \' 1 1 -1' 1 1 T I
S4 (M) Sl S13 P S4 (M) Sl S13 P
0.5 0.5 0.5 2 2 2
S4 (W) Sl S13 P S4 (W) Sl S13 P
I Triploid
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0.5 0.5 0.5 0.5 0.5 0.5
S4 (W) Sl S13 P S4 (W) Sl S13 P
0.5 0.5 0.5 0.5 0.5 0.5
FIG. 3. r-Protein synthesis rates predicted by both trans and cis regulation by r-protein S4. The first three
genes in the a operon are shown. M, mutant form of r-protein S4; W, wild-type S4; P, a promoter; .-, direction
of mRNA transcription and protein translation; a, segment corresponding to the presumed mRNA target site for
the binding of the wild-type S4; ---, interaction of the wild-type S4 with the target site(s) in a diploid strain if it
acts in trans (trans model) or strictly in cis (cis model). A more detailed explanation is given in the text.
In contrast, if the wild-type S4 functions as a not carry the distal part of the a operon, only the
translational repressor in trans as efficiently as synthesis rates of the first three r-proteins in the
in cis, the relative synthesis rate of the mutant a operon were considered in the predictions
S4 should be about 1 in the diploid strain and discussed above. The presence of A a-lac cI+
about 0.5 in the triploid strain. Both models prophage in N02514 or N02515 was confirmed
predict that the relative synthesis rates of S13 by the production of 0-galactosidase.
and S1I should be equal to the sum of the The relative synthesis rates of r-proteins in the
synthesis rates of both forms of S4. They should control N02514 and mutant N02515 haploids,
therefore be about 3 in both the diploid and diploids, and triploids were measured by pulse-
triploid strains if the cis model is correct, and labeling cells with [3H]lysine. Both mutant and
they should be about 2 and 1.5 in the diploid and wild-type haploid cells uniformly labeled with
triploid strains, respectively, if the trans model [14C]lysine were used as an internal standard for
is correct. comparison of the relative synthesis rates of r-
For actual experiments, haploid, diploid, and proteins (see footnote a of Table 2 for details).
triploid double lysogens of N02514 and N02515 The results are given in Table 2 as 3H/14C values
were constructed by using Xpapa, A trkA; Apapa, which have been normalized to the value for r-
A spc2; and A spc2, X a-lacl cI+, respectively. protein Li. In the control haploid, diploid, and
Figure 1 shows the structures of the specialized triploid strains, the relative synthesis rates of all
transducing phages A trkA, X spc2, and X a-lacl of the r-proteins measured were identical. The
cI+. It can be seen that X spc2 carries the entire absence of gene dosage effects in diploid and
spc and a operons with their bacterial promoters triploid strains confirms the results of previous
as well as the trkA+ and aroE+ genes. X trkA gene dosage experiments (12, 16, 31).
does not carry any r-protein genes, but does In the mutant strains, the results obtained are
have the bacterial trkA+ and aroE+ genes and very close to those predicted by assuming that
was used as a control for A spc2. A trkA and the wild-type S4 acts in trans as a translational
A spc2 lysogens were constructed and main- repressor. In the diploid strain N02515 (Xpapa,
tained by selecting AroE+ transductants. X a- A spc2), the relative synthesis rates of the wild
lacl cI+ carries the a promoter (Pa) and the type and mutant forms of S4 were identical and
genes for r-proteins S13, S11, and S4; it does not were both close to 1.0. The data for the relative
have the genes for the a subunit of RNA poly- synthesis rates of r-proteins Si1 and S13 were
merase or r-protein L17. Since X a-lac cI does also consistent with trans regulation by S4 since200 JINKS-ROBERTSON AND NOMURA J. BACTERIOL.
the predicted values of 2 were close to the rpsD mutant, we think it is significant because of
observed values of 1.7. In the mutant triploid the following observation. We have found that
N02515 (X a-lacl cI+, A spc2), the synthesis the synthesis rates of all a operon r-proteins
rates of the wild-type and mutant forms of S4 increase with increasing gene dosage when only
were also those predicted by the trans model. the mutant form of S4 is present, so that the
The relative synthesis rate of the mutant S4 was marginal overproduction of L17 seen in the rpsD
about 0.4, which is close to the predicted value mutant becomes significant (about 70%o) in an
of 0.5; the relative synthesis rates of S11 and S13 rpsDlrpsD merodiploid (our unpublished experi-
(about 1.3) were also close to the values of 1.5 ments). This demonstrates that L17 synthesis is
predicted by the trans model. Table 3 summa- affected by the S4 mutation, and we conclude
rizes these relevant data and compares them to that L17 is translationally regulated by r-protein
both the cis and trans model predictions. Based S4. An explanation as to why the increase in L17
on the results presented here, we conclude that synthesis is slight compared with that of S13,
the wild-type S4 acts in trans to regulate the Si1, and S4 in the rpsD mutant is as follows. The
translation of mRNA synthesized from the chro- exact nature of the rpsD mutation is not known,
mosomal a operon containing the rpsD allele. but the fact that the mutant S4 protein is missing
15% of the wild-type protein at the carboxy-
DISCUSSION terminal end suggests that premature termina-
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tion of translation occurs in the mutant. We
Role of S4 in regulating the synthesis of the a believe that this causes a polar effect on the
operon r-proteins. Previous in vitro and in vivo expression of the downstream L17 gene, leading
work has established that S4 is the translational to less efficient synthesis of L17 relative to S13,
repressor which regulates the synthesis of r- Sit, and S4.
proteins in the a operon: S13, S11, S4, and The results obtained with our rpsD mutant
probably L17 (8, 37). In agreement with the demonstrate that regulation by r-protein S4 is
regulatory role of S4, we have found that a essential for balancing the synthesis rates of r-
mutational alteration of this protein results in a proteins in the a operon with the synthesis rates
significant and specific increase in the synthesis of all other r-proteins under steady-state growth
rates of r-proteins S13, Sit, and S4. This finding conditions. A similar conclusion was reached in
confirms the earlier observation of Olsson and the analysis of a strain which is apparently
Isaksson that S4 and S13 are overproduced in deficient in the regulatory r-protein Li (21, 36).
rpsD mutants (33) and, in addition, demon- We have suggested that the capacity of r-protein
strates a comparable overproduction of r-protein synthesis from mRNA is in excess of that need-
S11. ed for ribosome assembly under the growth
In vivo induction experiments have indicated conditions used, and that this becomes evident
that L17 synthesis is regulated by r-protein S4 when the post-transcriptional regulatory mecha-
(8), but in vitro experiments have failed to nisms are disrupted as in the Li-deficient and
substantiate this (37). Assuming that the in vivo rpsD mutant strains. Consistent with this idea is
data accurately reflect the steady-state regula- the recent demonstration that the characteristic
tion of a operon r-proteins, we expected to see growth rate-dependent regulation of r-proteins
an overproduction of L17 comparable to that of synthesized from the a and spc operons is not
S13, Si1, and S4 in the rpsD haploid strain. determined by changes in transcription rates
Even though only a slight increase (about 20%) from these promoters and thus must be deter-
in the synthesis rate of L17 was observed in the mined by post-transcriptional mechanisms (25).
TABLE 3. cis-trans test of S4 repressor activity: comparison of the predicted and observed r-protein
synthesis rates in N02515 mutant strains
Synthesis rate of r-proteins
Diploid
DpodTili Triploid
r-Protein Haploid
Predicted Predicted
Observed Observed
cis trans cis trans
S13 2.0 3.0 2.0 1.6 3.0 1.5 1.3
Sl 2.0 3.0 2.0 1.7 3.0 1.5 1.3
S4w 1.0 1.0 0.9 1.0 1.0 0.9
S4m 2.0 2.0 1.0 0.8 2.0 0.5 0.4
All other 1.0 1.0 1.0 1.0 1.0 1.0 1.0
r-proteinsVOL. 151, 1982 TRANSLATIONAL REPRESSOR r-PROTEIN S4 201
Interaction of S4 with rRNA and mRNA. The The present study was designed to answer this
rpsD mutant strains studied by Olsson are tem- question of cis versus trans regulation. By using
perature sensitive due to a defect in ribosome an rpsD mutant strain as a background for
assembly at the restrictive temperature. S4 is constructing strains diploid and triploid with
one of the first r-proteins to bind to 16S rRNA in respect to the ax operon, it was possible to
in vitro reconstitution of 30S subunits (26). It has measure the relative contributions of r-proteins
been reported that a mutant form of S4, which is from different ot operons in a single cell. From
similar to those studied by Olsson and by us, has the experimental data presented in this paper,
a lower affinity for its binding site on 16S rRNA we conclude that S4 acts in trans as a transla-
than does the wild-type r-protein in vitro (17). tional repressor, and we assume that this is true
Consistent with the in vitro data is the finding of all other regulatory r-proteins. Presumably,
that only the wild-type form of S4 is incorporat- under steady-state growth conditions, small
ed into ribosomes in the rpsD/rpsD+ merodip- amounts offree regulatory r-proteins exist in the
loid strains (our data) (33), even though both cellular pool which can interact with either
forms of S4 are synthesized at the same rate. rRNA for ribosome assembly or with mRNA for
The presumed target site for S4 action on a- translational repression.
operon mRNA has structural homology with the We originally suggested that translational re-
S4 binding site on 16S rRNA, and it has been pression would occur mainly in cis (12). A cis-
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suggested that the same structural feature. of S4 type of autogenous regulation was considered to
is involved in the interaction with both rRNA explain data concerning the phenotypic domi-
and mRNA (29). Although this has not been nance of antibiotic-sensitive alleles over antibi-
proven, our data support this idea since muta- otic-resistant alleles in Strr/Strs or Spcr/Spcs
tional alterations in r-protein S4 appear to affect merodiploid strains (5, 35; see reference 12 for
the affinity of S4 for both 16S rRNA and the discussion). We now know, however, that re-
mRNA regulatory target site. pressor r-proteins function as efficiently in trans
Regulation in trans. The exact mechanism of as in cis, and we would predict that antibiotic
translational repression and its relief by compet- sensitive and resistant alleles should be ex-
ing rRNA synthesis is not known, but there are pressed equally in merodiploid strains. In view
two possibilities that should be considered. One of our expanded knowledge of the regulation of
is that, under steady-state growth conditions, r-protein synthesis, we think that the problem of
"free" repressor r-proteins are released from dominance deserves re-examination.
mRNA and then interact with either rRNA or The presence of a post-transcriptional mecha-
mRNA target sites, depending on the availability nism for regulating the synthesis of most of the r-
of rRNA. According to this model, repressor r- proteins in the str-spc and rif regions of the E.
proteins would be expected to function not only coli chromosome has been firmly established.
in cis but also in trans on any mRNA with the Some essential features of our regulatory model
proper target site. Another possibility is that, have been discussed in previous papers (29, 40)
upon completion of their synthesis, repressor r- and were used as assumptions in the present
proteins interact immediately with the target site study to predict the relative synthesis rates of oa-
on their own mRNA, and complete release of operon r-proteins in diploid and triploid strains
such r-proteins from their message requires cou- (Fig. 3 and above). The excellent agreement
pling with ribosome assembly. This second pos- found between the observed values and those
sibility implies that repressor r-proteins act only predicted by a trans model of repressor action
in cis and not in trans. gives further support to the validity of the essen-
From experiments in which r-protein genes tial features of the translational regulation mod-
have been fused to the lac or ara operon promot- el.
ers (8-10, 38, 41) on a multicopy plasmid, it is
clear that regulatory r-proteins can act in trans ACKNOWLEDGMENTS
to stop synthesis of r-proteins whose genes are This work was supported in part by the College of Agricul-
on the chromosome. In most of these cases, tural and Life Sciences, University of Wisconsin-Madison, by
however, the target sites for the regulatory r- Public Health Service grant GM-20427 from the National
Institutes of Health, and by grant PCM79-10616 from the
proteins are removed from the fused operon, National Science Foundation. S.J.-R. was supported by Pub-
resulting in an unregulated two- to fourfold lic Health Service predoctoral training grant GM-07133 from
overproduction of the induced r-protein. Trans- the National Institutes of Health.
lational repression under steady-state growth
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