JOURNAL OF BACTERIOLOGY, Aug. 1985, p. 476-4860021-9193/85/080476-11$02.00/0Copyright C 1985, American Society for Microbiology

Vol. 163, No. 2

Unbalanced rRNA Gene Dosage and its Effects on rRNA andRibosomal-Protein Synthesis

RICHARD J. SIEHNEL AND EDWARD A. MORGAN*Department ofExperimental Biology, Roswell Park Memorial Institute, Buffalo, New York 14263

Received 14 February 1985/Accepted 6 May 1985

The synthesis of rRNA was unbalanced by the introduction of plasmids containing rRNA operons with largeinternal deletions. Significant unbalanced synthesis was achieved only when the deletions affected both 16S and23S RNA genes or when the deletions affected the 23S RNA gene alone. Although large imbalances in rRNAsynthesis resulted from deletions affecting 16S and 23S RNA genes or only 23S RNA genes, excess 16S RNA anddefective rRNA species were rapidly degraded. Large imbalances in the synthesis of regions of rRNA did notresult in significantly unbalanced synthesis of ribosomal proteins. It therefore is probable that excess intact 16SRNA is degraded because ribosomal proteins are not available for packaging the RNA into ribosomes. DefectiveRNA species also may be degraded for this reason or because proper ribosome assembly is prevented by thedefects in RNA structure. We propose two possible explanations for the finding that unbalanced overproduc-tion of binding sites for feedback ribosomal protein does not result in significant unbalanced translationalfeedback derepression of ribosomal protein mRNAs.

The mechanism of regulation of ribosomal protein (r-protein) and rRNA transcription units is of considerableinterest because the synthesis of r-proteins and rRNA occurswith a precise stoichiometry, resulting in very small pools offree rRNA and r-proteins (9-11, 22, 37). The preciselycoordinated synthesis of ribosomal components is main-tained during wide variations in the synthesis rates of rRNAand r-proteins (9-11, 31).

Regulation of r-protein and rRNA operons probably in-volves a hierarchy of control mechanisms. In this hierarchy,as yet unknown mechanisms control the synthesis ofribosomal components in response to growth conditions byregulating (at least) the level of transcription of the sevenrRNA operons of Escherichia coli (24). The coordination ofr-protein synthesis to rRNA synthesis involves a transla-tional feedback mechanism first proposed by Nomura andco-workers (reviewed in reference 28). In generalized form,the r-protein translational feedback model proposes thatr-protein transcription units give rise to mRNAs that containone or more regulons. A regulon is defined as a contiguousgroup of protein-coding regions that are translationally regu-lated in unison. At least four individual regulons code forboth 30S and 50S proteins. A specific r-protein encoded ineach regulon binds to mRNA at the beginning of the regulononly when rRNA is not available for binding. The boundr-protein prevents translation initiation at the first codingregion, thereby also preventing the synthesis of proteinsfrom all other coding regions in the regulon because thetranslation of all but the first coding region in each regulon iscoupled to translation of the preceding coding region. Thetranslational feedback model in principle is sufficient toexplain how r-proteins are stoichiometrically synthesized inresponse to rRNA availability.

In vivo experimental support for r-protein translationalfeedback regulation comes largely from the finding thatrepression of r-protein synthesis results from the over-production of specific r-protein feedback repressors (re-viewed in reference 28). Consequently, the ability of r-proteins to translationally repress r-protein mRNAs is well

* Corresponding author.

established. However, it is not clear whether r-proteinmRNAs are significantly repressed under conditions of nor-mal growth. If all r-protein mRNAs are not subject tosignificant translational repression during normal growth,translational feedback repression might be physiologicallyimportant only when rRNA synthesis is precipitously re-pressed. For example, feedback repression could be impor-tant only during the dramatic reduction of rRNA synthesiscaused by the stringent response. Therefore, knowledge ofthe level of repression of r-protein mRNAs during steady-state growth is required to determine the physiologicalimportance of feedback regulation of r-protein mRNAs.One way to measure the level of repression of r-protein

mRNAs is to measure the level of derepression caused bythe overproduction of rRNA antirepressor. Overproductionor reduction of rRNA antirepressor is not easily achieved byalterations of the gene dosage of intact rrn operons becauserrn operons are directly or indirectly feedback regulated bythe ribosome content of cells (14). However, when rRNA isviewed as a linear array of nonoverlapping and overlappingfeedback r-protein repressor-binding sites, it is clear that theintroduction of rrn operons with internal deletions will resultin unbalanced synthesis of translational feedback r-protein-binding sites whether or not rRNA synthesis is feedbackregulated. The unbalanced synthesis of rRNA therefore hasthe potential to cause unbalanced synthesis of r-proteins.

In the experiments described in this paper, we cause largeimbalances in the synthesis of regions of rRNA by usingplasmids with internal deletions in rrn transcription units,and we examine the effects of these imbalances on rRNAstability, ribosome subunit ratios, and r-protein synthesis.

MATERIALS AND METHODS

Bacteria, phages, and plasmids. Strains EM2 (ilv-1 his-29pro-2 tsx trpA9605 trpR ara) and EM22 [ara(Am) galK(Am)galE lac(Am) trp(Am) tsx recA] have been described previ-ously (40). pCM-1 is a derivative of pBR322 that has achloramphenicol resistance gene inserted in the Tetr gene ofpBR322 (6). Unlike pBR322, pCM-1 does not have a func-tional rop copy number control gene. Therefore, plasmidsconstructed from pCM-1 exist in higher copy number than do

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UNBALANCED rRNA GENE DOSAGE 477

similar plasmids constructed from pBR322 (4). The single-stranded DNA phages M13mp8 and M13mp9 have beendescribed previously (2, 3). pSPC-1 and pERY-1 were de-rived from pLC7-21 by single point mutations in the genes for16S and 23S RNA, and they confer resistance to erythromy-cin and spectinomycin, respectively (41).

Construction of deletion plasmids. A single DNA fragmentcontaining rrnH and some flanking DNA was obtained bydigestion of pLC7-21, pERY-1, or pSPC-1 with BamHI andPstI. This fragment then was ligated into BamHI- andPstI-digested pCM-1. Deletions within rrnH then were con-structed with restriction nucleases. pCAS-000 and pCAS-230contain spontaneous deletions with imprecisely definedendpoints and were isolated during attempts to use restric-tion nucleases to construct plasmids with the structure ofpCAS-HP. pERC-1 and all deletion plasmids with intact 23SRNA genes were constructed with an erythromycin resist-ance mutation in the 23S RNA gene. All deletion plasmidswith an intact 16S RNA gene were constructed with aspectinomycin resistance mutation in the 16S RNA gene.pERC-1 and all deletion plasmids are described in Fig. 1 and2.

Preparation of RNA. [3H]uridine pulse-labeled RNA wasprepared by growing cells in MOPS medium (26) sup-plemented with 0.4% glucose, 1 ,ug of thiamine per ml, and50 ,ug of each the 20 amino acids per ml. A 20-p.g amount ofchloramphenicol per ml was added to overnight cultureswhen the cells contained plasmids. The overnight culturesthen were diluted to an optical density at 550 nm (OD550) of0.05 in medium without chloramphenicol. [3H]uridine wasadded at an OD550 of 0.5, followed 1 min later by the additionof three volumes of a hot 2:1 mixture of phenol/1% sodiumdodecyl sulfate-100 mM NaCI-8 mM disodium EDTA-20mM Tris chloride (pH 7.4). After 10 min of mixing at 100°C,the solution was cooled on ice, and the aqueous phase wasrecovered by centrifugation. The aqueous phase wasreextracted with cold phenol, and the RNA was recoveredby ethanol precipitation. [3H]uridine-steady-state-labeledRNA was similarly prepared, except that 10 ,uCi of[3H]uridine per ml was added to cells at an OD550 of 0.1.RNA was prepared from these cells after three cell doublingsin the presence of label. 32Pi-steady-state-labeled referenceRNA was similarly prepared, except that 100 ,uCi of 32p, per

Pst i

Bam Hi Cmr 1Kbi - I

FIG. 1. Structure of pERC-1. The shaded area derives frompCM1, thin lines are E. coli or ColEl DNA flanking rrnH which arederived from the parental plasmid pLC7-21, white areas indicaterrnH genes for rRNA and tRNAs, and black areas indicate rrnHprecursor-specific sequences. rrnH transcription is counter-clockwise in this drawing.

1 Kb

tRNAl tRNAcOSmol HindJ Smol Hpal PvuIE

HindIS~~satIl

.I23S

5S tRNA0sp

- ~A16S: 83-65O- 116S: 615-1384

A23S:2000bpL123S:1780bpA23S:608-1763

A16S-23S: 824-1345

8- 8- m - -83-614 877-1384 364-607 686-1345 1764-2203

pERC-1pERC- D2pERC-D4pCAS-OO0pCAS- 230pCAS-HPpASC- D1

PROBES

FIG. 2. Structure of rrnH on pERC-1 is given at the top. The firstsix black bars below this portion of the figure indicate the extent ofthe deletions in the six deletion plasmids. The bottom series of barsindicate regions of rrnH that were cloned to use as hybridizationprobes. The coordinates of nucleotides deleted or cloned are indi-cated. The numbering system that we used designates nucleotide 1as the first nucleotide coding for each mature RNA species. pERC-D2 was constructed by deletion of a HindlIl fragment, pERC-D4was constructed by deletion of a SmaI fragment, pCAS-000 andpCAS-230 were constructed by deletions with endpoint uncertain-ties indicated by thin lines, pCAS-HP was constructed by deletion ofa HpaI-PvuII fragment, and pASC-D1 was constructed by deletionof two Sall fragments. Probe 1, HindIII-SmaI fragment; probe 2,Sall-SmaI fragment; probe 3, Alul-HpaI fragment; probe 4, Sau3A-Sall fragment; probe 5, PvuII-HincII fragment. Probes 1, 2, and 5were cloned in M13mp8 and probes 3 and 4 were cloned in M13mp9in orientations that allow rRNA to hybridize to the strand packagedin phage particles.

ml was added to cultures of strain EM22 at an OD550 of 0.1and RNA was prepared after three cell doublings in thepresence of label. 32Pi-labeled tRNA for two-dimensional gelelectrophoresis by the method of Ikemura and Nomura (13)was prepared similarly, except that 1 mCi of 32p; per ml wasused to label cells for one doubling, starting at an OD550 of0.2.RNA hybridization. Hybridizations were performed in

triplicate by the method of Jinks-Robertson et al. (14).One-milliliter hybridization reactions contained equal countsper minute (approximately 2 x 105 cpm of each isotope) ofeither [3H]uridine-steady-state- or pulse-labeled RNA and32P,-steady-state-labeled reference RNA. Each hybridizationreaction contained two nitrocellulose filters (diameter, 6.5mm). A 3-,ug amount of M13mp8 single-stranded DNAprepared from phage particles was fixed to one filter, and 3,ug of single-stranded DNA from an M13mp8 or M13mp9clone of a region of rrnH (Fig. 2) was fixed to the other filter.After hybridization, the filters were washed and treated withRNase A by the method of Zengel et al. (45). Specifichybridization to a segment of rrnH was determined bysubtracting the radioactivity bound to the filter containingM13mp8 from the radioactivity bound to the filter with thecloned rrnH segment. Radioactivity bound to the filtercontaining M13mp8 was less than 10% of the radioactivitythat hybridized to rrnH sequences. The values for therelative abundance of rRNA regions are calculated by divid-ing the 3H/32P ratios by the 3H/32P ratio of RNA hybridizingto a single region of rrnH that was chosen for normalizationof the data. Synthesis rates were calculated with RNA

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478 SIEHNEL AND MORGAN

labeled with [3H]uridine for 1 min. Steady-state accumula-tion was calculated with RNA labeled for three cell dou-blings.

Labeling of ribosomal proteins. [3H]leucine-labeled refer-ence r-proteins were prepared from strain EM22 grown inMOPS (morpholine propane sulfonic acid) medium sup-plemented with 0.4% glucose, 1 ,ug of thiamine per ml, and40 ,ug each of tryptophan, isoleucine, and valine per ml.[3H]leucine (30 uCi/ml) was added at an OD550 of 0.1, and thecells were harvested after three doublings. 35S04-steady-state-labeled proteins were prepared by growing cells inMOPS minimal medium containing 54 ,uM K2SO4 sup-plemented with 0.4% glucose, 1 pLg of thiamine per ml, and50 ,ug of all 20 amino acids except methionine and cysteineper ml. Carrier-free 35S04 (0.5 mCi/ml) was added at anOD550 of 0.1, and labeling was terminated at an OD550 of 0.8.[35S]methionine-pulse-labeled proteins were similarly pre-pared in MOPS medium, except that 30 ,Ci of[35S]methionine per ml was added to cells at an OD550 of 0.5,followed 1 min later by a 90-s chase with 25 ,g of unlabeledmethionine per ml. In all experiments, labeling was termi-nated by pouring the cells over ice and washing the cells bycentrifugation. The cells then were frozen before the extrac-tion of r-proteins by the method of Lindahl and Zengel (19).

Sucrose gradients. Dissociated ribosomal subunits wereprepared from cells growing in MOPS medium. At an OD550of 0.5, 5 ml of culture was poured over an equal weight offrozen crushed buffer A (10 mM Tris chloride [pH 7.5], 0.1mM MgCl2, 30 mM NH4Cl, 1 mM P-mercaptoethanol),collected by centrifugation, suspended in 100 ,ul of buffer A,and incubated at 37°C for 5 min. A 10-,ul volume of 2 mg oflysozyme per ml was then added, and the cells were lysed bytwo cycles of freeze-thaw, followed by the addition of 10 pulof 10% Triton X-100 and 1 ,ul of 1 mg of DNase per ml. Thelysate was layered on a gradient of 15 to 30%o sucrose inbuffer A and centrifuged at 41,000 rpm for 7.5 h in aBeckman SW41 rotor. Absorbance profiles were recordedwith an ISCO UA-5 flow photometer.Polysomes were prepared from cells growing in MOPS

medium. A 200-,ug amount of chloramphenicol per ml wasadded at an OD550 of 0.5 to immobilize ribosomes on mRNA.The culture was then immediately poured over an equalweight of frozen, crushed TSM buffer (10 mM Tris succinate[pH 7.4], 10 mM MgCl2, 1 mM P-mercaptoethanol) contain-ing 200 pg of chloramphenicol per ml. The cells werecollected by centrifugation, suspended in TSM buffer pluschloramphenicol, and lysed by two cycles of freeze-thaw inthe presence of 200 ,ug of lysozyme per ml, followed by theaddition of 10 ,ul of 10% Triton X-100 and 1 ,ul of 1 mg ofDNase per ml. The lysate was layered on a gradient of 15 to30%o sucrose in TSM buffer containing 0.1 M KCl andcentrifuged at 41,000 rpm for 4.5 h in an SW41 rotor. Themobilities of 30S, 50S, and 70S ribosomes were determinedby centrifugation of authentic ribosome subunits and 70Sribosomes in parallel.

Electrophoresis of r-proteins. Two-dimensional acrylamidegel electrophoresis of r-proteins was done by the method ofKaltschmidt and Wittmann (17), except that first-dimensiontube gels (2.5 mm by 14 cm) contained 0.4% Nonidet P-40and second-dimension slab gels (1.5 mm by 16 cm by 12 cm)were polymerized from 20% acrylamide and 1.1% N,N-diallyltartardiamide. Samples for electrophoresis were pre-pared as described by Lindahl and Zengel (19) and contained2 x 107 cpm each of 35S- and 3H-labeled cell lysate orribosomes and 25 units (absorbancy at 260 nm) of unlabeled70S ribosomes. The r-proteins were located on Coomassie-

stained dried gels, and isotope ratios for each r-protein weredetermined after gel solubilization as described by Lindahland Zengel (19).

Plasmid copy number. Plasmid copy numbers were deter-mined by a modification of the method of Ikemura andNomura (13). A PvuII-EcoRI fragment of rrnH containing 5SrDNA and 23S rDNA downstream of position 1764 of the23S gene was labeled with 32P by nick translation (39) andhybridized by the method of Denhardt (39) to DNA fromplasmid-containing strains that had been isolated by themethod of Ikemura and Nomura (13), digested with BamHIto linearize the plasmids, denatured, and fixed to nitro-cellulose filters. A single 5-ml hybridization reaction con-tained 106 cpm of DNA labeled by nick translation, duplicateblank filters, and duplicate filters containing 1 ,ug of DNAfrom strain EM22(pCM1) and from EM22 containing eachplasmid used in this study. Since the DNA fragment labeledby nick translation can hybridize equally well to all seven E.coli chromosomal rrn operons and to all deletion rrn operonsused in this study, the radioactivity specifically bound tofilters containing DNA from strain EM22(pCM1) resultedfrom hybridization to seven rrn operons, and the copynumber of plasmids with partial or complete rrn operonscould be determined by the relative hybridization to DNAfrom cells containing these plasmids.

RESULTSConstruction of deletions. rrnH was cloned into pCM-1, a

high-copy-number derivative of pBR322 (Fig. 1). Internaldeletions in rrnH then were constructed with restrictionnucleases (Fig. 2). Plasmid constructions were designed sothat all plasmids with an intact 23S RNA gene have a singlebase change in the 23S gene that confers erythromycinresistance and all deletion derivatives with intact 16S geneshave single base changes in the 16S genes that conferspectinomycin resistance (41). Since all deletion plasmidswith intact 23S genes confer erythromycin resistance and alldeletion plasmids with intact 16S genes confer spec-tinomycin resistance (data not shown), it can be concludedthat all intact 16S or 23S genes in the plasmids used in thispaper contribute to functional ribosomes. It cannot be as-sessed firmly from these data alone whether rRNA synthe-sized from these plasmids is as efficiently assembled intoribosomes as is rRNA from intact chromosomal rrn operons.However, the deletions do not decrease the observed anti-biotic resistance, and in some cases they cause an increase inantibiotic resistance concomitant with an increase in plasmidcopy number (see below). It therefore is likely that a deletionin one rRNA gene does not affect significantly the synthesisof ribosomes from rRNA produced by the intact rRNA genesin the same operon.

Affects of deletions on growth rates. The doubling time ofbacterial strain EM22 is 50 min at 37°C in the medium usedin this study (MOPS medium supplemented with 20 aminoacids, glucose, and thiamine). When strain EM22 containsplasmid pERC-1, which carries an intact rrnH operon, thedoubling time increases to 65 min. The doubling times (inminutes) of strain EM22 containing the following deletionplasmids are: pERC-D2, 65; pERC-D4, 70; pCAS-000, 70;pCAS-230, 70; pCAS-HP, 200; pASC-D1, 60.

Plasmid copy numbers. Plasmid copy numbers were deter-mined by DNA-DNA hybridization as described above.DNA for these measurements was prepared from cellsgrowing in the same media as those used for labelingexperiments. These measurements show that the followingplasmids have the indicated copy numbers: pERC-1, 3.2;

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~~~~~~~UNBALANCED rRNA GENE DOSAGE 479

FIG. 3. Autoradiograms of small RNAs separated by two-dimensional gel electrophoresis. tRNAs were prepared from strain EM22

containing: A, no plasmid; B, pCM-1; C, pERC-1; D, pERC-D2; E, pERC-D4; F, pCAS-000; G, pCAS-230; H, pCAS-HP; I, pASC-D1. Small

RNAs are: 1, tRNAGIu; 2, tRNAfla'; 3 and 3', differently modified forms of tRNAIIe; 4, tRNAASP; 5, tRNATrP~; 6 and 6', all sequence variants

of 5S RNA.

pERC-D2, 4.6; pERC-D4, 6.4; pCAS-000, 33; pCAS-230, 27;

pCAS-HP, 32; pASC-D1, 29. Plasmids containing an intact

rrnH operon or rrnH operons with internal 16S deletions

exist in unexpectedly low copy numbers of 3 to 6 per cell.

Therefore, these plasmids do not cause large increases in

gene dosage. However, plasmids with deletions that include

part of the 23S gene have copy numbers of about 30 per cell.

The large variations in plasmid copy number due to these

small differences in plasmid structure are surprising. How-

ever, others also have shown that alterations of plasmids in

regions not directly involved in plasmid replication can

influence plasmid copy number (1).

tRNA and SS RNA synthesis. E. coli rrn operons contain

either tRNAGlu or both tRNAile and tRNAPAla genes between

16S and 23S RNA genes, and some operons contain

tRNAAsP or tRNATnP genes downstream of the 5S RNA

gene. The rrnH operon on the plasmids used in this study

contains tRNAVC, tRNAlBAa, and tRNAASPgenes. Since over-

produced tRNAs are not significantly degraded (13), meas-

urements of the overproduction of tRNAs and 55 RNA

synthesized from the plasmids can be used to measure RNA

synthesis rates. The relative abundance of 55 RNA and each

tRNA therefore was determined by separating small RNA

species by two-dimensional gel electrophoresis (Fig. 3, Ta-

ble 1). Although measurements of tRNAs from plasmids

with low copy numbers are imprecise due to incomplete

resolution of rrnH tRNAs from other tRNAs, these measure-

ments are consistent with the plasmid copy numbers deter-

mined by DNA-DNA hybridization and, together with meas-

urements of rRNA synthesis rates (see below), demonstrate

that rRNA synthesis from these plasmids is proportional to

plasmid copy number.

32 601

3~~~~~~~~~~4~~ ~ ~ ~~~~4

.......

... .. ....... .. ... .. ....p..

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480 SIEHNEL AND MORGAN

TABLE 1. Overproduction of small RNAs in plasmid-containing cellsaRelative overproduction in cells containing following plasmid

RNANone pCM-1 pERC-1 pERC-D2 pERC-D4 pCAS-000 pCAS-230 pCAS-HP pASCD1

tRNAIlBa 1.0 1.0 2.9 2.7 2.4 1.2 11 11 1.3tRNA 1le 1.0 0.9 1.8 1.8 1.6 5.1 5.5 5.2 1.7tRNAaSP 1.0 0.7 1.8 1.5 1.5 2.5 4.2 4.6 3.55S 1.0 1.3 1.4 1.3 1.3 3.3 3.6 3.6 3.4

a Overproduction was determined by excising regions from two-dimensional gels (Fig. 3) and quantitating the radioactivity in each small RNA. Overproductionis calculated by dividing the amount of radioactivity in each RNA species by the amount of radioactivity in tRNAcIu and tRNAT"P and normalizing this value bythe ratios obtained from tRNAs prepared from strain EM22 without any plasmids. The calculated value for relative overproduction is of course influenced by thenumber of genes on the chromosome coding for each tRNA species. There are three copies of tRNA1Aa and tRNAIIe genes, two copies of tRNAASP genes, andeight copies of 5S rRNA genes (24). All chromosomal copies of genes for these RNAs are located exclusively in rrn operons (3). The promoter strengths of all sev-en rrn operons have not been directly measured but are thought to be similar because of extensive promoter sequence homology (24, 38; unpublished data). Thedata presented are averages from at least two experiments. Individual experiments typically agree within 20%o.

rRNA synthesis and accumulation. Five different regions ofrrnH were cloned to enable hybridization measurements ofRNA synthesized from the corresponding regions of rrnoperons (Fig. 2). Synthesis rates of rRNA from each regionwere assessed by RNA-DNA hybridization with total RNAlabeled for 1 min before extraction from cells. The contribu-tion of the deletion plasmids to total rRNA synthesis can beassessed by these hybridization measurements because thesynthesis of rRNA from the deletion plasmids results inreduced relative synthesis of one or more of the five regionsof rRNA measured. The results show that the increases inrrn gene dosage due to plasmids with internal deletions inrrnH are directly reflected by the unbalanced synthesis ofthe five regions of rRNA (Table 2).Measurements of the steady-state accumulation of RNA

were made with RNA labeled for three cell doublings beforeextraction of the RNA. The results (Table 2) show that thereis some imbalance in the steady-state accumulation ofrRNA. However, the imbalance of accumulation is much

TABLE 2. Measurements of synthesis and accumulation of rRNAregions in cells containing plasmids with deletions of different

regions of rrnHaSynthesis rate for regions: Accumulation for regions:

Plasmid1 2 3 4 5 1 2 3 4 5

None 1.2 1.3 1.1 1.0 1.1 1.1 1.1 1.1 1.0 1.1pERC-1 1.2 1.2 1.2 1.0 1.0 1.0 1.1 1.1 1.0 1.1pERC-D2 1.0 1.6 1.6 1.4 1.5 1.0 1.0 1.2 1.1 1.3pERC-D4 1.8 1.0 1.6 1.4 1.5 1.0 1.0 0.88 0.88 0.98pCAS-000 5.0 5.0 1.2 1.0 4.4 1.7 1.8 1.0 1.0 1.2pCAS-230 4.1 4.1 1.2 1.0 3.8 1.6 1.8 1.1 1.0 1.2pCAS-HP 4.3 4.5 5.0 1.0 4.7 1.9 1.9 1.4 1.0 1.4pASC-D1 4.0 1.1 1.1 1.0 4.0 1.2 1.3 1.0 1.0 1.4

a Synthesis rates were determined by measuring labeled RNA extractedfrom cells 1 min after the addition of [3H]uridine. Accumulation was deter-mined by measuring labeled RNA extracted after three cell doublings in thepresence of [3H]uridine. For further details of rRNA measurement see thetext. The five cloned regions of rrnH used for these filter hybridizationmeasurements are described in Fig. 1. Rows 3 to 8 of this table give therelative abundance of rRNA complementary to five regions of rrnH. Theabundance of each rRNA region is normalized to the abundance of rRNAsynthesized from a single region of rrn operons that is intact in chromosomalrrn operons but is not present in the rrnH operon on the deletion plasmids. Ineach of these six rows the RNA abundance of the region chosen fornormalization of all other regions therefore is assigned a value of 1.0 which isunderlined. No deletions are present in rrn operons in the experiments in thefirst two rows, and region 4 was therefore arbitrarily used to normalize theother four regions. The mean values of several measurements are given.Standard deviations are less than 10%o of the mean value.

less than the imbalance of synthesis. It can be concluded thatdefective 16S and 23S rRNA molecules and normal 16SRNA molecules synthesized in excess of normal 23S RNAare degraded.RNA degradation rates. The degradation rates of excess

intact rRNA species or defective rRNA species can becalculated precisely from the data presented above, pro-vided that all rRNA not packaged into normal ribosomes ismonophasically degraded at a rate that is much faster thanthe negligible degradation rate of rRNA packaged intonormal ribosomes. The negligible degradation of rRNA inintact ribosomes is well documented (10, 31), and the rapiddegradation ofRNA not incorporated into normal ribosomesis supported by the results described above. Under theseconditions, the half-lives of degraded rRNA species can becalculated by the formula t112 = [y(x - 1)]/(z - x), where t112equals the half-life of a region of RNA, x equals the overac-cumulation of overproduced regions of rRNA, y equals thedoubling time of the culture, and z equals the rate ofsynthesis of RNA divided by the fraction of that RNAdestined for assembly into stable ribosomes. Values for y aregiven above, and values for x and z can be obtained directlyfrom Table 2. For example, when considering rRNA synthe-sized from region 1 of pCAS-HP (Fig. 2), x equals 1.9 (Table2), y equals 200 min (see above), z equals 4.3 (Table 2), andt1j2 therefore equals 75 min. The half-lives of all significantlyoversynthesized RNAs were calculated by this method(Table 3).Ribosome subunit ratios. To determine whether the un-

balanced synthesis of rRNA results in an unbalanced ac-cumulation of mature ribosome subunits, ribosome subunitratios were determined from OD profiles obtained by su-crose gradient centrifugation of dissociated ribosome

TABLE 3. Half-lives of RNAHalf-lives (min)a of RNA region:

Plasmid1 2 3 4 5

pCAS-000 15 17 NC NC 4pCAS-230 17 24 NC NC 5pCAS-HP 75 69 22 NC 24pASC-D1 3 NC NC NC 7

a Half-lives of regions of rRNA (Fig. 2) were calculated from the RNA-DNA hybridization measurements in Table 2. Values are presented only forthe plasmids which result in significant oversynthesis of rRNA. NC, Notcalculable because the internal deletion in rrnH includes a portion of theregion being measured.

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UNBALANCED rRNA GENE DOSAGE 481

Top DISTANCE BottonFIG. 4. Determination of ribosomal subunit ratios by sucrose

gradient centrifugation. A, Sucrose gradient profile of polysomesprepared from cells containing a complete rrnH operon on pERC-1;B, polysome profile from cells containing pCAS-HP; C, sucrosegradient profile of dissociated ribosome subunits from cells contain-ing pERC-1; D, profile of dissociated ribosome subunits from cellscontaining pCAS-HP.

subunits. The results (Fig. 4C and D, Table 4) show that,with the exception of cells containing pCAS-HP, no appar-ent subunit imbalance results from unbalanced rRNA syn-thesis. A more sensitive but qualitative measurement ofsubunit balance is achieved when residual free subunits wereanalyzed on sucrose gradients after 70S ribosomes wereimmobilized on mRNA by chloramphenicol before the cellswere lysed (Fig. 4A and B, Table 4). Again, only cellscontaining pCAS-HP exhibit any measurable apparentsubunit imbalance.When ribosomes are prepared from cells containing

pCAS-HP, there is a 2.4-molar excess of UV-absorbingmaterial at the position of 30S subunits (Fig. 4, Table 4). Thecomposition of RNA in material migrating at 30S and 50Stherefore was analyzed by hybridization to cloned DNAprobes from five regions of rrnH. The results (Table 5) showthat the 50S subunit region of sucrose gradients preparedwith cells containing pCAS-HP contains only normal 23SRNA, but approximately 20%o of the rRNA in the 30Ssubunit region is defective 23S RNA synthesized exclusivelyfrom the defective 23S RNA gene of pCAS-HP (exclusivesynthesis from pCAS-HP is proven by the absence ofhybridization to region 4). The rRNA and r-protein contentof this 30S region and the r-protein content of cells contain-ing pCAS-HP (see below) indicate that cells containingpCAS-HP have nearly equimolar levels of normal 30S and50S subunits. Therefore, the defective 23S RNA in the 30Sregion is insufficient to explain why the 30S region containsa 2.4-fold excess of UV-adsorbing material unless the defec-tive 23S RNA is part of a larger unprocessed precursor and

has an extinction coefficient somewhat greater than that of30S ribosomes. These properties are consistent with thesedimentation value of this abnormal RNA and the fact thatthe RNA probably is an unfolded conformation due to an

incomplete complement of r-protein (see also below).r-protein synthesis. The deletions in the plasmids used in

this paper delete regions of rRNA genes which code forRNA segments required for the binding r-proteins thatfeedback regulate r-protein synthesis (Fig. 5, Table 6).Therefore, in theory, the synthesis of rRNA from theseplasmids might cause unbalanced feedback derepression ofr-protein mRNAs. To determine whether unbalanced r-

protein synthesis was occurring, the synthesis rates ofindividual r-proteins were analyzed by two-dimensional gelelectrophoresis of r-proteins after labeling cells for 1 minwith [35S]methionine and chasing for 90 s with unlabeledmethionine. The r-protein synthesis rates in cells containingplasmids with intact rrnH operons or rrnH operons withinternal deletions are summarized in the top seven panels ofFig. 6. Leaving aside for the moment possible minor differ-ences in the r-protein synthesis rates obtained with cellscontaining the seven plasmids analyzed, two major conclu-sions may be drawn. The first conclusion is that low-copy-number plasmids without internal rrnH deletions (pERC-1)or with internal deletions in the 16S rRNA gene (pERC-D2and pERC-D4) do not cause significant imbalances in r-

protein synthesis (Fig. 6). This was not unexpected becausethese plasmids do not cause a significant imbalance in thesynthesis of binding sites for feedback regulatory r-proteins.The second, more significant conclusion is that high-copy-number plasmids with partial deletions of 23S but not 16Ssequences (pCAS-000, pCAS-230, and pCAS-HP) or withpartial deletions of both 16S and 23S sequences (pASC-D1)do not cause the synthesis of r-proteins to be unbalanced inproportion to the four- to fivefold unbalanced synthesis ofr-protein-binding sites which occurs in cells containing theseplasmids (Table 2, Fig. 6).

Close inspection of the r-protein synthesis rates presentedin Fig. 6 reveals that the high-copy-number plasmids withpartial deletions of 23S but not 16S sequences (pCAS-000,pCAS-230, and pCAS-HP) may cause minor differences inthe synthesis of some proteins. (When interpreting the datain Fig. 6, it should be remembered that synthesis rates are

arbitrarily normalized to the synthesis rate of L13, and theelevation of synthesis rates of one protein therefore isequivalent to the relative depression of the synthesis of L13.)The synthesis of r-proteins from the a operon appears to beelevated approximately 60% relative to L13 in cells contain-ing plasmids pCAS-000, pCAS-230, and pCAS-HP. How-ever, protein synthesis from the a operon is elevated to a

lesser extent relative to the synthesis of most other r-

TABLE 4. Ratio of ribosomal subunitsaSubunit

Plasmid imbalance in 30S:50S ratiopolysome profile

pERC-1 No 0.94:1.0pERC-D2 No 0.83:1.0pERC-D4 No 0.91:1.0pCAS-000 No 1.2:1.0pCAS-230 No 1.0:1.0pCAS-HP Yes 2.4:1.0

a The ratio of ribosomal subunits was determined as described in the legendto Fig. 4.

A p.RC-l30S 50S 70S PdY=M

B pCAS-HP

50S 70S

pERC-1 50S MOLM RAW

30SI 3OS:50S=

0.94: i.0

pCAS-HP MOLM RATK)30S 50S 3OS:50S=

2.4:1.0

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SIO SIO L3 L4 L23 L2 (L22, S19) S3 L16 L29 SI7

+

SPC. EL L24 ILS S14 8 L6 SS |L50 L5I Y x

ot +S11 E

+MMisc. 2Ts E1 trmoD L L1 LlI LI L21 @ L28L33

FIG. 5. Summary of r-protein operon structures. The three major r-protein operons analyzed are called the S10 operon, spc operon, anda operon. In each of these operons, the primary transcript is indicated by an arrow, and known feedback regulatory units within eachtranscription unit are indicated by bars below the arrow. The feedback regulatory r-protein that regulates each regulon is indicated by a +above the protein. r-proteins analyzed in this study are boxed. It is probable that L14 and L24 in the spc transcription unit feedback regulatetheir own synthesis (28). The miscellaneous category of transcription units are small transcription units dispersed on the chromosome. In mostcases, the feedback regulatory r-proteins for these operons (if any) are not known. The data summarized in this diagram have been reviewedpreviously (28).

proteins. It is possible that the overproduction of a operonproteins may be due to the relative oversynthesis of 16SRNA antirepressor, which derepresses the a operon bybinding to the feedback regulatory protein S4. However, itappears that the measured r-protein synthesis rates in cellscontaining pCAS-HP are significantly discoordinate evenwhen r-proteins coded in a single translational feedbackregulation unit are compared (Fig. 6). Similar discoordinater-protein steady-state accumulation also is measured withcells containing pCAS-HP (Fig. 6, bottom panel). Therefore,from these data alone, the apparent slight excess of proteinsfrom the a operon and other operons (Fig. 6) does notunequivocally result from an unbalanced derepression ofr-protein synthesis. We suggest that the apparent discoordi-nate synthesis and accumulation might be due to differencesin the r-protein extraction efficiencies from abnormal rRNAprotein complexes present in cells containing pCAS-HP. Our

TABLE 5. Relative molarity of rRNA regions in sucrose gradientpeaks

Plasmid Ribosomal subunit Relative molarity of rRNA region":peak 1 2 3 4 5

pERC-1 30S 1.1 1.0 0 0 0pERC-1 50S 0.06 0.03 0.96 1.0 0.96pCAS-HP 30S 1.2 1.0 0.24 0 0.20pCAS-HP 50S 0.14 0.12 1.1 1.0 1.1

a Ribosomal subunits from strains containing an intact rrnH operon inpERC-1 and an internal 23S deletion in pCAS-HP were separated by sucrosegradient centrifugation (Fig. 4), and the RNA content of the subunit peaksthen was analyzed by hybridization to DNA probes from five regions of rrnH(Fig. 2). In each row the molar abundancy of RNA complementary to -oneregion is normalized to 1.0 which is underlined. Thus, in 30S subunitsprepared from cells containing pCAS-HP (row 3), RNA from region 3 of 23SRNA has a molar abundancy 0.24 times that of RNA from region 2 of 16SRNA. For full details of the hybridization method see the text. Standarddeviations of the values are less than 10o of the mean.

use of a double-label protocol to correct for most differencesin r-protein extraction efficiencies cannot correct for differ-ences in extraction efficiencies from abnormal ribosomes, asthe control cells contain only normal ribosomes.The feedback regulation hypothesis, together with ribo-

some assembly maps (12, 36), predicts that unbalancedfeedback repression will result in unusually high pools ofcertain free r-proteins. Therefore, to determine whetherplasmids with internal deletions in 23S RNA genes cause aslight but significant imbalance in r-protein synthesis, experi-ments were performed (data not shown) to analyze ther-protein content of S-100 supernatants prepared from cellscontaining pCAS-HP or pERC-1. After freeze-thaw lysis of[35S]methionine-steady-state-labeled cells, S-100 super-natants were prepared with and without the addition of 2 MNH4Cl after lysis but before centrifugation (see above).NH4Cl (2 M) was added to reduce nonspecific binding ofr-proteins to high-molecular-weight material and to assist thesolubilization of proteins weakly bound to putative defective

TABLE 6. Presence of binding sitesPresence of":

PlasmidS4 S8 S9 S15 S16 S20 Li L4 L13 L19 L21 L25

pERC-D2 - - + - + + + + + + + +pERC-D4 + - ± - ± + + + + + + +pCAS-000 + + + + + + + - - ± - +pCAS-230 + + + + + + + - - ± - +pCAS-HP + + + + + + + - - ± ± +pASC-D1 + + ± + + + - - ± - +

a A summary of feedback regulatory or potential feedback regulatory r-protein-binding sites on rRNA synthesized from the deletion plasmids used inthis study is shown. Only r-proteins which potentially regulate transcriptionunits analyzed in this study are shown. ±, Deletion removes only part of thesmallest segment of RNA isolated bound the the r-protein or shown to becapable of rebinding the r-protein. The r-protein binding sites have beendetermined previously (2,5,7,8,21,25,30,33,34,43).

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UNBALANCED rRNA GENE DOSAGE 483

2

pERC-l

22

pERC-D2

22

pERC-D4

22

pCAS-OOO

22

pCAS-230 i

22

pCAS-HP

pASC-DI

pCAS-HPsteady-state

I

22

FIG. 6. Synthesis of individual r-proteins from cells containing various plasmids compared with synthesis of r-proteins from cells withouta plasmid. The upper seven panels are synthesis rates as determined by a short-term label, and the bottom panel is a measurement ofaccumulation as determined by long-term labeling. In each panel, L13 was used for normnalization, and the synthesis of this protein is therefore1.0 and indicated by a diamond. Bars extending above 1.0 indicate the relative overproduction of a protein from cells containing a plasmid(normalized moles of r-protein from cells with a plasmid/moles of r-protein from cells without a plasmid), whereas bars below 1.0 also arepositive in value and indicate relative overproduction of a protein from cells without the plasmid (normalized moles of r-protein from cellswithout a plasjnid/moles of r-protein from cells with a plasmid). Presentation of the data in this manner allows bars of equal length above andbelow 1.0 to be of equal significance. Thin vertical bars indicate 30S subunit proteins, and thick vertical bars indicate 50S subunit proteins.Standard deviations are indicated by short horizontal bars and were determined by running duplicate gels with r-proteins prepared fromindependent cultures. The boxes enclose different r-protein transcription units as described in Fig. 5.

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484 SIEHNEL AND MORGAN

ribosomal particles. r-proteins in the supernatants andribosomal pellets then were separated by two-dimensionalgel electrophoresis and analyzed for their protein contentsby autoradiography of the gels and by determinations of theradioactivity in each protein (Fig. 6). The results (not shown)revealed that the supernatants were depleted of 70% of ther-proteins in the extracts. The relative amounts of all super-natant r-proteins were similar for cells containing pERC-1and pCAS-HP (data not shown). It therefore is unlikely thatlarge amounts of free r-proteins result from unbalancedrRNA synthesis in cells containing pCAS-HP or other plas-mids with internal deletions in 23S RNA genes.Because cells containing pCAS-HP produce a defective

large subunit which sediments at 30S (Fig. 4, Tables 4 and 5),proteins present in this defective subunit conceivably mightbe r-proteins which are synthesized in excess but whichsediment with ribosomes during the preparation of ribo-some-depleted supernatants. The 30S regions of sucrosegradients prepared with cells containing pERC-1 and pCAS-HP (Fig. 4C and D) therefore were analyzed for r-proteincontent by methods detailed above for the analysis ofsupernatant r-proteins. The results (not shown) indicatedthat the 30S region of sucrose gradients prepared from cellscontaining pCAS-HP contains approximately 0.1 mol of the50S subunit proteins Li, L2, L3, L17, and L22 per mol of30S proteins. These proteins were not present in the 30Sregion of sucrose gradients prepared with extracts of cellscontaining pERC-1. The other 50S proteins analyzed in Fig.6 were not detectably bound to the defective large subunitproduced in cells containing pCAS-HP. Therefore, the ab-normal ribosome subunit produced in cells containing pCAS-HP does contain some large subunit r-proteins but does notcontain a large percentage of any 50S ribosome subunitr-protein.

In summary, all the data considered together indicate thatthe synthesis of r-proteins and ribosome subunits remainsbalanced or nearly balanced despite the large imbalances inthe synthesis of regions ofrRNA due to plasmids pCAS-000,pCAS-230, pCAS-HP, and pASC-D1.

DISCUSSIONOur results demonstrate that multicopy plasmids contain-

ing rrnH operons with large internal deletions in 23S RNAgenes or partial deletions of both 16S and 23S RNA genescan result in four- to fivefold imbalances in the rates ofsynthesis of rRNA regions which are required for the bindingof r-proteins that feedback repress r-protein mRNAs. Largeimbalances in rRNA synthesis by rrnH operons with internaldeletions in the 16S RNA gene were not achieved because oflow plasmid copy numbers. Antibiotic resistance mutationsin rRNA genes were used to qualitatively demonstrate thatthe 16S and 23S RNA genes left intact by these internaldeletions in rrnH contribute to a significant percentage of thefunctional ribosomes of the cell. The excess intact rRNAresulting from unbalanced rRNA synthesis and the defectiverRNA resulting from partial deletions of rRNA structuralgenes are rapidly degraded. No significant imbalances inr-protein synthesis result from the large imbalances in rRNAsynthesis achieved with plasmids with internal deletions in23S RNA genes. Stoichiometric amounts of apparently nor-mal 30S and 50S ribosomal subunits also accumulate despitelarge imbalances in the synthesis of binding sites for r-proteins that feedback repress r-protein mRNAs. One inter-nal 23S RNA deletion results in the accumulation of a smallamount of defective large subunit containing defective 23SRNA and a few r-proteins.

When intact 16S RNA is synthesized in four- to fivefoldexcess as the result of one of the three high-copy-numberplasmids with deletions in 23S RNA genes, the excess 16SRNA molecules are degraded with half-lives of 15 to 70 min.The instability of excess or defective RNA contrasts with thenearly complete stability of rRNA synthesized in rapidlygrowing wild-type cells (10, 31). These results suggest thatthe stability of rRNA in rapidly growing cells is the result ofthe complexing of rRNA with r-proteins. Rapid degradationof newly synthesized rRNA has been previously observedonly during very slow cell growth (10, 31).

If the many r-proteins which bind rRNA bind randomlyand irreversibly, a four- to fivefold overproduction of rRNAwithout overproduction of r-proteins will result in a partialcomplement of r-proteins in nearly all ribosome particles.Since the existence of substantial levels of defective ribo-some subunits is not consistent with our results obtainedwith plasmids with internal deletions in 23S RNA genes, it islikely that r-proteins associated with rRNA are reversiblybound until additional r-proteins complete ribosome as-sembly. Commitment of an rRNA molecule to assembly ordegradation also might result if a few r-proteins in limitedsupply are required to initiate the assembly of each subunit,as has been previously proposed (29, 32). As discussedbelow, the possibility that r-proteins become tightly boundonly after additional r-proteins complete assembly mayinfluence the way in which r-protein synthesis is regulated byfeedback r-proteins.The organization of r-protein genes and their method of

regulation by feedback r-proteins led to the prediction thatan unbalanced synthesis and accumulation of rRNA regionscontaining strong binding sites for feedback regulatory r-proteins (equivalent to an unbalanced synthesis of feedbackregulation antirepressors) will result in unbalanced r-proteinsynthesis. Our results obtained with plasmids with internaldeletions in 23S RNA genes demonstrate that a large im-balance in the synthesis of r-protein-binding sites on rRNAdoes not cause detectable imbalances in the synthesis ofr-proteins. These results mean either that the overproducedrRNA is not an effective antirepressor of r-protein synthesisor that r-protein mRNAs are substantially derepressed dur-ing steady-state growth. These possibilities are discussedbelow.rRNA overproduced during unbalanced rRNA synthesis

might not serve as an effective antirepressor if the strongbinding of r-proteins to excess rRNA is prevented by defectsin the assembly of excess rRNA into ribosomes. Thesedefects in assembly could result from the unbalanced r-protein synthesis that occurs because of deletion of somefeedback r-protein-binding sites. If this possibility is correct,our results indicate that defective assembly affects feedbackregulation when the pools of free r-proteins are increased byan amount too small to detect by the methods we used. Thisexplanation for our results also requires that r-proteinswhich feedback regulate r-protein operons can bind tightlyand irreversibly to rRNA only when ribosome assembly isdriven to completion by the full complement of other r-proteins. If these suppositions are correct, our results sug-gest that all r-proteins eventually become bound irreversiblyin the normal complement of complete ribosornal particlesand that the intact or defective excess rRNA is degradedbecause r-proteins are not available to participate in ribo-some assembly with excess rRNA. It is unlikely that thedegradation of excess rRNA per se prevents excess rRNAfrom serving as an antirepressor, as the half-life of excessrRNA is much longer than the time it takes free rRNA in

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UNBALANCED rRNA GENE DOSAGE 485

normal cells to be assembled into complete ribosomes (18).If the degradation of rRNA per se is the reason why excessrRNA cannot function as an antirepressor, rRNA synthe-sized in excess of r-proteins in wild-type cells also wouldprobably be degraded too rapidly to function as anantirepressor. We note that if cooperative ribosome as-sembly is required for feedback r-proteins to bind tightly torRNA, the synthesis rates of many r-proteins (includingthose which do not directly bind rRNA or r-protein mRNAs)may affect the pools of free feedback r-proteins and conse-quently indirectly regulate the synthesis of many r-proteins.Excess rRNA synthesis also might not significantly

derepress r-protein synthesis if r-protein mRNAs are largelyderepressed during normal growth. In vivo studies whichhave attempted to measure the level of repression of r-protein mRNAs have used r-proteins that either have defectsin both ribosome assembly or are always absent from theribosome (15, 16, 35, 42). These experiments indicate that(depending on the r-protein regulon examined) full derepres-sion may result in 20 to 100%o more r-protein synthesis fromthe fully derepressed operons than from operons regulatedby other feedback proteins. If the least derepressible r-protein mRNA limits the in vivo synthesis of all otherr-proteins (see also above and below), an upper limit for thein vivo further derepressibility of all r-protein mRNAs inwild-type cells is therefore under some conditions at most20% and likely to be less, since the 20% limit results fromanalysis of only two r-protein regulons in strains in whichdefective r-proteins may cause ribosome assembly defectsresulting in unusually large pools of feedback regulatoryr-proteins. These pools may result in an unusually high levelof feedback repression. Our results on the derepression ofr-protein synthesis in response to rRNA overproduction areconsistent with the limited capacity for translationalderepression during steady-state growth suggested by theseprevious experiments with defective feedback r-proteins.

It is possible that the r-protein mRNA content of cells maybe adjusted so that r-protein mRNAs always have a lowcapacity for feedback derepression. Previous measurementsof the r-protein mRNA content of cells suggest that ther-protein mRNA content of cells is adjusted by differences inmRNA synthesis or stability (10, 28, 44). A low level offeedback repression may in fact be advantageous, as theo-retical considerations based on the laws of mass actionindicate that feedback repression would be maximally ef-ficient if feedback repression is near the 50% level duringnormal cell growth and that a lesser degree of feedbackrepression during normal growth might be optimal if feed-back repression has a primary role in the reduction ofr-protein synthesis during precipitous declines in rRNAsynthesis caused by the stringent response. Mechanismssuch as mRNA degradation (10, 20, 27, 28, 31), transcrip-tional regulation (44), or alterations of mRNA translationalefficiency by mechanisms other than feedback regulation(20) theoretically are capable of maintaining a low level offeedback repression of r-protein mRNAs during all condi-tions of steady-state growth.

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