Molec. gen. Genet. 147, 1 7 9 - 1 8 8 (1976) © by Springer-Verlag 1976

Transcription of Escherichia coil Ribosomal DNA in Proteus mirabilis Edward A. Morgan and Sam Kaplan Department of Microbiology, University of Illinois, Urbana, Illinois 61801, U S A

Summary. Transcription of Escherichia coli ribosomal DNA introduced into Proteus mirabilis on F14 is described. We have developed an assay for E. eoli coded

ribosomal RNA involving fingerprinting of ribonuclease T1 digests of RNA isolated from ribosomal subunits. Sequence differences in the ribosomal RNA of the two species have allowed us to detect E. coli coded 16S, 23S, and 5S ribosomal RNA in ribosomal subunits of the E. coli-P, mirabilis hybrid. The proportion of E. coli coded rRNA in the hybrid is found at a level which is compatible with the number of E. coli (and P. mirabilis) ribosomal DNA sequences. The resulting ribosomal RNA appears in ribosomes in a form which indicates extensive compatibility of E. eoli coded ribosomal RNA with P. mirabilis ribosomal proteins and maturational factors.

Introduction

We have examined the transcription of E. coli ribosomal DNA (rDNA) in Proteus mirabilis, rDNA was introduced into P. mirabilis on E. coli episome F14 by Birnbaum and Kaplan (1971), who showed by saturation hybridization that F14 contributes about 30% of the ribosomal RNA (rRNA) gene dosage in the resulting intergeneric hybrid. Electron microscope heteroduplex mapping has revealed that there are two rRNA operons on F14 (Deonier et al., 1974). By use of two dimensional electrophoresis of RNase T1 digests of RNA isolated from the hybrid we have been able to detect~E, coli coded rRNA in this hybrid. Previous investigations of the expression of foreign genetic material in P. rnirabilis have involved R factors, F factors, or F' factors. Synthesis of proteins coded by these templates has been compared in E. coli and P. mirabilis (Falkow et al., 1964; Colby and Hu, 1968; Stubbs et al., 1973; Kontomichalou,

1967; Smith, 1969; Franklin and Rownd, 1973; Okamoto et al., t967; Baumberg and Dennison, 1975). These studies indicate that the regulation, transcription and translation of most E. coli templates is in some way deficient in P. mirabilis as compared to E. coli. These deficiencies are not, at least in one instance, a consequence of altered protein structure in P. mirabilis (Dale and Smith, 1971). In light of the findings referred to above, the synthesis of E. coli rRNA in P. mirabilis is interesting for several reasons. Unlike other systems, measuring a primary gene product is possible. Other studies have measured the net effect of transcription and translation. On the other hand, the newly transcribed rRNA precursor must undergo an extensive series of modifications and maturational events before packaging into mature ribosomes is complete (Schlessinger, 1974). Since F14 contains about 4 min of the E. coli chromosome, from 74' to 78' on the Taylor-Trotter map (Low, 1972), these events must be determined primarily, if not exclusively, by P. mirabilis gene products if such maturation is to occur. The establishment of a hybrid ribosome population in P. mirabilis, where a portion of the ribosomal RNA has undergone evolutionary sequence divergence in structural and perhaps regulatory portions of the operon, may allow an in vivo investig~ition of the importance of ribosomal RNA base sequence. The existance of this unique merodiploid may also help to clarify the regulatory mechanisms involved in the control of rRNA synthesis.

Materials and Methods Bacterial Stra&s. E. coli strains are K12 derivatives. Strain L M U R (lys-, u r a - , met , R N a s e 1-, rel ) was used as a source of 3H labelled R N A . E. coli 32p labelled 16S and 23S r R N A was prepared from AB1206 (thi-1, str8(R), his 4, gal z, T4-3R, lac y, pro A-2),

180

E.A. Morgan and S. Kaplan: Transcription of Escherichia coil Ribosomal D N A in Proteus mirabilis

a primary donor for F14. Strain W3110 was used for preparation of 32p labelled 5S rRNA. P. mirabilis strain PM14IV-4 is an isoleucine-valine requiring derivative of P M I 4 (Birnbaum and Kaplan, 1971). Strain F14/ PM14IV-4 was derived by Birnbaum and Kaplan (1971) by a mating of AB1206 with PM14IV-4. PM17 (lac-, gal-, nic-, t h y - , t r p - , u r a - ) was received from R. Rownd. Strain F 14/PM 14VI-4 was always maintained on media lacking isoleucine and valine. All stocks were at most two cultures removed from original frozen stocks of L. Birnbaum. Stocks for all strains were maintained in 10% glycerol at - 2 0 ° C. The presence of E. coli D N A in stocks of F14/PM14IV-4 was confirmed by CsC1 equilibrium density centrifugation and was consistent with the retention of one copy of F14 per copy of chromosome, as previously reported (Birnbaum and Kaplan, I971).

Labelling of Cultures. Cultures growing at 37 ° C with vigorous aeration were labelled with [3zp]-orthophosphate in the minimal low phosphate medium of Maaloe and Hanawalt (1961). Glucose was added to 0.4%, all required amino acids were added to a final concentration of 50 p~g/ml, pyrimidines to 10 Ixg/ml, and vitamins to 2 gg/ml. L M U R was grown in 20 ml of M9 salts media supplemented with 0.4% glucose, 1% casamlno acids, 50 gg/ml methionine and tryptophan, and 15 gg/ml uracil. 2 mC [3H]-uracil labelled in the 5-6 positions (NEN) was added to exponentially growing cells and aerated at 37 ° C until the onset of uracil starvation. An additional 15 gg/ml unlabelled uracil was then added, and the cells were grown another 15 rain to a final cell density of near 200 Klett units as measured with a Klett-Summerson colorimeter with a blue filter. R N A was prepared from PM17 growing in 20 ml of M9 salts media containing 0.4% glucose, 50 gg/ml lysine, 10 lag/ml thymine, 5 gg/ml uracil, and 1% casmino acids. 1 mC of [3H]-uracil was used to label the culture in a manner identical to that used for the preparation of R N A from L M U R .

Preparation of RNA. At the end of the desired growth period, the cultures were cooled by swirling on ice. All further operations were performed as rapidly as possible either on ice or in the cold. Cells were collected by centrifugation at 10,000 rpm for 10 min in the Sorval GSA or SS34 rotor and resuspended in 1.0 ml of Buffer A (10 m M Tris pH 7.5, 30 m M NH4CI, 10 -4 M MgC12). The cells were broken in a French Pressure cell into 1 mg of Macaloid. The debris was removed by centrifugation at 10,000 rpm for 10 min in the Sorvall SS34 rotor. The supernate was then layered on 12 ml, 15 30% sucrose (w/v) gradients in Buffer A. The gradients were centrifuged at 41,000 rpm for 71/2 h in the Spinco SW41 rotor. 30 drop fractions were collected from the bottom and portions spotted on filters and counted. The material was usually frozen at this point. The resulting ribosomal subunits were usually well separated from each other and from low molecular weight material. The pooled subunit fractions were diluted with an equal volume of A E N buffer (0.1 M NaC1, 0.01 M Na Acetate, 0.001 M EDTA pH 5.3) and extracted with an equal volume of 1 x SSC saturated phneol. Two volumes of ethanol were added to the aqueous phase and the R N A collected by low speed centrifugation after storage overnight at - 20 ° C. The RNA was lyopholized to dryness, resuspended in 0.5 ml of A E N buffer and layered on 12 ml, 15-30% sucrose (w/v) gradients in A E N buffer. After centrifugation for 151/2 h at 41,000 rpm in the SW41 rotor, 30 drop fractions were collected from the bottom of the gradients and the R N A located by counting portions of each fraction. Regions of each gradient corresponding to 16S, 23S, or 5S r R N A were pooled, an equal volume of AEN added, and the R N A precipiated by addition of two volumes of ethanol. 16S and 23S r R N A were washed three times with 80% ethanol, lyopholized to dryness, resuspended in a small volume of water, and the specific activity determined. Samples containing 5S r R N A were lyophilized to dry-

ness, resuspended in E buffer (0.04 M Tris, 0.02 M Na acetate, 0.001 M EDTA, pH 7.2 with acetic acid) plus 0.2% SDS and 10% glycerol, and layered on 0.8 x 10 cm 6% acrylamide gels with a 1 cm 2.8% acrylamide cap. Gels were electrophoresed 6-71/2 h at 4 mamp/tube. The gels were frozen, sliced in 1 mm slices using a Mickel gel slicer, and each slice placed in a scintillation vial containing 0.3 ml of A E N buffer and counted by Cerenkov counting. The samples containing 5S r R N A were pooled and shaken overnight. Acrylamide and precipitated SDS were removed by centrifugation at 0 ° C. The R N A was precipiated by addition of three volumes of ethanol, washed three times with 80% ethanol, lyopholized to dryness, and resuspended in a small volume of water. R N A from FI4/PM14IV-4 was additionally purified on one occasion by electrophoresis on 0.8 x I0 cm 12% acrylamide gels for 151/2 h at 4 mamp/tube. 0.8 x 10 cm 2.8% acrrylamide gels were routinely used to assay 16S and 23S rRNA. Electrophoresis was 6 8 h at 4 mamp/tube.

Digestion and Chromatography of RNA. 16S r R N A was digested with RNase T1 (Worthington) or with RNase T1 and E. coli alkaline phosphatase (Worthington) as described (Sanger and Brownlee, 1967). Second dimensions of 16S r R N A digested with RNase T1 only were electrophoresed in 6.5% formate, 0.1 M pyridine as described (Uchida et al., 1974). Second dimensions of RNase T1 and alkaline phosphatase digested 16S and 23S r R N A were electrophoresed in 6.5% formate as described (Sanger and Brownlee, 1967). 5S r R N A was digested with RNase T1 only, and the second dimension was electrophoresed in 6.5% formate. All first dimensions were as described (Sanger and Brownlee, 1967). Analysis of RNase A (Worthington) digestions of RNase T1 digestion products of 5S r R N A used a pyridine-formate buffer system (Uchlda et al., 1974). Digestion mixtures for initial digestion by either RNase T1 or RNase T1 and alkaline phosphatase typically contained 2-5 x 103 cpm of [32p] and 0.5-2x 103 cpm of [3H]r R N A per base of the r R N A species being digested, so that oligonucleotides derived by digestion of all R N A species had similar amounts of radioactivity.

Analysis of Chromatograms. 32p was located by autoradiography using Kodak No-screen X-ray film. Coincidence of a2p and aH was determined by counting portions excised from the paper. 3H non-coincident with 32p was located by excising 1 cm squares of paper in areas where the 3H was known to run. Radioactivity was determined using a Nuclear Chicago scintillation counter and employing toluene based scintillation fluid. A comparision of chromatograms of 16S r R N A with those of Fellner et al. (1970) and Uchida et al. (1974), and of 5S r R N A with those of Jarry and Rosset (1971) and Sogin et al. (1972) facilitated analysis of chromatograms.

Results

Purity o f R N A . A n a l y s i s o f a c r y l a m i d e

gels and fingerprints of RNase T1 digests indicated that RNA species isolated as in Materials and Methods are in p u r e f o r m . R N A e x t r a c t e d f r o m 5 0 S s u b u n i t s is f r e e from tRNA as indicated by analysis on 12% acrylam i d e gels. S i m i l a r l y , a n a l y s i s o f 5S r R N A on 6% acrylamide gels as indicated in Materials and Metho d s is s u f f i c i e n t t o r e s o l v e 5S r R N A f r o m o t h e r c o m ponents. Throughout the purification, ribosomal subu n i t s o f all s t r a i n s b e h a v e a s h o m o g e n o u s entities

E.A. Morgan and S. Kaplan: Transcription of Escherichia coli Ribosomal D N A in Proteus mirabilis Table 1. Fraction of E. coli coded 16S r R N A calculated using the 32P/3H ratio for individual E. coli unique oligonucleotides as discussed in the text. Spot refers to designations in Figure 1. Sequence designations are those of Fellner et al. (1970) and are assigned as discussed in the text. Values in colums A D and F G are obtained from c h r o m a t o g r a m s of R N A which have been digested by R N a s e T1 and alkaline phosphatase, Each column refers to R N A obtained by different methods of purification. Column A : Total R N A from the 30S ribosomal subunits of F14/PM14IV-4. Column B: Intact 16S r R N A isolated from 30S subunits of F14/ PM14IV-4, discarding small a m o u n t s of smaller molecular weight R N A present in R N A purified as in Column A. Columns C, D: Two independent preparations of R N A isolated from 30S subunits of F14/PM14IV-4 which have been purified by pelleting. Column E." Intact 16S r R N A isolated from 30S subunits of F14/PM14IV-4 and digested by R N a s e T1 only. Column F." R N A from mixtures of cultures AB1206 and PM14IV-4 in the ratio (by optical density of cultures) of I : 1 and (Column G) 1 : 3. For all c h r o m a t o g r a m s the following oligonucleotides~:which are present in equimolar concentrations in PM14IV-4 and AB1206, were used as references for the 32P/3H ratio of the R N A digested: Spots 1. ( U C U U G ) , 5 [(U, C, C) A C A A C A U G ] , 8 ( A C C C A C U G ) and 11 (UG) Spot Sequence

A

B

C

D

E

F

G

2 3 4 6 7 9 10

0.26 0.26 0.42 0.20

0.34 0.38 0,41 0.25 -

0.30 0.31 0.47 0.25 0.28 0.34

0.20 0.29 0.30 0.25 0.26 -

0.31 0.20 0.18 0.31

0.69 0,76 0,83 0.70 0,59 0,56 0,73

0.42 0.46 0.48 0.35 0.43 0.41 -

ACCCUCAUAAG AUUAAACG pAAAUUG ACCUUCG UCUCG CAUAACG UAACG

-

of the RNA coded by E. coIi rDNA in F14/PM14IV-4 is intact, and that we have not enriched for either E. coli or P. mirabilis coded rRNA from ribosomes during isolation. E. coli coded rRNA in the hybrid must be similar in size and form ribosomes grossly similar to the ribosomes of P. rnirabilis. Identification of Species-specific Oligonucleotides. E. coli [3H]-rRNA was mixed with E. coli or P. mirabilis [32p]-rRNA, digested, and fingerprinted. From the resulting fingerprints, the location, approximate molarity, and pyrimidine/phosphate ratio of oligonucleotides resulting from 16S, 23S, and 5S rRNA could be determined. It was assumed that 3H labeling occurs equally well in cytosine and uracil residues as uracil auxotrophs were used. This appears to be correct as indicated by the distribution of radioactivity in oligonucleotides of known sequence. The distribution of E. coli and P. mirabilis oligonucleotides is depicted in Figures 1-3. [32p]-rRNA from F14/PM14IV-4 was mixed with E. coli [3H]-rRNA, digested, and fingerprinted. The resulting autoradiograms are presented in Figures 4-8. As can be seen, E. coli unique oligonucleotides are found in fingerprints of F14/PM14IV-4

~ in sucrose gradients. Total cell RNA of all strains, when analysed on gels, yields no significant species which might be atrributed to primary rDNA transcripts or maturational precursors, other than 16S, 23S, or 5S rRNA (unpublished data). As the separation of relevant components during purification was nearly complete, recovery of material was high, and it was unlikely that selective enrichment of E. coli or P. mirabilis coded rRNA in F14/PM14IV-4 occurred. Total RNA of 30S subunits of F14/ PM14IV-4 has also been recovered from ribosomes pelleted through 10- 2 M Mg + +. RNA has also been extracted from subunits purified as in Materials and Methods but without further purification of the RNA on sucrose gradients containing AEN buffer. RNA purified by either of these procedures yields qualitatively and quantitatively similar fingerprints as does intact 16S rRNA purified as described in Materials and Methods (Table 1). RNA purified from ribosomes which have been pelleted underwent extensive nucleolytic cleavage in the RNase ÷ wild type cells used here, but not when LMUR was employed as a source of RNA (unpublished data). RNA purified as in Materials and Methods is largely undegraded when isolated from either the RNase + or RNasestrains used here. We therefore feel that the bulk

181

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Fig. 1 a and b. A schematic diagram indicating the distribution of oligonucleotides resulting from digestion of a mixture of E. coli and P. mirabilis 16S r R N A . E. coli unique oligonucleotides (black circles), P. mh,abilis unique oligonucleotides (gray circles), and spots containing oligonucleotides of both species (open circles) were determined as described in the text. N u m b e r s refer to Table 1. a Fingerprint resulting from RNase T1 and alkaline phosphatase digestion. Oligonucleotides containing no uracil residues are not included in this diagram, b Fingerprint resulting from RNase T1 digestion

182

E.A. Morgan and S. Kaplan: Transcription of Escherichia coli Ribosomal DNA in Proteus mirabilis

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Fig. 2. A schematic diagram indicating the distribution of oligonucleotides resulting from RNase T1 and alkaline phosphatase digestion of a mixture of E. coli and P. mirabilis 23S rRNA. E. coli unique oligonucleotides(black circles), P. mirabilis unique oligonucleotides (gray circles), and spots containing oligonucleotides of both species (open circles) were determined as described in the text. Numbers refer to Table 2. Oligonucleotides containing no uracil residues are not included in this diagram Fig. 3. A schematic diagram of the distribution of E. coli and P. mirabilis oligonucleotides resulting from RNase T1 digestion of a mixture of the 5S rRNA of two species. Open circles indicate oligonucleotides common to both species, black areas indicate E. coli unique sequences, and shaded areas indicate P. mirabilis unique sequences

rRNA. That these spots are due to E. coli unique sequences is confirmed by the recovery of expected amounts of 3H label in corresponding portions of the chromatogram. Several oligonucleotides, not apparent in the pictures presented here, were partially or wholly resolved in original autoradiograms. Tables 1-3 indicate the fraction of each R N A species which is E. coli coded, as calculated from the 32p/3H ratio observed for each oligonucleotide. The blank spots in these and subsequent tables indicate that the designated oligonucleotide was not well resolved in the experiment in question. In these calculations sequence and molarity data of authors cited in the Analysis of Chromatograms section of Materials and Methods were used where possible. The 32p and 3H labelled 23S r R N A as discussed above were used to determine the required pyrimidine/phosphate ratios for this r R N A species. The values presented in Tables 1-3 are corrected for background and spillover of 32p into the 3H channel and are calculated using Formula 1: 1. The fraction of r R N A which is E. coli coded = A/B. For E. coli unique oligonucleotides; A = 3 2 p cpm/3H c p m . n u m b e r of pyrimidines/number of phosphate atoms. For reference oligonucleotides; B = average 32p cpm/3H c p m . n u m b e r of pyrimidines/ number of phosphate atoms. Reference oligonucleotides are oligonucleotides present in equimolar quantities in both P . m i r a b i l i s and E. coli and which serve as a source of internal standardization in each chromatogram for: 1. the ratio of [32p]-rRNA and [3H]-

b

Fig. 4a and b. Fingerprints of oligonucleotides containing uracil derived from RNase Tl-alkaline phosphatase digestion of a P. mirabilis 16S rRNA and b. F14/PM14IV-4 16S rRNA

E.A. Morgan and S. Kaplan: Transcription of Escherichia coli Ribosomal DNA in Proteus mirabilis

183

Fig. 5. RNase T1 fingerprint to F14/PM14IV-4 16S rRNA. The arrow points to the expected location of the 5'-terminal oligonucIeotide of E. coil 16S rRNA (pAAAUUGp) if it did not contain a 5' phosphate

Fig. 6. Fingerprint of oligonucleotides containing uracil resulting from RNase Tl-akaline phosphatase digestion of E. coli 23S rRNA

r R N A digested, and 2. an internal correction for differential counting efficiency of 32p and 3H.

et al., unpublished data). Sequence assignments for almost all oligonucleotides can be reliably m a d e for 16S r R N A on the basis of published data and mobility. Since the use of R N a s e T1 alone or in conjunction with alakaline phosphatase yield similar results with E. coli 16S r R N A , (Table 1), we conclude that the values obtained by either m e t h o d are reliable.

1 6 S r R N A . The techniques e m p l o y e d have revealed most o f the sequence differences o f R N a s e T1 derived oligonucleotides for 16S r R N A of E. coli and P. m i r a bilis (Fellner et al., 1970; U c h i d a et al., 1974; Woese

b

Fig. 7a and b. Fingerprints of oligonucleotides containing uracil resulting from RNase Tl-alkaline phosphatase digestion of a P. mirabdis 23S rRNA and b F14/PMI4IV-4 23S rRNA

184

E.A. Morgan and S. Kaplan: Transcription of Escherichia coli Ribosomal DNA in Proteus mirabilis

a

b

Fig. 8a and b. Fingerprints of oligonucleotides resulting from RNase T1 digestion of a P. mirabilis 5S rRNA and b. F14/PM14IV-4 5S rRNA

Table 2. Data from chromatograms of 23S rRNA. Spot refers to designations in Figure 2. The pyrimidine: phosphate ratio for these E. coli unique spots was determined by co-digestion of E. eoli 3zp labeled and 3H labeled 23S rRNA. Columns A and B: The fraction of 23S rRNA which is E. coli coded calculated using individual E. eoli unique oligonucleotides derived from digestion of F14/PM14IV-4 23S rRNA and not corrected for overlap. Column C: Ratios of 3zp/3H for these oligonucleotides when a mixture of P. mirabilis 3H labeled and F14/PM14IV-4 32p labeled RNA is digested. Values are corrected for the pyrimidine/phosphate ratio and for the ratio of 3H and 32p labeled RNA digested. In Column C, a value of 1.0 indicates that the 3zp is derived from P. mirabilis 23S rRNA and an infinitely large value indicates the 32p is E. eoli 23S rRNA derived and no overlap of 3zp from neighboring P. mirabilis oligonucleotides is occurring. Intermediate values reflect overlap of P. mirabilis oligonucleotides into regions containing E. coli coded sequences, and therefore observed values are likely between 30 50% inflated Spot

pyrimidine ratio phosphate

A

B

C

12 13 14 15

0.75 0.46 1.0 0.26

0.262 0.515 0.593 0.350

0.296

2.31 3.00 -

0.606 0.337

C o l u m n s F and G o f Table 1 are independent determinations, based u p o n the a / p / 3 H ratio for each E. coli-specific oligonucleotide tested, of the fraction o f E. coli 16S r R N A derived f r o m 1 : 1 and 1 : 3 mixtures of E. coli and P M 14IV-4 cells, respectively, prior to breakage. The fraction o f E. coli 16S r R N A in the cell mixtures is on the basis o f R N A per Klett

unit of cells and not R N A per cell for each cell type. The average ratio o f the 1:1 to 1:3 values is 1.67 as c o m p a r e d to an expected ratio o f 2. This indicates that averaging of the values for the fraction o f r R N A which is E. coli coded m a y result in an inflated estimate of the E. coli r R N A due to overlap with neighboring P. mirabilis derived oligonucleotides. This error o f course varies with the resolution of the selected oligonucleotides and this is reflected in the data of Table 1. As expected, the oligonucleotides which yield the lowest fraction o f E. coli r R N A in the mixing experiment give ratios closest to the expected ratio o f 2. These oligonucleotides tend to yield a value near 0.2 for the fraction o f E. coli 16S r R N A present in the hybrid. Even when we consider all o f the values determined, the fraction o f E. coli 16S r R N A in the hybrid is approximately 0.25. rRNA. C h r o m a t o g r a m s o f 23S r R N A are congested because o f the large n u m b e r o f oligonucleotides and therefore permit only a fraction o f the species specific oligonucleotides to be determined by our techniques. We have co-digested mixtures o f F14/ P M 1 4 I V - 4 [32p]-23S r R N A and P M 1 7 [3H]-23S r R N A because only a few, well isolated, E. coli unique oligonucleotides derived f r o m 23S r R N A were located. E. coli coded sequences should give rise to oligonucleotides with a 32p/3H ratio too high to easily be accounted for by P. miirabilis derived sequences. Ideally this ratio should be infinity. The resulting fingerprints reveal oligonucleotides which are E. coli 23S

E.A. Morgan and S. Kaplan: Transcription of Escherichia coli Ribosomal DNA in Proteus mirabilis

unique as determined by both mobility and 32p/3H ratios (Table 2). The fraction of the 23S rRNA which is E. coli coded, as calculated from the 3 2 p / B H ratio of individual E. coli unique oligonucleotides arising from co-digestion of F 14/PM 14IV-4 [32p] and LMUR [3H] labeled 23S rRNA, is given in Table 2. Correction for spillover of the magnitude indicated in the legend to Table 2 as determined by co-digestion of F 1 4 / P M 1 4 I V - 4 [32p] and PMI7 [3H]-23S rRNA indicates that roughly 0.2 of the 23S rRNA of F14/ PM14IV-4 is E. coli coded. 5 S rRNA. The technique which we have employed has revealed all the species specific oligonucleotides of P. mirabilis and E. coli 5S rRNA as indicated by the extensive sequence analysis of Jarry and Rosset (1971) arts Sogin et al. (1972). Values for the fraction of E. coli coded 5S rRNA in F14/PM14IV-4 obtained from oligonucleotides present in all E. coli 5S rRNA sequences are near 0.2 (Table 3). Overlap of P. mirabilis oligonucleotides into E. coli unique oligonucleotides does not significantly affect these values due to the good resolution of the oligonucleotides which derive from this small RNA. Many E. coli 5S rRNA fractional sequences are dealt with below, in a separate section. Counting Error. We find that for co-digests of E. coli [32p] and [3H]-rRNA or P. mirabilis [32p] and [3H] rRNA that relative counting efficiencies of 32p and 3H vary according to sequence. If, in RNase Tl-alkaline phosphatase patterns, the 32p/3H ratio for UG is normalized to 1.0, the ratio for UUG is 1.1 and for UUUG is 1.2. As these oligonucleotides are very well isolated this suggests that, on DEAE paper, base sequence and oligonucleotide length may be affecting our data to a measurable extent. Counting efficiency of 3H may be lower in long oligonucleotides due to increases in internal quenching with oligonucleotide length. Heterogeneity o f R N A . Some RNase T1 produced oligonucleotides derived from 16S, 23S, or 5S rRNA occur in molarities much less than one. The simplest interpretation is that they are derived from heterogeneous sequences within the rDNA complement of the organism. The heterogeneous sequences of E. coli 5S rRNA have been extensively studied by Jarry and Rosset (1971). They have mapped oligonucleotide UCUCCUCAUG on the region of the E. coli chromosome included in F14 (Jarry and Rosset, 1973). Their data proved that this sequence is present in only one copy of the 5S rRNA genes in E. coli K12, and that there are at least two 5S rRNA operons on F14, possibly corresponding to the location of

185

Table 3. E. coli 5S rRNA sequences in F14/PM14IV-4. Spot refers to designations in Figure 3. The sequence and molarity of oligonucleotldes in E. coli 5S rRNA are taken from Jarry and Rosset (1971). Numbers in Column A refer to the fraction of 5S rRNA which is E. coli coded in F14/PM14IV-4 and calculated as described in the text. Numbers in parenthesis are corrected for their partial molarity in E. coli and their suggested representation on F14 (see text). Spots 18 and 21 are proposed to be P. mirabilis fractional oligonucleotides, not reported by Sogin et aI. (1972). Oligonucleotides AAACG (Spot 25), A A C U G (Spot20), and UCUCCCCAUG (Spot 17) were used as internal standards for the ratio of 32p to 3H labeled RNA digested (see text) Spot

Sequence

Molarity in E. coli

A

16 19 22 23 26 27

UCUCCUCAUG ACCCCAUG CCAUG CCUG CCAO CAG

0.15 0.81 0.27 0.98 0.91 0.49

0.60 (0.18) 0.15 0.37 (0.20) 0.28 0.18 0.46 (0.23)

the 16S-23S rRNA operons detected by electron microscope heteroduplex mapping (Deonier et al., 1974). It should be noted that formula 1 does not apply for oligonucleotides which occur in partial molar yields in E. coli. An estimate of the expression of F14 in P. mirabilis (numbers in parentheses in Table 3) can be derived by multiplying: the value obtained by use of formula 1 by the proposed molarity of the fractional oligonucleotide in E. coli and further, by either 2 or 1, depending on whether the oligonucleotide is thought to occur within one or both respectively, of the E. coli 5S rRNA regions on F14. For oligonucleotides UCUCCUCAUG, CCAUG and CAG the appropriate corrections which we need to use are: (0.15 M) (2), (0.27 M) (2) and (0.49 M) (1) respectively. Proper correction of formula 1 by this method gives values of 0.18-0.23 for the fraction of 5S rRNA which is E. coli coded in F14/PM14IV-4. Because Jarry and Rosset (1973) established that oligonucleotide UCUCCUCAUG is present on only one of the two 5S rRNA cistrons on F14, and because it yields a corrected value of 0.18 in Table 3, which is essentially the same as that (0.2) determined for oligonucleotides present in all 5S rRNA cistrons in E. coli, we conclude that both 5S rRNA cistrons on F14 are transcribed equally. By similar considerations, we propose that fractional oligonucleotide (Jarry and Rosset, 1973) CAG (0.49 molar) is present on both copies of the 5S rRNA genes of F14. E. coli 5S rRNA oligonucleotide CCAUG (0.27 molar) appears to be present on one copy of the 5S rRNA genes of F14, but due to variable production of this oligonucleotide in our hands, we feel we cannot comment conclusively as to its presence on F14. We have

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E.A. Morgan and S. Kaplan: Transcription ofEscherichia coli Ribosomal DNA in Proteus mirabilis

found an oligonucleotide (Number 24 in Fig. 3) in F14/PM14IV-4 but not in PM14IV-4. This oligonucleotide is also present in the chromatograms presented by Jarry and Rosset (1971) but they do not include it in their E. coli 5S rRNA catalog. Due to its presence in F14/PM14IV-4 and the fact that it contains 3H activity when 3H-labeled E. coli 5S rRNA is included in the digest indicates it is a true E . coli partial. However, in our hands it is produced in variable amounts when E. coli 5S r R N A is examined so we cannot exclude the possibility that it represents an enzyme cutting error. E. coli 16S rRNA oligonucleotide C A U A A C G has been proposed to be a fractional oligonucleotide (Fellner et al., 1972) but this has been debated (Uchida et al., 1974). This oligonucleotide is E. coli unique in F14/PM14IV-4 and is therefore localized on F14 (Table 1). From its 3Zp/3H ratio, we cannot distinguish between the presence of C A U A A C G on one copy of the 16S rRNA genes ofF14 and its presence in one half of the 16S rDNA or the alternate model where C A U A A C G is present on both copies of the 16S rDNA of F14 and, is present on all 16S rDNA of E. coli. Discussion Sequence Similarities. The structural relation between E. coli and P. mirabilis r R N A requires comparison

in order to comment upon the role of R N A sequence in ribosome assembly and function. To some extent sequence must influence maturational events of the rRNA, such as nucleolytic cleavage, methylation, and the binding of ribosomal proteins. The 5' ends of mature E. coli and P. mirabilis 16S rRNA do not yield the same RNase T1 derived oligonucleotide. An oligonucleotide corresponding to the E. coli 5' terminus ( p A A A U U G . . . ) (Fellner et al., 1970) is present on fingerprints of F14/PM14IV-4 16S rRNA which have been digested with RNase T1 and alkaline phosphatase (Fig. 1). The 5' end of E. coli 16S rRNA migrates in a heavily congested region in chromatograms of RNase T1 digested R N A and was not located in the hybrid. From this we conclude that maturational cleavage of E. coli 16S rRNA occurs at its usual location (leaving its usual 5' phosphate) in F14/PM14IV-4 although cleavage in P. mirabilis usually occurs in a region which must contain a sequence dissimilarity within six bases of the cleavage point as RNase T1 digestion of P. mirabilis 16S rRNA does not yield this hexanucleotide. Maturational cleavage must take place on the precursor 16S rRNA since the species has oligonucleotides not found in mature 16S rRNA at both the 5' and 3' ends (Sogin et al., 1971 ; Brownlee et al., 1971 ; Lowry

et al., 1971; Hayes et al. 1971). Although sequence of the 5' end of P. mirabilis 16S rRNA has not been determined, the specificity for this cleavage could still reside in a common base sequence, that is, a sequence which does not include the areas of sequence divergence. Alternatively, cleavage may be determined by either gross secondary structure, or cleavage may be determined by ribosomal proteins normally bound at the time cleavage occurs in vivo (Nashimoto and Nomura, 1970). All RNase T1 derived oligonucleotides containing post-transcriptionally modified bases as well as the 3' terminal oligonucleotide of E. coli 16S r R N A are represented by identical sequences in P. mirabilis, as determined by co-migration of isotope. This has been substantiated by further sequence analysis (C.R. Woese, unpublished data). This is in accord with the known evolutionary conservation of methylated sequences in prokaryotes (Sogin et al., 1972) and with the proposed functional role of the 3' end of 16S rRNA (Shine and Dalgarno, 1974), which would impose considerable evolutionary restriction on this sequence. Oligonucleotides known to be present in E. coli precursor 16S rRNA but not in mature 16S rRNA were not detected on the chromatograms of F14/ PM14IV-4 16S r R N A although many of them resolve well in the systems used here. Since the 5' end of E. coli coded 16S rRNA in the hybrid is the same as in E. coli, and mobilities on gels are indistinguishable, we conclude that proper maturational cleavage is occuring for E. coli coded 16S r R N A in the hybrid. Mobilities on gels also appear identical for P. mirabilis and E. coli coded 5S and 23S rRNA sequences in the hybrid and no precursor-specific sequences known to be present in E. coli 5S rRNA (Forget and Jordan, 19969) were detected in the digests. We therefore believe that normal or near normal maturation of all E. coli coded rRNA species is occuring in F14/PM14IV-4. We could detect no oligonucleotides in any rRNA species in F14/PMi4IV-4 that were not present in a mixture of E. coli and P. mirabilis rRNA. Therefore any gross alteration in the structure of either E. coli or P. mirabilis rRNA in F14/PM14IV-4 is unlikely. No aberrations in sedimentation of ribosomes and no accumulation of precursors were noted under the steady-state conditions employed in the study (unpublished data). Conclusion

Synthesis of E. coli ribosomal R N A from an F14 template has been detected in F14/PM14IV-4, an E. coli-P, mirabilis hybrid. The percent of the r R N A

E.A. Morgan and S. Kaplan: Transcription of Escherichia coli Ribosomal DNA in Proteus mirabilis which is E. coli coded is approximately 20-25 percent when considering 16S, 23S, or 5S r R N A . This is near the 30 percent of the r D N A which is of E. coli origin in this strain as determined by saturation hybridization (Birnbaum and Kaplan, 1971). A more accurate estimate of the percent of the r R N A which is E. coli Coded can be made. Since F14 is known to contain two r R N A templates (Deonier et al., 1974; Jarry and Rosset, 1973), E. coli has 6-8 r R N A templates, and P. mirabilis and E. coli have similar r D N A dosages (Birnbaum and Kaplan, 1971; Jarry and Rosset, 1973), it can be concluded that F14/PM14IV-4 has 20 25 percent E. coli r D N A on this basis. This is very close to the percent of the r R N A which is E. coli coded in F14/PM14IV-4. These results are therefore consistent with the proposal that E. coIi r D N A is transcribed as well as P. mirabilis r D N A in this strain, that equal packaging of the products into ribosomes occurs, and that the resulting hybrid ribosomes are stable. Appreciable packaging deficiency of E. coli coded r R N A may possibly exist in F14/PM14IV4, but lack of detectable precursor R N A under steady state growth argues against this, as maturation of r R N A has been found to be dependent on packaging events (Nashimoto and N o m u r a , 1970). These results are therefore consistent with the proposal that E. coli r D N A is transcribed as well as P. mirabilis r D N A in this strain, that equal efficiency of packaging of the products into ribosomes occurs, and that the resulting hybrid ribosomes are stable. This is in contrast to the generally much lower levels of E. coli coded protein products which have been found in similar hybrids (Falkow et al., 1964; Colby et al., 1968; Colby and Hu, 1968; Stubbs et al., 1973; Baumberg and Dennison, 1975; Kontomichalou, 1967; Fraklin and Rownd, 1973; O k a m o t o etal., 1967; Smith, 1969) and which may be due to lower levels of transcription or translation, as discussed by Baumberg and Dennison (1975). As we find efficient r R N A transcription in F14/PM14IV-4, we suggest a translational deficiency of E. coli message as the cause of lowered protein synthesis in these other studies in these hybrids. We feel that the close correspondence of the percent of r D N A and r R N A of E. coli origin in this hybrid indicates transcriptional and perhaps regulatory similarity of the r R N A of P. mirabilis and E. coli. The discovery that all three r R N A species are present in this strain in similar amounts is totally consistent with the proposed sequence of maturation by which all three r R N A species are derived from a single transcript which undergoes packaging and maturational cleavage in a cooperative process (Nashimoto and N o m u r a ; Nikolaev et al., 1975; Ginsberg and Steitz, 1975).

187

It has been demonstrated for t R N A that extensive evolutionary conservation of function and of secondary and tertiary structure can exist despite primary sequence divergence. The base paired regions of the molecule apparently contribute a structural framework within which the base sequence is free to fluctuate while sequences involved in recognition and maintaining three dimensional structure are highly conserved (Kim et al., 1974). 5S r R N A seems to follow these restrictions as far as it structure and function can be determined (Fox and Woese, 1975). We therefore feel that the compatibility of E. coli r R N A with P. mirabilis ribosomal protein evident in the hybrid despite substantial divergence of the primary structure of the r R N A of the two species in no way indicates that ribosomal R N A is not intimately involved in the structure and function of ribosomes. Experiments in progress indicate that ribosomes in F14/PM14IV-4 containing E. coli r R N A possess many, if not all, functional activities of normal ribosomes. Acknowledgement. This research was supported by N.I.H. grant

HD-03521. E.M. was supported by N.I.H. pre-doctoral training grant GM-00510. We would like to acknowledge C.R. Woese and E. Reichmann for use of space and equipment.

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Communicated by H.G. Wittmann

Received February 17, 1976/March 18, 1976