These re- suits are consistent with the ideas that the level of expression of the genes ...... Blumenthal, R.M., Reeh, S., Pedersen, S. : Regulation of transcrip-.
Molec. gen. Genet. 165, 79-86 (1978) © by Springer-Verlag 1978
Gene Expression in Escherichia coli B/r During Partial Rifampicin-Mediated Restrictions of Transcription Initiation Robert M. Blumenthal and Patrick P. Dennis Department of BiochemistryUniversity of British Columbia, Faculty of Medicine, 2075 Wesbrook Place, Vancouver, British Columbia V6T 1W5, Canada
Summary. The antibiotic rifampicin inhibits transcription initiation, but not the elongation and completion of nascent R N A transcripts. Addition of low concentrations of rifampicin only partially blocks initiation but at the same time specifically alters the general pattern of transcription in the culture. The transcription of the genes specifying the /3 and /3' subunits of R N A polymerase, and to a lesser extent of the genes specifying the R N A and protein components of the ribosome, was specifically stimulated relative to total transcription. In contrast, the transcription of the lactose operon was selectively reduced. These resuits are consistent with the ideas that the level of expression of the genes specifying the /3 and /3' subunits is sensitive to the general rate of R N A synthesis in the culture, and that the expression of the /3 and /3' R N A polymerase genes is related to the expression of ribosome component genes.
Introduction
RNA polymerase is a central component in gene expression; the bulk of all systems regulating gene expression in bacteria seem to act at the level of transcription initiation. RNA polymerase is structurally one of the most complex enzymes in Escherichia coli, being a heteromultimer of molecular weight 5 x 105 (Burgess, 1969). The process of transcription alone would seem to require much less complexity since the bacteriophage T7-induced and mitochondrial RNA polymerases are single peptide chains of molecular weight 1 x 105 and 6 x 104, respectively (Kuntzel and Schafer, 1975; Studier and Maizel, 1969; Chainberlin etal., 1970). This complexity of the E. coli For offprints contact. P.P. Dennis
R N A polymerase may provide the enzyme with allosteric properties and allow some heterogeneity in promoter selection (Travers, 1976). The antibiotic rifampicin binds to the /~ subunit of E. coli RNA polymerase with a binding constant of 1 - 2 x 10 9 M at 25 ° C (Heft and Zillig, 1970; Stender et al., 1975; Yarbrough et al., 1976), and inhibits R N A chain initiation without interfering with elongation and completion of nascent RNA transcripts (Sippel and Hartmann, 1968). When bacterial cultures were treated with rifampicin, a transient increase in the rates of synthesis of the c~, /3, /3' and a subunits of R N A polymerase was observed (Hayward et al., 1973; Hayward and Fyfe, 1978; Nakamura and Yura, 1976). These studies employed high levels of rifampicin added to E. coli merodiploid strains containing both a rifampicin-sensitive and rifampicin-resistant rpoB gene. In these strains RNA synthesis in the presence of rifampicin continued because a fraction of the cell's R N A polymerase was resistant to the drug. Similarly, a restriction on RNA synthesis in conditionally lethal RNA polymerase mutants also stimulated the production of RNA polymerase subunits (Dennis, 1977a; Kirschbaum, 1978). The results of these and many other experiments suggest that the expression of the rpoB and C genes is related in some way to the transcription capacity of the cell. In an attempt to understand this relationship we have added rifampicin at low concentrations to a wild-type strain of E. coli to partially restrict the initiation of transcription, and have examined the expression of genes specifying the /3 and /3' subunits of R N A polymerase and the enzyme /3-galactosidase at the levels of transcription and of translation of the respective mRNAs. Our results indicate that transcription of the genes specifying the /3 and /3' subunits is stimulated whereas transcription of the lactose operon is reduced following the imposition of a partial rifampicin-mediated restriction on R N A
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R.M. Blumenthal and P.P. Dennis: E. coli Gene Expression During Rifampicin-Mediated Restrictions
synthesis. It was further observed that transcription of genes specifying the RNA and protein components of the ribosome was enhanced somewhat by the partial restriction.
Materials and Methods Bacterial Strains The bacterial strains used in this work are derivatives of E. coli B/r. Strain NC3 is hsr- and strain NC83 is leuAt~, trp£TM, lacI, and lacL37UV5 (Wanner et al., 1977). The lacI and lacL37UV5 lesions make transcription of the lactose operon in strain NC83 both "constitutive" and insensitive to catabolite repression. In media containing L-tryptophan and L-leucine, the growth rates of strains NC3 and NC83 were indistinguishable at all temperatures tested. Both NC3 and NC83 possess single, wild-type (rifampicinsensitive) rpoB genes.
Growth Conditions and Growth Analysis The medium used was MOPS (Neidhardt et al., 1974) with glucose or glycerol added to final concentrations of 0.2%. In the experiment involving strain NC83, the medium was further supplemented with L-trypthophan (50 gg/ml) and L-leucine (50 gg/ml). All cultures were grown at 37 ° C in water bath shakers. When used, isopropyl/LD-thiogalactoside (IPTG) was added to a concentration of 1.0 mM. Rifampicin was stored in methanol a - 2 0 ° C as a 5 mg/ ml stock solution. The rate of accumulation of cellular mass was determined by measuring the absorbance of the culture at 420 nm (A420) using a Gilford spectrophotometer with a 1 cm light path. Where appropriate, these values were corrected for the absorbance due to the rifampicin. The rate of accumulation of RNA was determined as the A260 of the acid-insoluble, alkali labile fraction or culture mass (Bremer et al., 1973).
Labeling and Analysis of Proteins Protein labeling was achieved by adding [3SS]methionine (specific activity 609 Ci/mmol; 1 gCi/ml) for 1 rain, and then adding excess unlabeled methionine and incubating for an additional 4 min. Labeled cells were centrifuged, washed, and resuspended in SDS lysis buffer (Laemmli, 1970). These suspensions were placed in boiling water for 2 rain, and stored at - 20 ° C. The total radioactivity of these preparations was determined in quadruplicate by coprecipitating portions of each sample with bovine serum albumin in trichloroacetic acid. The precipitates were collected on nitrocellulose membrane filters, washed with trichloroacetic acid and with ethanol, dried, and the radioactivity was determined in a liquid scintillation counter. The radioactivity associated with particular polypeptides was determined by subjecting equal amounts of radioactivity from the labeled preparations to electrophoresis on a 1.5 mm x 12 cm 7.5% SDS-polyacrylami~te gel with a 3 cm 3% stacking gel (Laemmli, 1970). After fixing and drying, an autoradiogram was prepai'ed and subjected to densitometry using a Quick-Scan Jr. (Helena Instruments, Beaumont, Texas) equipped with an integrator. The activity of/Lgalactosidase was assayed using the standard colorimetric assay for hydrolysis of o-nitrophenyl-/?-D-galactopyranoside (ONPG). Culture samples were disrupted by CHC13 and SDS (Miller, 1972). When appropriate, absorbance due to rifampicin was corrected for.
Labeling and Hybridization of RNA RNA labeling was achieved by adding [3H]uracil (specific activity 26 Ci/mmol; 10 gCi/ml) for 1 min. Samples were harvested by centrifugation and RNA was prepared (Dennis and Nomura, 1975). The RNA preparations were diluted, if necessary, to an A260 of 1.0, and stored at - 2 0 ° C. The total radioactivities of these preparations were determined in duplicate by spotting samples on nitrocellulose membrane filters, drying the filters, and counting by liquid scintillation. The radioactivity associated with particular mRNA species was determ!ned by hybridization to excess denatured DNA immobilized on nitrocellulose filters (Dennis and Nomura, 1975). The hybridization probes employed are described in detail elsewhere (Dennis, 1977b). Briefly, hybridization to ,~dspclDNA corrected for hybridization to 2dtrk represents mRNA homologous to a cluster of 15 ribosomal protein genes and the gene specifying the e-subunit of RNA polymerase (aggregate protein molecular weight of 300,000) located near 72 min (Bachmann et al., 1976). Hybridization to pcc720 DNA represents mRNA homologous to the rpoB and C genes specifying the/~ and/?' subunits of RNA polymerase (molecular weight of 320,000) and located near 88 min. The difference in hybridization to pcc 703 and pcc 720 represents mRNA homologous to a cluster of 4 ribosomal protein genes (molecular weight of 67,000) located adjacent to the rpoB and C genes. Hybridization to 2plac5 DNA represents lactose operon mRNA (molecular weight of 110,000) (Shapiro et al., 1969). Ribosomal RNA was assayed by hybridization to ,~ilv5 DNA, and the efficiency of this hybridization was monitored using a 32P-labeled rRNA standard (Collins et al., 1975; Little and Dennis, manuscript in preparation).
Results Effects o f Low Concentrations o f Rifampicin on Cell Growth T h e g e n e r a l effects o f a d d i n g l o w c o n c e n t r a t i o n s o f r i f a m p i c i n t o a s t e a d y - s t a t e c u l t u r e o f E. coil B / r ( s t r a i n NC3) growing in glucose minimal medium were determ i n e d (Fig. 1). T h e r a t e s o f m a s s a c c u m u l a t i o n a n d of RNA accumulation were reduced by about 50% following addition of rifampicin to a final concentrat i o n o f 10 g g / m l , a n d w e r e r e d u c e d b y a b o u t 7 5 % f o l l o w i n g a d d i t i o n o f 20 g g r i f a m p i c i n p e r ml. T h e s e reduced rates were maintained without significant change for more than one hour. Similar kinetics were observed for cultures growing in glycerol minimal medium. The continued growth of the cultures in t h e p r e s e n c e o f l o w c o n c e n t r a t i o n s o f r i f a m p i c i n , alt h o u g h a t r e d u c e d r a t e s , m a k e s it p o s s i b l e t o e x a m i n e t h e effects o f a p a r t i a l r e s t r i c t i o n o f R N A c h a i n i n i t i a t i o n o n t h e e x p r e s s i o n o f v a r i o u s c l a s s e s o f genes.
Effects o f Rifampicin on the Synthesis o f R N A Polymerase and fl-galactosidase The synthesis of the/~ and/~' subunits of RNA polymerase has been shown to increase, relative to total
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Fig. 4.A-D Rates of synthesis of specific m R N A species before and after rifampicin addition. Rifampicin was added to glycerolIPTG or glucose cultures, at various times samples were removed and pulse-labeled with [3H]uracil, and the labeled RNA was prepared for hybridization. A The steady-state glucose minimal culture of strain NC3; B the glucose culture 30 min after the addition of 20 pg rifampicin per ml; C the steady-state glycerol-IPTG minimal culture of strain NC3 ; and D the glycerol-IPTG culture 30 min after the addition of 15 lag rifampicin per ml. The RNA preparations were adjusted to a concentration of about 50 lag/ml and 50, 100, 150 and 200 111 were hybridized to excess amounts of specific and nonspecific D N A species immobilized on nitrocellulose filters. Each assay contained one blank filter, 2 2 D N A filters, 22dtrk filters, 42dspcl filters, 2 pcc720 filters and 2,tplac5 filters, and in a separate series one blank filter, 22DNA filters, and 2 pcc703 filters. The D N A filters contained 167 fmoles of denatured DNA. The average radioactivity per filter, corrected for the background and non specific hybridization (to 2 or Zdtrk DNA) is illustrated. The total radioactivity per 50 111 of RNA in these assays was A) 1.8x105cpm, B) 9.7x10 ¢cpm, C) 1,1x105cpm, and D) 4.4 x 10¢ cpm. The difference in radioactvity associated with,tdspc~ and 2dtrk filters represents m R N A from the ribosomal protein gene cluster near 72 min (o). The difference between pcc720 or 2plac5 and 2 filters represents, respectively,/~ and/3' RNA polymerase subunit m R N A (n) and lactose m R N A (m). The difference between pcc703 and 2 filters represents m R N A corresponding to the /3 and /3' subunits of RNA polymerase and four ribosomal proteins whose genes are adjacent to those for the 13 and/3' subunits (e). The difference between pcc703 and pcc720 filters represents the m R N A corresponding to those four ribosomal proteins. The ordinates have been adjusted to compensate for differences in the total radioactivity incorporated into the respective R N A preparations, so the slopes of these respective curves reflect the synthesis rates of the various m R N A species relative to total RNA synthesis
R.M. Blumenthal and P.P. Dennis: E, coli Gene Expression During Rifampicin-Mediated Restrictions
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TIME (MIN AFTER RIF ADDITION) Fig.5. A-H. Relative rates of synthesis of various R N A species following rifampicin addition. Portions of a steady-state culture of strain NC3 growing in glucose minimal medium (panels A D) were pulse-labeled with [3H]uracil before or after the addition of rifampicin to 10 ~tg/ml (m,[]) or 20 lag/ml ( e , o ) . A steady-state culture of strain NC3 growing in glycerol-IPTG minimal medium (panels E-H) was pulse labeled before or after the addition of rifampicin to 15 gg/ml (A,zx). The R N A preparations were subjected to hybridization analysis as described in the legend to Figure 4. All of the m R N A synthesis rates are expressed relative to the pre-rifampicin rate of synthesis of m R N A complementary to 2dspcI D N A (100%). The rRNA synthesis rates are normalized to their own pre-rifampicin synthesis rates (100%). The broken lines represent the pre-rifampicin rates of synthesis of the various R N A species. "/~fl'" m R N A (A, E) is R N A hybridizing to pcc720 D N A ; " L A C " m R N A (B, F) is R N A hybridizing to 2plae5 D N A ; r-protein m R N A (C, G) is R N A hybridizing to 2dspcl D N A (spc) or to the ribosomal protein genes adjacent to rpoB and C (703 720)
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