Regulation of the expression of ribosomal protein genes in Escherichia coli

J. MoL BioL (1975) 97, 61-76

Regulation of the Expression of Ribosomal Protein Genes in Escherichia call PA~R~CXP. D~.~Ist AND

I~.ASAYASU NOMU~A

Institute for Enzyme Research Departments of Geneticz and ,Biochemistry University of Wisconsin Madison, Wisc. 53706, U.S.A. (Received 3 March 1975) The in v~o expression of genes coding for ribosomal proteins has been examined by using a quantitative R N A - D N A hybridization assay. Total cellular RNA isolated from exponential-phase cultures of E a c / ~ c h ~ coli growing at rates between 0-67 and 2-10 doublings per hour and from cultures following a nutritional shift-up was hybridized to DNA from a A transducing phage carrying at least 22 ribosomal protein genes. These r-protein genes cede for about 400,000 daltens of protein or about half of the total protein per ribosome. The results indicate t h a t the fraction of the total cellular RNA which is homologous to the r-protein genetic region on the transducing phage, s, increases with increasing growth rate such that s ---- 0.0016 ~ + 0"0006, where g is the steady-state growth rate in doublings per hour. Comparison of the rate of synthesis of the r-proteins coded for b y genes on the phage and the level of r-protein messenger RNA homologous to these genes during steady.state and transition conditions indicates that the expression of r-protein genes is controlled prlm~rily b y a mechanism which regulates the amount of m R N A for r-protein which is available for translation. This control m a y be exercised either at the level of initiation of transcription at the promoters for r-protein genes or at the level of the stability of the r-protein mRNA. The results further suggest the existence of a second control mechanism which influences the translation of r-protein mRNA. 1. I n t r o d u c t i o n I n the bacterium Escherichia cell, the rate of ribosome production is dependent upon the nutritional composition of the growth medium. During exponential-phase growth the synthesis rates of the ribosome components are balanced so t h a t neither free ribosomal R N A nor free ribosomal proteins accumulate (Maal~e & Kjeldgaard, 1966; Dennis, 1972; Kjeldgaard & Gausing, 1974; Dennis & Bremer, 1974a; Schleff, 1967; Gausing, 1974a,b). Co-transcription of the 16 S, 23 S and 5 S r R N A contn'butes to the co-ordinate production of the three species of r R N A (Doolittle & Pace, 1970,1971 ; Pettijohn et al., 1970; D u n n & Studier, 1973; Nikolaev et al., 1973). Stilloit remains uncertain how the production of all the ribosomal proteins is co-ordinated, how the production of the r R N A s and r-proteins are balanced to each other and how the overall rate of ribosome synthesis is regulated. I t seems clear however, t h a t the tPresent address: Dept. of Microbiology, University of British Columbia, Vancouver, B.C., Canada. 61

62

P.P.

D E N N I S AND M. NOMURA

expression of both r R N A and r-protein genes is subject to the influence of the r d gene control system (Dennis & Nomura, 1974,1975). The regutatio~a of the expression of r-protein genes could be mediated either a t the level of transcription or a t the level of translation of the p r i m a r y transcripts from the r-protein genes. Recent isolation of lambda transducing phages carrying m a n y rprotein genes (Jaskunas e£ al., 1975) has provided a means to estimate directly the a m o u n t of r.protein m R N A using D N A - R N A hybridization techniques. I n this paper we have examined the nature of r-protein gene expression b y determining the a m o u n t of m R N A coding for the r-protein under a variety of steady-state and transition conditions. The results indicate t h a t the level of r-protein gene expression is controlled primarily b y a mechanism which regulates the a m o u n t of m R N A for r-protein which is available for translation. Presumably, this control is exercised a t the level of initiation of tramscription a t the promoters for r-protein genes, although a mechanism influencing the stability of r-protein m R N A cannot be excluded. I n addition, evidence is presented which suggests the existence of a second mechanism capable of influencing the translation of r.protein m R N A . This secondary translational control mechanism m a y be the fine control which ensures balanced production of r R N A and r-protein during a variety of steady-state and transition conditions.

2. Materials and M e t h o d s (a) Bacteria~ strair~, medium and conditions of growth The bacterial strains used were NF522, a leu- p y r B - derivative of E. cell B AS19 and U-32, a pyrimidinc-requiring derivative of E. coZi B/r (ATCC 12407) obtained following u.v. irradiation and penicillin selection (Davis, 1949). Bacteria were grown at 37°C in minimal MOPS medium (Neidhardt et al., 1974) eonta~n~ug [5-SH]uracil (3 ~g/ml; 10 ~Ci/ml) and leucine (20 ~g/ml) when required for growth and supplemented with one of the following energy sources: succinate (0"4%) ; ribose (0.2%) ; glycerol (0.2%) ; glucose (0.2%); glucose plus 17 amino acids (the synthetic mixture of Novick & Mass (1961), devoid of leucine, tyrosine and phenylalanine) ; glucose plus 17 amino acids plus hypoxanthine (20 pg/ml). Growth was monitored as absorbance at 460 nm in a Zeiss PMQII speetrophotometer with a 1 cm light path. Uracil supplementation to a concentration of 3/%g/ml was sufficient to support exponential-phase growth of a requiring strain to an A4eo of 0.6 to 0.7. The exponential-growth rates of the pyrimidine-requiring and parental strains were indistinguishable and growth of the requiring strain ceased inunediately upon exhaustion of the exogenous pyrimidine. Experimental cultures were started by at least a 104-fold dilution of a fresh culture grown to early stationary phase in MOPS medium supplemented with 20 ~g of nonradioactive uracil/ml. All experiments were terminated when the A4eo of the cultures was between 0.2 and 0.3 (i.e. at least one cell-doubling prior to exhaustion of the exogenous uracil). Nutldtional shift-up was accomplished by addition of glucose phm 17 amino acids to bacteria growing in succinate medium at an A460 of approx. 0.20. (b) Labeling and extraction of R1VA The pyrimidine nucleotides in RNA were radioactively labeled to homogeneity by allowing the pyrimidine-requiring bacteria to grow exponentially for at least 10 celldoublings in medium containing [5-3H]uracil as the sole pyrimidine source. Samples were harvested b y rapid cooling to 0°C in ethanol/CO2 and simultaneous addition of crushed frozen medium and sodium azide to a final concentration of 0-01 ~. Cells were concentrated by centrifugation (10,000 g, 10 min, at 0°C) and resuspended in 1 ml of lysis buffer C (containing per 1:2 g NH4C1, 6 g Na2HPOa, 3 g I~-I2PO4, 3 g NaC1 and 0.65 g sodium azide; p H 6"9).

RIBOSOMAL PROTEIN

GENE EXPRESSION

63

Lysis of the bacteria was accomplished b y transferring t h e suspension (1 ml) into a t u b e containing 1 m l of sodium dodecyl sulfate lysis m e d i u m (0.5% of sodium dodecyl sulfate, 0-1 M.NaC1, 0.01 M-disodium E D T A ) p r e h e a t e d a t 100°C (Bremer & Yuan, 1968}. The m i x t u r e was k e p t a t 100°C for a b o u t 20 s or until the suspension became clear. The lysates were cooled to 30°C a n d i m m e d i a t e l y e x t r a c t e d 3 times with equal volumes of fresh redlstiUed phenol s a t u r a t e d with T r i s / a z i d e / E D T A buffer (0.1 M-Tris, 0.01 M-sodium azide a n d 0.001 M-EDTA to p H 8.1 with HC1). The phenol phases were combined a n d re-extracted with 2 ml of Tris/azide]EDTA buffer. The NaC1 concentration of t h e combined aqueous phases was a d j u s t e d to 0.2 M a n d the nucleic acids were p r e c i p i t a t e d b y t h e a d d i t i o n of 2 vol. absolute ethanol a t --20°C. Following centrifugation t h e nucleic acids were resusp e n d e d in 2 ml Tris/azide/EDTA buffer a n d the residual phenol was removed b y extraction with ether. The Mg 2+ concentration was a d j u s t e d to 5 mM a n d t h e D N A was solubilized b y digestion with 10 ~g of D N A a s e (RNAase free} for 20 m i n a t 23°C. Disodium E D T A was a d d e d to a final concentration of 5 mM along with 100 ~g of Pronase B (Calbiochem; self-digested b y incubating in Tris/azide/EDTA buffer for 2 h a t 37°C a n d 2 m i n a t 80°C) a n d incubated for 1 h a t 37°C. R N A was then recovered following extraction with phenol a n d precipitation with ethanol as described above. The R N A was resuspended in 2 ml 2 X SSC (0"3 M-NaCI, 0"03 M-SOdium citrate} a n d e x t r a c t e d with ether. The specific radioa c t i v i t y of the R N A p r e p a r a t i o n s (cts/min per A26o nm) was determined a n d the concentration a d j u s t e d to approx. 50 ~g/ml (an A260 of 1.0 was assumed to be equivalent to 50 ~g of R N A ] m l l . R a d i o a c t i v i t y was determined b y s p o t t i n g a p o r t i o n of t h e R N A solution directly on a nitrocellulose m e m b r a n e filter (0-45 ~m pore-slze; Millipore Corp.} a n d counting in 5 ml of toluene base scintillation fluid. Portions of the R N A p r e p a r a t i o n were used in the hybridization assays described below. (e) Preparation of phage DNA D N A was p r e p a r e d from bacteriophage A a n d transducing phages ~dtrk a n d ~dspcl. Growth a n d properties of the phages have been described (Jaskunas et al., 1975}. P h a g e particles were purified b y two successive rounds of centrifugation in CsC1 density gradients. The D N A s were obtained from the phage particles b y extraction with phenol according to t h e m e t h o d described b y Z u b a y et al. (1970). F o r hybridization, the D N A s were diluted to a concentration of 10 ~g/ml in 2 X SSC buffer, d e n a t u r e d b y a d d i t i o n of N a O H to 0.3 M a n d incubated a t room t e m p e r a t u r e for 1 h. The p H was r e a d j u s t e d to 7.0 with HC1 and t h e d e n a t u r e d D N A s were immobilized b y filtration onto a nitrocellulose m e m b r a n e filter (0.45 p m pore-size; Milllpore Corp.} which h a d been soaked in 2 × SSC buffer. The filters were air dried for 1 h a n d then placed into an e v a c u a t e d dessicator a n d b a k e d a t 80°C for 3 h. These filters containing t h e immobilized phage D N A were used in the hybridization assays. (d) Hybridization assay Filters containing no D N A or D N A from bacteriophages ~, 2dtrk or ~dspcl were placed into h y b r i d i z a t i o n vials containing [SH]RNA in 2 m l 2 × SSC buffer. The vials were incu. b a t e d a t 67°C for 18 to 20 h with gentle rotation. The filters were then removed, washed with 3 × 20 ml 2 × SSC buffer a n d digested with a m i x t u r e of R N A a s e T1 a n d R N A a s e A for 40 min a t 26°C as described previously b y Pedersen & K j e l d g a a r d (1972). The filters were again washed with 4 × 20 mi 2 × SSC buffer, dried a n d counted in 5 mi scintillation fluid in a liquid scintillation counter.

3. R e s u l t s (a) Specific ribosomal protein messenger R N A during steady.state growth T h e a m o u n t o f m R N A for r - p r o t e i n i n b a c t e r i a g r o w i n g u n d e r a v a r i e t y o f p h y s i o logical c o n d i t i o n s w a s d e t e r m i n e d using a D N A - R N A h y b r i d i z a t i o n a s s a y . A uracilr e q u i r i n g m u t a n t o f E . coli B / r was g r o w n e x p o n e n t i a l l y for a t l e a s t t e n cell-doublings in f o u r different m e d i a c o n t a i n i n g [SH]uracil as t h e sole source o f p y r i m i d i n e in o r d e r

64

P. P. DENNIS

A N D M. N O M U R A

to radioactively label the pyrimidine nucleotides in RNA to a constant specific radioactivity. A constant amount of the total RNA isolat~ from each of these cultures was hybridized to various amounts of DNA prepared from bacteriophage A and transducing phages Adtr/c and Ad81vcl (Fig. 1). The Adtr/c phage carries the aroE and tr/cA bacterial genes but no detectable r-protein genes, whereas the Ad~10cl phage carries the aroE and tr/c~l genes in addition to approximately 22 r-protein genes (o )

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Fza. 1. Hybridization of constant amounts of t o t a l R N A to increasing a m o u n t s of A, Adtr/c a n d A d ~ c l DNA. Total cellular R N A was isolated from E. coZi B / r U-32 growing in media supplem e n t e d with [gH]uraoil a n d sueeinate (•), glycerol (b), glueose (e) or glueose plus a m i n o acids (d). G r o w t h rates in doub]Lings/h were 0-65, 1-05, 1.36 a n d 2.0 in t h e 4 respective growth media. T h e speo. aet. of t h e R N A preparations were 6 . 8 0 x l 0 s ets/min p e r ~12e0 a n d t h e i n p u t R N A radioactivities per hybridization assay were 1.72x 106, 1.20x l0 s, 1.80x l 0 s a n d 1.30x l 0 s ets/min

RIBOSOMAL P R O T E I N GENE E X P R E S S I O N

65

(Jaskunas et al., 1975). The proteins coded for by the genes known to be on the phage include $3, $4, $5 (spc gene product, $7, $8, S l l , S13, S14, L5, L6, L l l , L13, L14, L15, L16, L17, L18, L19, L22, L23, L24, and L30). This represents about 400,000 daltons of protein or about one haft of the total ribosomal protein. The amount of RNA hybridizing to the ~dtrb DNA was only slightly greater than the amount hybridizing to the ~ DNA indicating that the aroE-trbA region of the bacterial chromosome has a relatively low transcriptional activity. In contrast, the amount of RNA hybridizing to )~dspcl DNA was considerably greater t h a n the hybridization to ~dtrb DNA and indicates that the unique DNA carried by the ~dspcl phage has a relatively high transcriptional activity. Presumably, this active material represents exclusively r-protein genes. Thus the difference between hybridization to ~ d ~ c l DNA and to )~dtrb DNA represents in all likelihood the m R N A homologous to the r-protein genes carried b y the transducing phage (see below) and will be termed "specific r-protein m R N A " in this paper. As mentioned above, specific r-protein m R N A probably represents about haft of the total mRNA for r-protein present in the RNA preparation. The final values of the plateaux reached at infinite DNA concentrations represent the fractions of the respective total RNAs which are homologous to the r-protein genes carried on the phage (i.e. the specific r-protein m R N A fraction). These values were determined by extrapolation to infinite DNA concentration and gave values for the specific r-protein mRNA fraction of 0.235~, 0.304~/o, 0.370~ and 0.480~/o for bacteria grown in the succinate, glycerol, glucose or glucose-amino acids media respectively. A more accurate determination of the relative relationship between the specific r-protein m R N A fraction and the bacterial growth rate was obtained b y hybridizing increasing amounts of RNA to a constant and excess amount of DNA (see Appendix and Fig. 2). The specific hybridization was again obtained as the difference between radioactivity hybridized to ~dspcl DNA and to ~dtr~ DNA. The relative fraction of specific r-protein m R N A in the RNA prepared from bacteria growing at the four respective growth rates was obtained from the slopes of the specific hybridization curves (see Appendix and legend to Table 1). I t was found t h a t the fraction of the total RNA which is specific r-protein m R N A increases from 0.172 to 0.370 over the range of steady-state growth rates from 0.65 to 2.0 doublings per hour (Table 1). Similar experiments were carried out using RNAS, which were also homogeneously labeled with [all]uracil, from both E. cell B/r-U23 and 1~F522 growing in a variety of nutritionally supplemented growth media which supported rates of steady-state

for the 4 respective growth conditions. The hybridization assays (left panels) were done in a total eel. of 2 ml of 2 × SSC and contained a blank filter (subtracted from other values) and separate filters containing equal amounts of ADNA (C)), ~td~rbDNA (@) and ~tdspcl DNA ( × ). The specific hybridization (right panels) was calculated as the difference between radioactivity hybridized to ~depcl DNA filters minus radioactivity hybridized to ~tdtrb DNA filters. The specific r-protein mRNA fraction of the total RNA was determined from the double reciprocal plot of I/specific hybridization and 1/DNA concentration. The radioactivity in specifichybrids at infinite DNA concentration was estimated to be 400, 364, 666 and 625 ots/min and corresponds to 0.234%, 0.304%, 0.370% and 0.48% of the total radioactivity in the respective input RNAs. A t a concentration of 20 pg of Adspcl DNA per filter, the radioactivities in Adspcl specific hybrids in the 4 respective input RNAs were about 300, 230, 500 and 470 ors/rain and correspond to 75% of the radioactivity in specific hybrids observed at infinite DNA concentration.

P. P. DENNIS

66

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Fz6. 2. Hybridization of increasing amounts of total R N A to constant amounts of ~, ~dtr~ and ~dapcl DNA. Increasing amounts of the [SH]RNA preparations described in the legend to Fig. 1 were hybridized to constant amounts of ~, ~dtr~ and ~dalvcI DNA. The A~e o of R N A preparations from bacteria grown in sueeinate (a), glycerol (b), glucose (c) and glucose plus amino acids (d)

RIBOSOMAL PROTEIN GENE EXPRESSION

67

TABL~ 1

De~e~vaination of the fraction of the total input R N A which specifically hybridizes to the ribosomal protein genes carried by the Adspcl transducing

phage Growth medium

Suoclnate Glycerol Glucose Glucoseamino acids

Growbht rate

Specific hybrids$ (cts/min)

0.65 1.05 1,36 2.0

1940 2440 3480 4260

Specific Input RNA~ Specific hybrids/§ r-proteinll Total input (cte/min × 10 -6) RNA ( X 10 -s) mRNA(%) fraction 1.50 1.39 1.60 1.54

1.29 1.76 2.18 2.77

0.172 0.234 0.290 0.370

t Growth rates in doubling per hour. ~:Ct~/min in specific hybrids, H, and eta/rain in input RNA, R, calculated for an input of 250 tzl of RNA/assay (Fig. 2). The A2eo of the RNAs prepared from bacteria grown in suceinate, glycerol, glucose and glucose-amino acids were 0.890, 0.825, 0.951 and 0-912, respectively, while the specific radioactivity of the RNAs was 6.80 × 106 eta/rain per A28o. § The ratio of the radioactivity in specific hybrids to total input radioactivity gives the relative fraction of specific r-protein mRNA in the various preparations. That is, according to equation (4d) of the Appendix, 1 H 8-K R' where H is radioactivity in ~dspcl-specifie hybrids, R is radioactivity in input RNA, 8 is th0 fraction of the total input RNA which is specific r-protein mRNA, K is a constant, the numerical value of which depends on the DNA concentration used in the hybridization assays. The value of the constant K was taken to be 0.75 since, in the experiment illustrated in Fig. 1, 75% of the specific r-protein mRNA was hybridized at a hdspcl DNA input of 20 ~g/assay. However, in separate experiments the value of K was estimated to be between 0-86 and 0.88 by carrying out a second round of hybridization with the supernatant RNA not initially hybridized by 2, ~dtrk and hdspcl DNAs, as described in the legend to :Fig. 2. Thus, our values for the fraction of specific r-protein mRNA may contain a 10 to 20% systematic overestimation error. s is the specific r-protein mRNA fraction of the total input RNA. No attempt has been made to correct for possible differences in the pyrimidino content of total RNA and specific r-protein mRNA.

gro~rth r a n g i n g from 0.48 to 2.10 d o u b l i n g s per hour. T h e results are s u m m a r i z e d i n F i g u r e 3. T h e observed g r o w t h r a t e d e p e n d e n c y of s, t h e f r a c t i o n of t h e t o t a l R N A which is specific r . p r o t e i n m R N A , c a n be described b y t h e r e l a t i o n s h i p : s = 0.0016/~ + 0.0006, where/~ is t h e g r o w t h r a t e i n d o u b l i n g s per hour. T h u s a t a growth r a t e of 1-0 d o u b l i n g per h o u r specific r - p r o t e i n m R N A a c c o u n t s for 0-22o//0 o f t h e t o t a l cellular R N A , a n d t o t a l r - p r o t e i n m R N A a c c o u n t s for a b o u t 0-44% of t o t a l cellular R N A . was 0.890, 0.825, 0-915 and 0-912, respectively, and the spee. act. of the RNAs was 6.80× 106 ctsflnin per Aa6o. Each hybridization assay was done in 2 ml 2 × SSC and contained 10 filters: 2 blanks (subtracted from other values), 2 containing 5 t~g A DNA (O), 2 0ont~inlng 5 pg Ad~rk DNA (@) and 4 containing 5 ~Lg Adspel DNA (x). The specific hybridization (- . . . . ) was obtained as the difference between hybridization to Ad~rb filter and to ~ p c l filter. The total radioactivity in specific hybrids was 4 times the difference (i.e. accounting for the 4 separate Adapcl filters each containing 5 pg DNA).

68

P. P. D E N N I S

AND

M. N O M U R A

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FrG. 3. Specific r-protein mRNA fraction as a function of the steaxly-state growth rate. The fraction of the total RNA which is specific r-protein mRNA was determined as described in the legends to Fig. 2 and Table 1. RNA was prepared from exponential-phase cultures of E. coli B/r growing at rates ranging from 0-65 to 2-1 doublings/h ( A, O)- Independent pre-shfft and 100-rain post-shift RNA samples from exponential.phase cultures growing in succinate medium and shifted to glucose plus amino acids medium are also given ([]). Similar experiments using strain NF522, growing at steady-state rates ranging from 0.48 to 2.0 doublings/h are also included ( X ). In control experiments it has been demonstrated t h a t the homology between Adspcl DNA and total ell-labeled stable RNA (rRNA and t R N A ; bacteria were labeled with [ell]uracil for one cell-doubling and chased with an excess of non-radioactive uracil for two cell.doublings) is less than 0.003~. This amount of hybridization with stable RNA is at least 50-fold less than the corresponding specific hybridization observed with total [SH]RNA (rRNA, t R N A and mRNA) isolated from bacteria growing in succinate minimal medium (F = 0.67 doubling/h). The small amount of residual hybridization most probably represents homology with bacterial DNA fragments from the r-protein genes which survive the I)NAase treatment and contaminate the RIqA preparations, rather than homology with stable RNA species. Additional experiments using DNA from the Adspc2 phage (Jaskunas e~ at., 1975) and a deletion m u t a n t of Ad8toc2, Adspc2-A9, which removes about 60~/o of the r-protein genes normally carried on the parental phage (Jaskunas & Nomura, unpublished data), have been carried out. The amounts of specific hybridization to these phages (i.e. RNA hybridized to these phage DNAs, corrected for RNA hybridized to Adtrk DNA) increase linearly ~4th increasing growth rate and extrapolate to a positive value on the abscissa at zero growth rate. A similar linear relationship with growth rate has also been observed with respect to the amount of RNA homologous to the I)NA deleted by the A9 deletion (i.e. the amomlt calculated as the difference between RNA hybridized to Adspc2 DNA and that to AdsTc2-A9 DNA). The /19 deletion is in the middle of the r-protein gene cluster, and has r-protein genes remaining on both sides of the deletion. The physical size of the deletion is approximately what one would expect from the number of r-protein genes deleted (Jaskunas & Nomura, unpublished data) and thus the RNA homologous to the deleted DNA segment almost

RIBOSOMAL P R O T E I N GENE E X P R E S S I O N

69

certainly represents exclusively r-protein mRNA. These control experiments thus confirm the general growth-rate dependency of the a m o u n t of specific hybridization with the 2dspcl DNA observed in Figure 3. Furthermore, the results also support the assumption t h a t R N A analyzed b y the specific hybridization method using ~dspcl phage is almost entirely r-protein mRNA. (b) Specific ribosomal protein messenger RNA following a shift.up A nutritional shift-up from succinate to glucose plus amino acids medium results in a rapid induction in the synthesis of ribosomal protein (Schleif, 1967; Gullov et aL, 1974; Dennis, 1974b; Gansing, 1974b). T h a t is, the differential synthesis rates of r.protein, ~r (synthesis rate of r-protein/synthesis rate of total protein), increases approximately twofold or more within the first five to ten minutes following the shift-up. The level of specific r-protein m R N A was examined during this transition period to determine if induction after a shift-up correlates with a simultaneous increase in the amount of r-protein mRNA. Bacteria were grown in succinate minimal medium (/~ = 0.67 doubling/h) containing [3H]uracil in order to label homogeneously the pyrimidine nucleotides in RNA. At zero time glucose plus amino acids were added and at subsequent times samples were removed for R N A preparation and hybridization. As shown in Figure 4, the fraction of specific r-protein m R N A was observed to increase gradually from the pre-shfft value of approximately 0.17°/o to a value of approximately 0'38~/o b y five to six minutes after the shift-up. After six minutes, the amount of specific r-protein m R N A remained essentially constant or decreased slightly. Thus, by five to six minutes

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70

P . P . D E N N I S AND M. NOMURA

following the shift, the level of specific r-protein m R N A has reached a value a t or near the final post-shift steady-state level of 0-37 to 0-41%. (In these experiments the post-shift steady-state growth rate was 2.1 doublings/h.) The relative differential synthesis rates of individual r-proteins at various times following the nutritional shift-up have been d e t e r m i , e d (Dennis, 1974b). B y combining the measurements for the specific r-proteins coded for b y the bacterial genes carried on the Ad~sloclchromosome, the relative differential synthesis rate of specific r-protein, was obtained (Mg. 5). Following a lag of about one minute, this differential rate increased rapidly to a maximum value nearly 2.6.fold above the pre-shift level before dec]Jnlng to a value near the post-shift steady-state level. The observed differences between the differential synthesis rate of specific r-protein coded for b y genes carried on the Adspcl phage (Fig. 5) and the fraction of specific r-protein m R N A (Fig. 4) during the transition period immediately following the shift-up could possibly be the result of some unl~nown anomaly which interferes with the accuracy of the measurements. Conversely the differences m a y be of physiological significance (see Discussion, section (c)). //

/'\

Suc¢inate Glucose-amlnoacids

-

2.5

0.20 A

2.0

/

0.15 -

°o o

0'I0 -- - -

,nu

.e~ e

•

I'0 ~

0'05 -

o

0"5

o

I 2

!

I

I

4 6 8 Time after shifl-up (rain)

I I0

/

!

o

F m . 5. The differential synthesis rate of specific ribosomal protein following a nutritional shiftup. The relative differential synthesis rates of the specific r-proteins coded for by genes carried on the phage Adspcl (@) were calculated by s,mm~tion of the contribution of each of the individual r-protein ~ values at the various times after the shift-up (data from Dennis, 1974a) and weighed for molecular weight and mole fraotion of leueine differences (Dzionara e~ a2., 1970; Kaltsehmldt e~ ed., 1970). Also included are the steady-state ~r values ( n ; Dennis & Bremer, 1974a) and the pre-shift and 100-rain post-shift relative = values (A; Dennis, 1974b).

4. D i s c u s s i o n (a)

Ribosomal protein messenger RNA fraction during steady-state growth

The hybridization experiments summarized in ~ g u r e 3 indicate t h a t the fraction of the total RNA which is specific r-protein mRNA, s, varies as a linear function of the growth rate,/~. T h a t is, 8 ---- 0.0016/~ + 0.0006. These measurements are somewhat lower than the values obtained from the less precise experiments illustrated in Figure 1 and in addition, m a y contain a systematic error if the proportion of specific

RIBOSOMAL

PROTEIN

GENE

71

EXPRESSION

R N A entering into D N A - R N A complexes is either greater or less t h a n 75~/o under these conditions of hybridization (see the footnote to Table 1). Throughout these experiments we have not attempted to distinguish between the structural and functional activity of r-protein m R N A ; t h a t is, we have assumed t h a t the m R N A which hybridizes to ;~dspcl DNA is also active as a template in protein synthesis. The specific r-protein m R N A represents about one half of the total r-protein mRNA. I f all mRNAs are translated indiscriminately and have the same average life-time at a particular growth rate, the amount of total m R N A can be calculated directly from the differential rate of r-protein synthesis, ~r, and the fraction of specific r-protein m R N A (Table 2; Dennis & Bremer, 1974a; Dennis, 1974a). The estimates o f the amount of m R N A range from about 3.5 to 4"5~o of the total cellular RNA.

TABLE 2

Estimation of the fraction of the total cellular RNA which is messenger RNA Growth medium Growth rate (doublings/h) Fraction of spee. r-protein mRNA~ (% of tot~l RNA) Diff. rate of r-protein synthesis (~r)$ Total mRNA (% of total RNA) §

Succinate

Glycerol

Glucose

Glucoseamino acids

0.67

1.05

1.30

2.i0

0-167

0.227

0-278

0:396

0.092

0-106

0-122

0.189

3.63

4.20

4.56

4.17

The fraction of specific r-protein mRNA was obtained from Fig. 3. ~:The differential rates of r.protein synthesis ~ (synthesis rate of r-protein/synthesis rate of total protein} is from the data of Dennis & Bremer (1974a). § Total mRNA was calculated from the fraction of total RNA which is specific r-protein mRNA, and the differential rate of total r-protein synthesis and assuming that specific r-protein accounts for 50% of the total r-protein and that all mRNA (r-protein mRNAs as well as nonr-protein mRNAs) are translated with equal efficiency at a particular steady-state growth rate. ~ (b) Regulation of ribosomal protein gone exlrression

during steady.state growth I n the bacterium E. coli B/r, the amount of stable R N A per genome increases in proportion to the steady.state growth rate and rRNA represents a nearly constant fraction of the total cellular R N A (Maaloe & Kjeldgaard, 1966; Dennis, 1972; Dennis & Bremer, 1974b; Kjeldgaard & Gansing, 1974). This means t h a t the amount and the rate of synthesis of rRNA must increase, respectively, in proportion to the growth rate and in proportion to the second power of the growth rate. Furthermore the specialized transducing phage ~dspcl carries at least 22 r-protein genes (Jaskunas etal., 1975) which account for an aggregated mass of about 400,000 daltons of protein or half of the total protein per 70 S ribosome. I t is thus possible to ealcu], ate both the synthesis rate of ribosomes and the synthesis rate of specific r-protein from the ratio of RNA/DNA (Table 3). Comparison of the specific r-protein synthesis rate and the amount o£ spe¢iltle

P. P. DENNIS

72

A N D M. N O M U R A

TABLE 3

Synthesis rate of specific ribosomal protein and the amount of 8pecifio ribosomal protein mRI~A Growth medium

Succinate

Glycerol

Glucose

Glucoseamino acids

G r o w t h r a t e (doublings]h) RNA/DNA ~ (nucleotides/genome) Ribosome synthesis rate b (ribosomes]rain p e r genome) Spee. r-protein synthesis r a t e ° (amino acids]min per genome) F r a c t i o n of spec. r-protein mRNA d ( % of total RNA) A m o u n t of spee. r-protein mRNA • (nueleotides/genome) ( m R N A eopies/genome) Translation]spat. m R N A f (assuming a n av. life-time of 2 min) Av. life.time of spot. r-protein m R N A (rain) ~ (assuming 20 translation/spot. mRNA)

0.67 2-14 × l 0 T

1.05 3.35 × l 0 T

1-36 4-34 x l 0 T

2-10 6.71 × 10 ~

30.4

74.4

1.09 x 16s

2.68 × l 0 s

4-50 x 105

10.7 × 105

0.167

0.227

0.278

0.396

3.57 X 104 3.25

7.61 x 104 6.92

12.1 X 104 11.0

26.6 × 104 24.2

18.4

21-2

22.4

24.2

2.16

1.90

1.79

1.66

125

297

a I n E. co//B]r t h e R N A / D N A ratio increases in proportion to the growth rate, p, in doublings/h. The factor of proportionality was estimated to be 4.2 (Dennis, 1972; Dennis & Bremer, 19745). Furthermore, a genome equivalent of D N A contains 7.6 x 106 nucleotides. Thus as a sample of t h e calculation for bacteria growing in succinate minimal m e d i u m (p = 0.67), t h e R N A ] D N A is 4.2 X /~ x 7.6 X 10e=2"14 X 107 nucleotides]genome. The fraction of t h e t o t a l R N A which is r R N A is a nearly c o n s t a n t fraction (,-,0.85) independ e n t of the growth rate (Dennis, 1972; Dennis & Bremer, 1974b) a n d t h e 70 S ribosome contains a b o u t 4650 nucleotides- of RNA. Thus for p ~- 0.67, t h e ribosome synthesis rate is 2 . 1 4 x l 0 T x 0-85 x (ln 2]60) X p X (114650) = 30.4 ribosome/rain per genome. QThe synthesis rate of specific r-protein was o b t a i n e d from t h e ribosome synthesis rate a n d t h e assumptions t h a t specific r-protein accounts for approximately h a l f of t h e t o t a l r-protein (i.e. approx. 400,000 daltons o u t of 800,000 daltons of t o t a l r-protein) a n d t h e average molecular weight of t h e amino acid In r-protein is 112 daltons. Thus t h e synthesis r a t e of specific r-protein is 30-4x800,000 x ( 1 1 2 ) x (11112) ~ 1-09 X l 0 s amino acids/rain per genome a t / ~ = 0-67. T h e fraction of t h e t o t a l R N A which is r-protein-specific m R N A , s, is obtained from t h e relationship: s ~ 0-0016 p ~- 0.0006 (Fig. 3). F o r p ~- 0.67, e = 0-167%. e T h e a m o u n t of specifie r-protein m R N A is t h e p r o d u c t of t h e R N A / D N A ratio (nucleotides] genome) a n d t h e fraction of t h e specific r-protein m R N A . T h a t is t h e a m o u n t of specific r-protein m R N A is 2-14x l 0 T x 0.00167= 3.57 X 104 nueleotides]genome. F u r t h e r m o r e , since 11,000 bases In m R N A are required to code for t h e 400,000 daltons of specific r-protein, t h e n u m b e r of specific m R N A copies a t p = 0-67 is 3.57 × 104x (1]11~000) = 3.3 m R N A copies/genome. t The translational efficiency of specific r-protein m R N A is t h e p r o d u c t of synthesis r a t e of specific r-protein x 3 x average life-time of r-protein-specific m R N A × ( I / a m o u n t of r-proteinspecific m R N A ) (Dennis & Bremer, 1974b). F o r a n average life-time of 2 rain t h e translational effieiensy a t p ---- 0.67 is 1-09 × l0 s X 3 × 2 X (113.57 × 104) = 18.4 translations per copy of specific r-protein m R N A . Alternatively, if t h e average life-time of t h e m R N A is variable a n d t h e n u m b e r of translations per r-protein m R N A during its life-time is c o n s t a n t (assumed to be 20 translations] m R N A ) t h e same relationahlp can be used to calculate t h e average life-time. T h a t is, the average life-time is 3.57 × 10 ~ x 20 × 1](1.09 × 105 × 3) ~ 2,16 rain. Neither t h e n u m b e r of translations n o r t h e average life-time of t h e specific m R N A is known; only t h e a m o u n t of r-protein-specific m R N A is known. Thus it remains unclear whether one, or both, of these u n k n o w n parameters is ~ubject to growth-rate-related variations.

RIBOSOMAL PROTEIN GENE EXPRESSION

73

r-protein mRNA (Table 3) clearly indicates that the level of expression of the specific r-protein genes is regulated primarily by a mechanism which determines the amount of r-protein-specific mRNA. That is, over this growth-rate range, where the specific r-protein synthesis rate per genome equivalent of DNA increases 9.7-fold, the level of specific r-protein mRNA per genome equivalent of DNA increases 7.5-fold. The increase in the amount of specific r-protein mRNA at the higher growth rates results from either an increase in the frequency of initiation of transcription at r-protein gene promoters or from an increase in the stability of specific r-protein mRNA or both. The difference between the 9.7-fold increase in the synthesis rate of specific r-protein per genome and the 7.5-fold increase in the level of specific r-protein mRNA per genome over this growth-rate range suggests the existence of a secondary mechanism which also influences the level of r-protein gene expression. This secondary mechanism influences either the average life-time or the translational efAcieney (the number of times an mRNA is translated during its life-time) of the specific r-protein mRNA or both (Table 3). I t should be noted however, that the need to postulate a secondary control mechanism affecting the translation of r-protein mRNA would be e]jmluated if our measurements of the specific r-protein mRNA fraction contain an overestimate amounting to 0.0006 of the total cellular RNA under all conditions (i.e. 8 ---- 0.0016/~ rather than the observed relationship: 8 -----0.0016/~ ~- 0.0006). (c) Ribosomal proSein ~nessenger R.tCA frac~or~ d~ring transitio~

between steady states of growth Within minutes following a nutritional shift-up from succinate to glucose plus amino acids medium, the exponential synthesis rate of ribosomes, and therefore that of rRNA and r-protein, increases about threefold to a value at or near the final post-shift steady-state rate (Maal~ & Kjeldgaard, 1966; Schleif, 1967; Dennis & Bremer, 1974c; Gausing, 1974b). The exponential synthesis rate is defined simply as the percentage increase in the respective cellular component per unit time; for a more rigorous definition, see Dennis & Bremer (1974v). The increased level of expression of the r-protein genes following the shift-up results primarily from an increase in the fraction of the t o t ~ RNA which is mRNA for r-protein, although a small increase in the stability and]or translational efficiency also makes a contribution. This means that the threefold increase in the exponential synthesis rate of r-protein is accompanied by a 2.3-fold increase in the fraction of specific r-protein mRNA (Fig. 4) and that the r-protein mRNA is translated more efficiently in the post-shift growth medium (see Discussion, section (b)). Again, the increase in the amount of specific r-protein mRNA would reflect an increase in the frequency of initiation of transcription at r-protein gene promoters if the average life-time of the r-protein mRNA is essentially independent of the bacterial growth rate. During the transition period following the shift-up, the ribosome efficiency and consequently the total protein synthesis rate increases 30 to 40% (Dennis & Bremer, 1974a; Gausing, 1974b). Coupled with a 2-1-fold increase in ~r from 0.09 to 0.19, this accounts for the approximately threefold increase in the exponential sydthesis rate of r-protein in the post-shift growth conditions. However, as described in Results (see Fig. 5), the relative ~ values for the specific r-proteins coded for by genes carried on the )tdspcl phage increase to a greater extent than the amount of specific r-protein mRNA during this period and reach a maximum nearly 2.6-fold above the pre-shift

74

P.P.

DENNIS AND M. NOMURA

value before declining again to near the post-shift steady-state value of 2.1. Oscillatory patterns in the ~ values for total r-protein and individual r-proteins following nutritional shift-up have also been observed (Gausing, 1974b; Dennis, 1974b). In contrast, the fraction of specific r-protein mRNA seems to increase rapidly to near the post-shift steady-state value and remain essentially constant thereafter. These observations suggest that the specific r-protein mRNA is transiently translated more efficiently during the brief interval between four and six minutes following the shift-up. The nature of this phenomenon is unclear; possibly it may be related to a mechanism required for maintenance of a balance between the synthesis rates of rRNA and r-protein. For instance, if rRNA synthesis responds more rapidly than r-protein synthesis (due to a lag either in the small increase in the ribosome efficiency or in the response of ~r), the pools of free r-protein would be depleted. The balance could be restored by a mechanism which senses the level of free r-protein and/or free rRNA and which actively promotes ribosome binding to specific r-protein mRNA. Alternatively, the phenomenon may result simply from a transient decrease in the level of total mRNA during this period, and hence a transient increase in the frequency of translation of all mRNA including r-protein mRNA. In summary, the level of expression of r-protein genes during steady-state growth and following a nutritional shift-up is determined primarily by a control mechanism which regulates the amount of r-protein mRNA. Presumably this mechanism operates at the level of initiation of transcription at the r-protein gene promotors, although it cannot be excluded that the mechanism may operate at the level of mRNA stability. We are currently investigating these alternatives. The data also suggests an additional control mechanism which affects the translation of r-protein mRNA. The influence of this secondary mechanism(s) seems to be exemplified during the transition period following a nutritional shift-up and may possibly be of importance in maintaining the fine balance between rRNA and r.protein production.

APPENDIX L e t D ----concentration of DNA; R ----concentration of RNA; s R ----concentration of specific RNA (i.e. s is the fraction of the total RNA which specifically hybridized to the DNA); and H = concentration of the observed DNA-RNA hybrid. The rates of formation and dissociation of the DNA-RNA complex are given by the relationships:

At equilibrium vl

=

v2

v~ = k x ( D - - H ) ( S R - - H )

(1)

v2 = k2(H).

(2)

and ( D -- H ) ( s R -- H ) =

~sH.

(3)

Under the conditions of hybridization used here, the DNA is present in such an excess over RNA that the amount of I)NA in the hybrid is negligibly small compared to the amount of free DNA. That is D~_D--H. Using this relationship, equation (3) simplifies to the form D'sR

-- D'H

= k3H

(4)

RIBOSOMAL P R O T E I N GENE E X P R E S S I O N

75

and rearranging 1

1 ks = a"R ~ D ' s R "

(4a)

Thus from equation (4a) it is clearly evident t h a t a double reciprocal plot of 1/1t versa8 1/19 should give a linear function t h a t extrapolates to a value of 1/sR as 1) -> oo (and 1/1) -~ 0). F r o m a plot of this type it is also possible to obtain the fraction of the specific R N A which is present in D N A - R N A hybrids at various DNA concentrations. Equation (4) can also be written

I n the experiments illustrated in Figure 2 (main text) 1) is held constant at 20 pg of ~dspcl DNA per assay and in large excess over sR, and only R is varied. Thus equation

(4b) can be rewritten (4c)

H = K'sR,

where K is a constant whose numerical value depends on the concentration of DNA used in the experiments. The observed linear relationship between H and ~ supports the validity of the above equation and the initial assumption stated in equation (3) (i.e. t h a t DNA is present in excess). ~rom the experimental results shown in Figure 1, (main text), it is estimated t h a t at 20 ~g of ~dsl~cl DNA per assay, approximately 75% of the specific r-protein mRNA is in D N A - R N A hybrids at equilibrium; t h a t is, the value of the constant K is 0.75 under these conditions. Thus the fraction of the total R N A which is specific for the ~dslacl DNA, 8, can be calculated from the slope ( H [ R ) of the curves in Figure 2, according to the relationship: 1 H 8=~.~=0.75

1

H R

(4d)

We thank KaCZaleenRyan for her assistance in preparing the h transducing phages and Dr John Ingversen for his helpful suggestions in setting up the DNA-RNA hybridization assay. We also thank Drs J. Dahlberg, L. Lindahl and S. R. Jaskunas for their valuable suggestions. This work was supported in part by research grants, GM20427 from the National Institute of General Medical Science (U.S. Public Health Service) and GB31086 from the National Science Foundation. This is a Laboratory of Genetics publication, i.e. publication no. 1828 from the Laboratory of Genetics, University of Wisconsin, Ma~iison. REFERENCES Bremer, H. & Yuan, D. (1968). J. Mol. Biol. 38, 163-180. Davis, B. (1949). Proc. Nat. A c ~ . Sci., U.S.A. 35, 1-10. Dennis, P. (1972). J. Biol. Ghem. 247, 2842-2845. Dennis, P. (1974a). J. MoL Biol. 88, 25-40. Dennis, P. (19745). J. MoL Biol. 89, 223-232. Dennis, P. & Bremer, H. (1974a). J. Mol. Biol. 84, 407-422. Dennis, P. & Bremer, H. (19745). J. Bacteriol. 119, 270-281. Dennis, P. & Bremer, H. (1974c). J. Mol. Biol. 89, 233-239. Dennis, P. & Nomura, M. (1974). Proc. Nat. Acad. Sci., U.S.A. 71, 3819-3823. Dennis, P. & Nomura, M. (1975). Nature (London), 255, 460-465. Doolittle, W. & Pace, N. (1970). Nature (London), 228, 125-128. Doolittle, W. & Pace, N. (197t). Proc. Nat. Acad. Sci., U.S.A. 68, 1786-1790.

76

P.P. DENNIS

AND

M. N O M U R A

Dunn, J. & Studier, F. W. (1973). Proc. Nat. Acad. Sci., U.S.A. 70, 3296-3300. Dzionara, M., Kaltschmidt, E. & Wittmann, H. (1970). Proc. Nat. Acad. Sci., U.S.A. 67, 1909-1913. Gausing, K. (1974a). Mol. Gem Genet. 129, 61-75. Gausing, K. (1974b). Degree of Licentiatus Scientarum Thesis, University of Copenhagen. GLfllsv, K., Meyenburg, K. & Molin, S. (1974). Mol. Gem Gent. 130, 271-274. Jaskunas, S. R., Lindahl, L. & Nomura, M. (1975). Pros. Nat. Aead. S0i., U.S.A. 72, 6-10. Kaltschmidt, E., Dzionara, M. & Wittmann, H. (1970). Mol. Gen. Genez. 109, 292-302. Kjeldgaard, N. & Gausing, K. (1974). I n Ribosomes, Gold Spring Harbor Laboratory, Cold Spring Harbor, New York. Maaloe, O. & Kjeldgaard, N. (1966). In Gontrol of ~lacromolecular Synthesis, Benjamin, New York. Neidhardt, F., Bloch, P. & Smith, D. (1974). J . Bact~riol. 119, 736-747. Nikolaev, N., Silengo, L. & Schlossinger, D. (1973). Proc. Nat. Acad. Sol., U.S.A. 70, 3361-3365. Novick, R. & Maas, W. (1961). J . Bact~riol. 81, 236-240. Pedersen, S. & Kjeldgaard, l~. (1972). lllol. Gen. Genet. 118, 85-91. Pettijohn, O., Stonington, O. G. & Kossmann, C. (1970). Nature (London), 228, 235-239. Schleif, R. (1967). J . Mol. Biol. 27, 41-55. Zubay, G., Chambers, D. & Cheong, L. (1970). I n The Lactose Operon, Cold Spring Harbor Laboratory, Gold Spring Harbor, New York.