Vol. 129, No. 2 Printed in U.S.A .
JouItNAL oF BACT3i1OLOGY, Feb. 1977, p. 580-688 Copyright 0 1977 American Society for Microbiology
Influence of the Stringent Control System on the Transcription of Ribosomal Ribonucleic Acid and Ribosomal Protein Genes in Escherichia coli PATRICK P. DENNIS Department of Microbiology, University of British Columbia, Vancouver, British Columbia, Canada V6T 1 W5 Received for publication 30 August 1975
The fraction of the total ribonucleic acid (RNA) synthesis rate that is messenger RNA (mRNA) for ribosomal protein (r-protein) and ribosomal RNA (rRNA) has been estimated in valS(Ts) rel+ stringent and valS(Ts) relAl relaxed strains of Escherichia coli during a partial inhibition of valyl-transfer RNA aminoacylation. The partial inhibition was accomplished by shifting the strains from the permissive growth temperature of 29.50C to the semipermissive temperature of 35.50C. The RNA synthesized at the elevated temperature was pulse labeled with [3H]uracil. The fraction of the total incorporated 3H radioactivity in r-protein mRNA or in rRNA was estimated by specific hybridization to the transducing phages Aspcl, which carries about 15 r-protein genes, and Xilv5, which carries an rRNA transcription unit. The results clearly demonstrate that the rel gene influences the fraction of the total RNA synthesis rate that is rprotein mRNA and rRNA; in the rel+ strain these fractions are significantly decreased, and in the relAl strain they are significantly increased relative to control cultures. This indicates that the expression of the genes coding for the RNA and protein component of the ribosome are most likely regulated at the level of transcription. Furthermore, it appears that the distribution of functioning RNA polymerase between rRNA genes, r-protein genes, and other types of genes is influenced by the rel gene control system; presumably this influence is mediated through the unusual nucleotide guanosine tetraphosphate. In the bacterium Escherichia coli, the stringent control system regulates the production of the ribonucleic acid (RNA) and protein components of the ribosome (6, 8, 25). Amino acid or charged transfer RNA (tRNA) deprivation of rel+ stringent strains results in accumulation of guanosine tetraphosphate (ppGpp) and in reductions (i) in the rate ofaccumulation of stable RNA, (ii) in the amount of ribosomal protein (rprotein) messenger RNA (mRNA), and (iii) in the differential rate of r-protein synthesis (defined as r-protein synthesis rate/total protein synthesis rate). In contrast, amino acid deprivation of relA1 strains results (i) in a failure to accumulate ppGpp, (ii) in unabated accumulation of stable RNA, and (iii) in an elevation in the amount of r-protein mRNA and in the differential rate of r-protein synthesis (4, 6, 8, 14, 25). The precise mechanism whereby the stringent control system regulates RNA synthesis is the subject of controversy (11). Initially it was assumed that amino acid control over RNA synthesis was attributable to an arrest of initiation 580
that affected both stable RNA and mRNA transcription. It was subsequently shown that the synthesis of general and specific mRNA's (i.e., trp mRNA and lac mRNA) was essentially normal during amino acid deprivation in rel+ and relAl strains (13, 15, 19, 22, 23). Conversely, the accumulation of r-protein mRNA, like the accumulation of stable RNA, has recently been shown to be subject to stringent control (8). The influence of the amino acid control system on the accumulation of these RNAs could conceivably be mediated at the level of transcription as originally suggested or at the level of stability of the respective transcripts (10). In this paper, the effect of the stringent control system on the expression of rRNA and rprotein genes is examined by using RNA-deoxyribonucleic acid (DNA) hybridization (7, 9). The study uses isogenic rel+ and relAl strains carrying a temperature-sensitive mutation in the valyl-tRNA synthetase gene. A partial restriction of valyl-tRNA aminoacylation (and protein synthesis) results in elicitation of either the stringent or relaxed response and is
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achieved by shifting the respective rel+ and relAl cultures from a permissive growth temperature to a semipermissive growth temperature (6, 14). The rate of rRNA and r-protein accumulation and consequently ribosome biosynthesis is reduced in the rel+ strain at 35.50C relative to the control cultures; the rate of rRNA and r-protein accumulation and consequently ribosome biosynthesis is increased in the relAl strain relative to the control cultures. After these initial adjustments in the rates of ribosome biosynthesis, the temperature-sensitive strains maintain a more or less balanced condition of growth for some time at the semipermissive growth temperature of 35.50C, and protein synthesis continues at about 60 to 70% of the non-temperture-sensitive control rate (6). These conditions thus minimize any secondary and nonspecific effects on the expression of stable RNA and mRNA genes that might result from prolonged periods of severe amino acid deprivation. The transcriptional activity of rRNA and r-protein genes was estimated under these conditions by measuring the fraction of a pulse label that enters into rRNA and r-protein mRNA, respectively. (The transcriptional activity of a gene or group of genes is here defined and used to mean the fraction of the total RNA synthesis rate [nucleotides polymerized per unit time] that is transcribed from that gene or set of genes. For rRNA genes, it is [rate of rRNA synthesis/rate of total RNA synthesis]; for r-protein genes, it is [rate of r-protein mRNA synthesis/rate of total RNA synthesis].) The results clearly demonstrate that the transcriptional activity of rRNA and r-protein genes is reduced in the rel+ strain and increased in the relAl strain. It appears likely that the stringent control system influences directly the patterns of transcription of the genes coding for the RNA and protein components of the ribosome and consequently the global distribution of RNA polymerase between different classes of genes on the E. coli chromosome. MATERIALS AND METHODS Conditions of growth. The bacterial strains used were the parental strain NF314 (leu- valS+ rel+) and isogenic derivatives stringent NF536 [leu- valS(Ts) rel+] and relaxed NF537 [leu- valS(Ts) relA1I]. The strains were derived from E. coli B AS19 and were obtained from N. Fiil. Cultures were grown in minimal MOPS medium containing 0.2 mM phosphate (10yCi of 32p per ml) and supplemented with 0.2% glucose and 20 ,mg of leucine per ml (6). Growth was monitored as absorbance at 460 nm. The permissive growth temperature was 29.5°C, and the semipermissive temperature was 35.5°C. The experimental cultures were begun
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by a 10-5 dilution of a fresh stationary-phase culture into 32P-labeled growth medium. When the cultures reached a density of 0.3, they were shifted from 29.5 to 35.5°C. After 15 min, [5-3H]uracil (25 Ci/mmol; 20 ,uCi/ml) was added. Incorporation was terminated after 1 min by pouring the cultures over ice, and RNA was extracted and purified as described (7). DNA-RNA hybridization. The 3H and 32p radioactivities in r-protein mRNA and in rRNA were assayed by hybridization to DNA from transducing phage Aspcl (17), carrying about 15 r-protein genes (obtained from M. Nomura), and Xilv5 (5), carrying rRNA genes (obtained from N. Fiil), respectively. The details of DNA preparation and the hybridization assay have been described (7, 9). Competitor RNA was obtained from strain NF314 grown in MOPS supplemented with 0.2% glucose and 0.4% vitamin-free Casamino Acids. Cultures were exposed to 50 ,ug of rifamycin per ml for 30 min before harvesting. This results in virtually complete decay of unstable RNA (24). At an input of 100 ,ug per hybridization assay, this rifamycin RNA was unable to compete r-protein mRNA. RNA concentrations were determined as absorbance at 260 nm (A260), using a Gilford spectrophotometer with a 1-cm light path. An A2680 of 1.0 is equivalent to about 50 ug of RNA per ml. Radioactivity was determined by spotting a measured portion of RNA solution onto a nitrocellulose filter and counting as described (7). The 32p specific activities of the RNAs were about 1.2 x 107 cpm/A260, and the 3H specific activities were: NF317 (valS+ rel+), 3.89 x 10 cpm/A260; NF536 [ualS(Ts) rel+], 8.79 x 105 cpm/A260; and NF537 [valS(Ts) relAl], 1.73 x 107 cpm/A2.. The difference in the incorporation of [3H]uracil during the 1-min pulse period reflects the influence of the stringent control system on the utilization of exogenous pyrimidine during amino acid deprivation rather than differences in the synthesis rate of RNA (12, 22). All data have been corrected for spillover of 32p into the 3H counting channel.
RESULTS Labeling of cellular RNA with [32P]orthophosphate and [3Hluracil. The effect of the stringent control system on the transcriptional activities of genes coding for RNA and protein components of the ribosome was examined by RNA-DNA hybridization. Cultures of the parental valS+ rel+, the stringent valS(Ts) rel+, and the relaxed valS(Ts) relAl strains were grown in medium containing [32P]orthophosphate to homogeneously label cellular nucleic acid. At zero time, the cultures were shifted from the permissive temperature of 29.50C to the semipermissive temperature of 35.50C. The temperature shift partially inhibited aminoacylation of valyl-tRNA and consequently resulted in a 30 to 40% reduction in the rate of protein synthesis in the valS(Ts) strains relative to the non-temperature-sensitive control strain (6); this resulted in the elicitation
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of the stringent or relaxed response. Nucleic acids synthesized at the elevated temperature were labeled after 15 min at 35.5°C by a brief 1-min exposure to [3H]uracil, and RNA was prepared. Nucleic acids synthesized in the 29.5°C control cultures (i.e., no temperature shift and therefore no inhibition of protein synthesis) were also labeled with [3H]uracil for 1 min, and RNA was prepared. The distribution of 3H and 32p radioactivities into the rRNA and r-protein mRNA in the respective RNA preparations was analyzed by hybridization to DNA from X transducing phages containing either rprotein genes from the "str-spc" region (17) or rRNA genes from the "ilv" region (5) of the bacterial chromosome. The amount of 32P radioactivity hybridizing to the respective DNAs gives the amount of r-protein mRNA or rRNA; the amount of 3H radioactivity gives an estimate of the transcriptional activities of r-protein genes and rRNA genes. It should be emphasized that each RNA preparation contained both 3H and 32p radioactivities. The hybridization of these radioactivities was determined in a single set of experiments but described separately; 32P hybridization data and the 3H data are presented below. Hybridization of 32P radioactivity. The hybridization of 32P-labeled RNA (prepared from
the three cultures that had been shifted to 35.5°C and pulse labeled with [3H]uracil) to Xtrk and Xspc1 DNA is illustrated (Fig. 1). Both Xtrk and Xspcl phages carry the aroE and trkA genes; in addition, the Xspcl phage carries about 15 r-protein genes. This represents about one-third to one-half of the total r-protein DNA on the bacterial chromosomes (7, 17). The fraction of the total 32p radioactivity that is r-protein mRNA and hybridizes to the Xspcl DNA is obtained from the slope of the Xspcl hybridization curve corrected for nonspecific hybridization to Xtrk DNA. It has been shown previously that X DNA and Xtrk DNA hybridize approximately equal amounts of input radioactivity; therefore, most or all of the radioactivity associated with Xtrk DNA is nonspecific (7, 8). The results of this experiment corroborate previous measurements (8) and indicate that r-protein mRNA hybridized to Xspcl DNA accounts for 0.175, 0.049, and 0.432% of the total RNA input radioactivity from the parental, the rel+, and the relAl strains, respectively (see legend of Table 1). Thus the amount of r-protein mRNA is subject to the influence of the stringent control system. Using RNA prepared from cultures grown at permissive temperature, the fraction of 32P radioactivity in r-protein mRNA was constant (Table 2; 8). Also note that the amount of
BiNFp536
A NF 314
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FIG. 1. Hyridization of [32P]RNA to Xtrk and Xspcl DNA. The RNA was prepared for hybridization from cultures that had been grown in 32P-labeled growth medium at 29.5°C, shifted to 35.5°C for 15 min, and pulse labeled with [3H]uracil for 1 min. Hybridization of 32p ratioactivity to DNA from Xtrk and Aspcl is illustrated in: (A) the parental strain; (B) the valS(Ts) rel+ strain; and (C) the valS(Ts) relAl strain. Specific hybridization of r-protein mRNA is obtained as the difference in radioactivity associated with Xspcl filters (0) and Xtrk filters (-). Radioactivity associated with Xtrk filters is more than 90% nonspecific (7, 8). The 32p specific activity of the input RNA was about 1.22 x 107 cpmlA260, and the input radioactivity (cpm) per 50 pi was: NF314, 5.82 x 105; NF536, 5.97 x 105; and NF537, 5.43 x 105. Each assay contained 50 to 200 pi of input RNA and six DNA filters (two containing 5 pg each of Xtrk DNA and four containing 5 pg each of Xspcl DNA) in a final volume of 2 ml of 2 x SSC (SSC = 0.15 M NaCl + 0.015 M sodium citrate). The average values from each assay are illustrated; the individual filters varied by less than +5% of the average values. The hybridization of the 3H radioactivity in the RNA preparation is illustrated in Fig. 4.
TRANSCRIPTION OF rRNA AND r-PROTEIN GENES
VOL. 129, 1977
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TABLE 1. Hybridization of [32P]RNA to Xspcl DNA and Ailv5 DNAa
Hybridization DNA Input radioactivity (cpm)
Strain
Hybridizedra-
~~~~(cpm)
H/I (%)
NF314 (vals+ rel+)
Aspcl Ailv5
5.82 x 105 3.10 x 103
1,016 1,256
0.175 (0.167)b 40.5 (42.0)
NF536 [valS(Ts) rel+]
Xspcl Xilv5
5.97 x 105 3.18 x 103
292 1,234
0.049 (0.056) 38.6 (38.2)
NF537 [valS(Ts) relAl]
Xspcl 5.43 x 105 2,348 0.432 (0.368) Ailv5 2.88 x 103 1,168 40.5 (37.6) a The data were obtained from the experiments illustrated in Fig. 1 and 2. The input radioactivity and the hybridized radioactivity are calculated at an input of 50 ,ul of RNA per assay. The hybridized radioactivity is four times greater than the values shown on the ordinates of Fig. 1 and 2 and accounts for the fact that each assay contained four specific DNA filters. The specific activity of 32P in the RNA preparations was about 1.22 x 107 cpm/A m. The ratio of radioactivity in specific hybrids to input radioactivity (H/I) is proportional to the fraction of the total input RNA that is homologous to the bacterial DNA carried on the Xspcl or Ailv5 DNA. The factor of proportionality is 1.33 and accounts for the fact that about 75% of the specific RNA enters into specific RNA-DNA hybrids at equilibrium under these assay conditions (7, 9). Thus 1.33 x 0.175% = 0.232% of the total input radioactivity is mRNA homologous to the 15 r-protein genes carried on the Aspcl DNA in the control NF314 culture at 35.5°C. This represents about one-third to one-half ofthe total r-protein mRNA. The RNA preparations contain about 20, 15, and 3 to 5% of the 32P radioactivity in DNA, tRNA, and mRNA, respectively. The hybridization efficiency of rRNA to Ailv5 DNA is again about 75% under these assay conditions. Therefore, at most about 50% of the input radioactivity should hybridize to Xilv5 DNA. b Values from a duplicate set of experiments. TABLE 2. Hybridization of [3HI32P]RNA labeled at the permissive temperature to Aspcl DNA and Xilv5
DNAa Percent radioactivity in specific hybrids (H/I) Strain
32P radioactiv- 3H radioactivity ity hybridized to DNA
Aspcl NF314 (valS+ rel+) 0.161 NF536 [valS(Ts) rel+] 0.159 NF537 [valS(Ts) 0.137 relA ]
Ailv5 40.0 40.5 35.7
hybridized to DNA
Aspcl 1.29 1.25 1.17
Ailv5 15.0 14.4 15.0
a The data were obtained from experiments similar to those described in detail in the legends to Fig. 1, 2, 4, and 5 and Tables 1 and 3. The RNA was prepared for hybridization from cultures grown in ['2P1 growth medium at 29.5°C and pulse labeled for 1 min with [3Mfuracil.
r-protein mRNA did not change appreciably after the temperature shift in the parental strain (Tables 1 and 2; 8). The specific hybridization of the 32P radioactivity to excess Xilv5 DNA indicated that at least 38 to 41% of the input radioactivity from each of the three respective RNA preparations was homologous to the bacterial substitution (Fig. 2 and Table 1). Similar results were obtained in control cultures that were not shifted to the elevated temperature (Table 2). The segment of bacterial DNA carried on the Xilv5 phage contains single copies of the 16S, 238, and 5S rRNA genes, and also a 4S gene coding
for tRNAlle and probably one additional tRNA gene (5, 21; unpublished data). About 95% of the 32P radioactivity hybridized to Xilv5 DNA could be competed by addition of 10 jig of competitor stable RNA (Fig. 3). Extrapolation to infinite competitor concentration indicated that virtually 100% of the 32P radioactivity could be displaced from hybrids. This means that virtually all of the 32p radioactivity hybridized to Xilv5 DNA was stable RNA and, as expected, the RNAs prepared from the three strains contained approximately equal proportions of rRNA. Hybridization of [3H]uracil pulse-labeled RNA. The RNA synthesized at the semipermissive temperature of 35.5°C was pulse labeled for 1 min with [3H]uracil. The fraction of the total 3H radioactivity in a given class of RNA thus reflects the transcriptional activity of the genes coding for that RNA relative to the total transcription occurring on the bacterial genome. The transcriptional activities of r-protein and rRNA genes were estimated by hybridization to Xspcl DNA and Xilv DNA, respectively (Fig. 4 and 5). From the results, it is clearly apparent that the stringent control mechanism influenced the transcriptional activities of these important genes coding for the RNA and protein components of the ribosome. Using RNA from the parental valS+ rel+ strain, 1.69 and 18.5% of the total 3H radioactivity hybridized specifically to Xspcl DNA and Xilv5 DNA, respectively (Table 3). In the
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FIG. 2. Hybridization of [32P]RNA to X and Xilv5 DNA. The RNA was prepared for hybridization from cultures that had been grown in 32P-labeld growth medium at 29.5°C, shifted to 35.5°C, and pulse labeled for 1 min with [3H]uracil. Hybridization of 32p radioactivity to DNA from X and Xilv5 is illustrated in: (A) the parental strain; (B) the valS(Ts) rel+ strain; and (C) the valS(Ts) relAl strain. Specific hybridization of rRNA is obtained as the difference in radioactivity associated with Xilv5 filters (a) and X filters (a). The 32p specific activity of the input RNA was about 1.22 x 107 cpm/A260, and the input radioactivity (cpm) per 50 p1 was: NF314, 3.10 x 103; NF536, 3.18 x 103; and NF537, 2.88 x 103. Each assay contained 50 to 200 of input RNA and six DNA filters (two containing 5 pg each of XDNA and four containing 5 pg each of Xilv5 DNA) in a final volume of 2 ml of2 x SSC. The average values from each assay are illustrated; the individual filters varied by less than +5% of the average. The hybridization of the 3H radioactivity in the RNA preparation is illustrated in Fig. 5. A
;
NF 314
{B
C
NF 536
NF 537
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FIG. 3. Hybridization of [32P/3H]RNA to Xilv5 DNA in the presence of nonradioactive stable RNA. The RNA was prepared for hybridization from cultures that had been grown in 32P-labeled growth medium at 29.5°C, shifted to 35.5°C for 15 min, and pulse labeled for 1 min with [3H]uracil. Competition of 32p (U) and 3H (0) radioactivity from hybridization with Xilv5 DNA by 0 to 10 pg of stable RNA is illustrated in; (A) the parental strain; (B) the valS(Ts) rel+ strain; and (C) the valS(Ts) relAl strain. Each assay contained three DNA filters (one containing 5 pg of X DNA and two containing 5 pg each of Xilv5 DNA), a constant amount of[32P13H]RNA, and 0 to 12 pg of nonradioactive competitor stable RNA. The input radioactivity (cpm) was: NF314,1.24 x 104 32P and 3.12 x 103 3H; NF536,2.54 x 104 32P and 1.83 x 103 3H; and NF537,1.15 x 104 32P and 1.64 x 104 3H. In the absence ofcompetitor, the radioactivity (cpm) hybridized specifically to the two Xilv5 DNA filters was: NF314, 4.57 x 103 32p and 5.32 x 102 3H; NF536, 8.87 x 103 32p and 8.8 x 10' 3H; and NF537, 4.19 x 103 32p and 4.50 x 103 3H. The zero competition values are the average ofthree identical assays that varied by less than ±+5% of the average. All other points are from single assays. The efficiency of hybridization of stable RNA in these assays is slightly less than in Fig. 2 and 5 because the amount ofspecific Xilv5 DNA was reduced to 10 pAg per assay. By replotting the data as the reciprocal of the percentage of radioactivity competed from hybrids versus reciprocal ofthe RNA competitor concentration and extrapolating the linear curve to infinite competitor, it becomes apparent that virtually 100% of the 32P radioactivity and 85 to 95% of the 3H radioactivity can be competed.
stringent valS(Ts) rel+ strain, the values were decreased to 0.81 and 7.01%, respectively. In the relaxed valS(Ts) relAl strain, the values were increased to 2.06 and 30.5%, respectively.
The distributions of 3H radioactivity in RNA prepared from 29.5°C control cultures were identical (Table 2). Thus these results clearly demonstrate that the stringent control system
TRANSCRIPTION OF rRNA AND r-PROTEIN GENES
VOL. 129, 1977 A NF 314
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r NF 536
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input RNA (microliters)
FIG. 4. Hybridization of [3H]RNA to Xtrk and Aspcl DNA. The RNA was prepared and hybridized to Xtrk and Xspcl DNA as described in the legend to Fig. 1. The 32p specific activity ofthe RNA was about 1.22 x 107 cpm/A260, and the 3H input radioactivity (cpm) per 50 1 was: NF314, 1.47 x 105; NF536, 4.30 X 104; and NF537, 7.71 x 105. Hybridization of the 3H radioactivity in the RNA prepared from the parental strain (A), the valS(Ts) rel+ strain (B), and the valS(Ts) reIAl strain (C) to Xtrk filters (U) and Aspcl filters (-) is illustrated. Hybridization of the 32p radioactivity in the input RNA to Atrk and Aspcl DNA is illustrated in Fig. 1. Further details are given in Table 3.
input RNA (microliters)
FIG. 5. Hybridization of [3H]RNA to A and Xilv5 DNA. The RNA was prepared and hybridized to A and Ailv5 DNA as described in the legend ofFig. 2. The 32P specific activity of the RNA was about 1.22 x 107 cpml A260, and the 3H input radioactivity (cpm) per 50 i1 was: NF314, 7.30 X 102; NF536, 228 x 102; and NF537, 4.10 x 103. Hybridization of 3H radioactivity in the RNA prepared from the parental strain (A), the valS(Ts) rel+ strain (B), and the valS(Ts) reLAU strain (C) to X filters (-) and Xilv5 filters (-) is illustrated. Hybridization of the 32P input radioactivity is illustrated in Fig. 2. Further details are given in Table 3.
influences the expression of rRNA and r-protein genes. Presumably this occurs at the level of transcription (see Discussion). Competition with nonradioactive stable RNA indicated that more than 80% of the 3H
radioactivity hybridized to Xilv5 DNA was rRNA (and a small amount of tRNA). The remaining 10 to 20% represented spacer sequences present in the 6,000-nucleotide transcript from rRNA genes that are degraded
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DENNIS
TABLE 3. Hybridization of 13H]RNA to Xspcl DNA and Xilv5 DNAa Strain
Hybridization DNA
Hybridized radioInput radioactivity activity (cpm) (cpm)
H/I (
NF314 (valS+ rel+)
Xspc1 Xilv5
1.47 x 105 7.80 x 102
2,488 144
1.69 (1.53)' 18.5 (18.2)
NF536 [valS(Ts) rel+]
Aspc1 Xilv5
4.3 x 104 2.28 x 102
384 16
0.81 (0.89) 7.01 (6.60)
2.06 (1.99) 15,900 7.71 x 105 Xspc1 4.10 x 103 30.5 (24.8) Xilv5 1,252 a The data were obtained from the experiments illustrated in Fig. 3 and 4. The input radioactivity (I) and the hybridized radioactivity (H) are calculated for an input of 50 ,ul of RNA per assay. The specific activities of [3H]RNA from the three strains were: NF314, 3.66 x 106 cpm/A2,0; NF536, 8.78 x 105 cpm/A260; and NF537, 1.74 x 107 cpm/A20. The specific activity of 32P in the three preparation was 1.22 x 107 cpm/A260. The differences in the 3H radioactivity incorporated during the 1-min pulse-labeling period illustrate the effect of the stringent control system on the uptake of exogenous pyrimidine during amino acid deprivation (11, 22). For further details see the legend of Table 1. b Values from a duplicate experiment.
NF537 [valS(Ts) relAl]
during maturation and assembly of ribosomal particles. In addition, about 1% or less of the noncompetable material was ilv mRNA (5, 28). DISCUSSION Turnover of [3HIRNA during the labeling period. During a 1-min pulse label, the average incorporated radioactive nucleotide is in RNA for considerably less than 30 s. This is because of the short lag (about 5 s) in the uptake of exogenous uracil from the growth medium and the dilution effect caused by the intracellular nucleotide pools. If the half-life of an RNA sequence is 1 min and decay is exponential, less than 25% of the incorporated nucleotides will be degraded by the end of the labeling period. The uptake lag, the pool dilution effects, and a longer half-life will reduce this percentage such that under normal growth conditions a negligible fraction of the pulselabeled RNA will be degraded. However, because of this small amount of turnover of labeled mRNA during the pulse period, the estimates will be slightly underestimated for the transcriptional activities of mRNA genes and slightly overestimated for stable RNA genes. Inhibition of protein synthesis could conceivably alter the half-life of unstable RNA sequences. For example, chloramphenicol treatment appears to stabilize mRNA (20). The experimental conditions used here, however, inhibit protein synthesis in the temperature-sensitive strains by only about 30% relative to the non-temperature-sensitive control strain. It is probable, therefore, that the stability of mRNA is not significantly altered. The stringent control system influences the expression of genes coding for the RNA and protein components of the ribosome (8). The
results summarized in Table 3 suggest that rRNA and r-protein genes are regulated at the level of transcription because the fraction of the 3H radioactivity entering into rRNA and rprotein mRNA in the rel+ strain was significantly less than in the control cultures; in this instance the amount of r-protein mRNA available for translation and the rate of accumulation of rRNA were reduced. In the relAl strain the opposite result was observed. The fraction of the 3H radioactivity entering into rRNA and r-protein mRNA was greater than in the control cultures, and the amount of r-protein mRNA available for translation and the rate of accumulation of stable RNA were elevated. Stabilization of mRNA by slight inhibition of protein synthesis would also contribute to the large accumulation of r-protein mRNA under these conditions. With regard to rRNA synthesis, the 3H radiaoctivity that cannot be competed from Xilv5 DNA hybrids using stable RNA represents, almost exclusively, the unstable spacer sequences from the 30S rRNA transcript (5, 9, 28). In all strains pulse labeled at 35.5°C, this material represents about 10% of the radioactivity hybridizing to Xilv5 DNA. This suggests that the maturation and stability of the 30S rRNA precursor in these strains are not significantly perturbed by the experimental conditions used. Stringent control and regulation of cell growth. The product of the rel gene is a protein, the stringent factor, which is associated with the bacterial ribosome (3). During limittion of aminoacylated tRNA, the stringent factor-ribosome complex produces ppGpp. This unusual nucleotide has been implicated both in vivo and in vitro as a negative effector in
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TRANSCRIPTION OF rRNA AND r-PROTEIN GENES
the control of transcription of rRNA genes (2, 14, 27) and r-protein genes (18; L. Lindahl and M. Nomura, unpublished data) and as a positive effector in the control of in vitro transcription of the trp and his operons (26, 29). Bertrand et al. (1) suggest that ppGpp might act at the attenuator site in the leader region of the tryptophan operon. The results summarized in Table 3 demonstrate a direct or indirect involvement of the rel gene product on the in vivo transcriptional activities of rRNA and r-protein genes. This involvement is presumably mediated by the nucleotide ppGpp at the level of transcription of rRNA and r-protein genes. In the valS(Ts) rel+ strain, ppGpp accumulates at 35.50C (6, 14) and transcription of rRNA and r-protein genes relative to total transcription is restricted; in the valS(Ts) relAl strain, ppGpp fails to accumulate and transcription of rRNA and r-protein genes is enhanced. In both strains the instantaneous rate of total RNA synthesis is about the same as in the parental control culture. [The relative instantaneous rates of total RNA synthesis in the three cultures can be roughly approximated from the relative rates of RNA accumulation and the fraction of the 3H radioactivity homologous to Xilv5 DNA. The relative rates of RNA accumulation in a similar experiment were: NF314, 1.40; NF536, 0.64; and NF537, 1.52 [6]. The estimates of the relative instantaneous rates of total RNA synthesis were calculated as follows: NF314, (1.40)(1/0.183) = 7.65; NF536 (0.64)(1/0.068) = 9.42; and NF537, (1.52)(1/ 0.276) = 5.51. These estimates are accurate only within a factor of two, but do indicate that the instantaneous rates of total RNA synthesis are approximately equal in the three respective cultures at 35.50C.] In total, these observations seem to point to the stringent control system as a major regulatory system in bacteria. The system is probably involved in the maintenance of a balanced rate of ribosome production in a given nutritional environment. The stringent factor protein serves as a sensor of the availability of high-energy phosphates and amino acids necessary to support the biosynthesis of proteins in that environment. A reduction in the charging levels of tRNA due to energy or amino acid limitation results in idling of ribosomes and accumulation of ppGpp. The nucleotide presumably has a negative effect on the transcription of genes coding for RNA and protein components of the ribosome and probably other factors required for protein synthesis (16; unpublished data). This allows the cell to conserve vital energy and to utilize limited
587
amino acids for the synthesis of necessary and important proteins. The nucleotide has a positive effect on the in vitro transcription of amino acid biosynthetic operons (26); translation in vivo of these RNAs into protein could potentially restore the proper levels of amino acids necessary for protein biosynthesis and exponential-phase growth. In addition, there are certainly other regulatory systems in the bacterium, some of which have not yet been fully characterized, that also influence the expression of these different classes of genes. ACKNOWLEDGMENTS Support for this work was obtained from the National Research Council of Canada. I thank Anita Quail for assistance in preparing the X transducing phages. LITERATURE CITED 1. Bertrand, K., L. Korn, F. Lee, T. Pratt, C. Squires, and C. Yanofsky. 1975. New features of the regulation of the tryptophan operon. Science 189:22-26. 2. Block, R. 1976. Synthesis of ribosomal RNA in a partially purified extract from Escherichia coli, p. 226240. In 0. Maaloe and N. Kjeldgaard (ed.), Alfred Benzon Symposium IX, Control of ribosome biosynthesis. Munksgaard, Copenhagen. 3. Block, R., and W. Haseltine. 1974. In vitro synthesis of ppGpp and pppGpp, p. 747-761. In M. Nomura, A. Tissieres, and P. Lengyel (ed.), Ribosomes. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 4. Cashel, M., and J. Gallent. 1969. Two compounds implicated in the function of the RC gene in Escherichia coli. Nature (London) 221:838-841. 5. Collins, J., N. Fiil, P. Jorgenaen, and J. Friesen. 1976. Gene cloning of Escherichia coli chromosomal genes important in the regulation of ribosomal RNA synthetases, p. 356-369. In 0. Maaloe and N. Kjeldgaard (ed.), Alfred Benzon Symposium IX, Control of ribosome biosynthesis. Munksgaard, Copenhagen. 6. Dennis, P., and M. Nomura. 1974. Stringent control of ribosomal protein gene expression in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 71:3819-3823. 7. Dennis, P., and M. Nomura. 1975. Regulation of the expression of ribosomal protein genes in Escherichia coli. J. Mol. Biol. 97:61-76. 8. Dennis, P., and M. Nomura. 1975. Stringent control of the transcriptional activities of ribosomal protein genes in E. coli. Nature (London) 225:460-465. 9. Dennis, P., and D. Nordan. 1976. Characterization of the hybridization between purified 168 and 23S ribosomal ribonucleic acid and ribosomal deoxyribonucleic acid from Escherichia coli. J. Bacteriol. 128:2834. 10. Donini, P. 1972. Turnover of ribosomal RNA during the stringent response in Escherichia coli. J. Mol. Biol. 72:553-569. 11. Edlin, G., and P. Broda. 1968. Physiology and genetics of the ribonucleic acid control locus in Escherichia coli. Bacteriol. Rev. 32:206-226. 12. Edlin, G., and J. Neuhard. 1967. Regulation of nucleotide triphosphate pools in Escherichia coli. J. Mol. Biol. 24:225-230. 13. Edlin, G., G. Stent, W. Baker, and C. Yanofsky. 1968. Synthesis of a specific messenger RNA during amino acid starvation of E. coli. J. Mol. Biol. 37:257-268. 14. Fill, N., K. Meyenburg, and J. Frieser. 1973. Accumulation and turnover of guanosine tetraphosphate in Escherichia coli. J. Mol. Biol. 71:769-783.
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15. Forchasmer, J., and N. Kjeldgaard. 1968. Regulation of meenger RNA synthesis in Escherichia coli. J. Mol. Biol. 37:245-256. 16. Furano, A., and F. Wittel. 1976. Synthesis of elongation factors Tu and G are under stringent control in Escherichia coli. J. Biol. Chem. 251:898-901. 17. Jaskuna, S. R., L.Lindahl, and M. Nomura. 1976. Specialized transducing phages for ribosomal protein genes of Echerichia coli. Proc. Natl. Acad. Sci. U.S.A. 72:8-10. 18. Johnsen, M., and N. Fil. 1976. Synthesis of ribosomal protein L71L12 in vitro, p. 221-225. In 0. Maalee and N. Kjeldgaard (ed.), Alfed Benzon Symposium IX, Control of ribosome biosynthesis. Munksgaard, Copenhagen. 19. Lavelle, R., and G. Deliauwer. 1968. Messenger RNA synthesis during amino acid starvation in E. coli. J. Mol. Biol. 37:269-288. 20. Levinthal, C., D. Fan, A. Higa, and L. Zimmerman. 1963. The decay and protection of messenger RNA in bacteria. Cold Spring Harbor Symp. Quant. Biol. 28:183-187. 21. Lund, E., J. Dahlberg, L. Lindahl, S. R. Jaskunas, P. P. Dennis, and M. Nomura. 1976. Transfer RNA genes between 16S and 238 rRNA genes in rRNA transcription units ofE. coli. Cell 7:166-177. 22. Morris, D., and N. 0. Kjeldgaard. 1968. Evidence for the non-oordinate regulation of ribonucleic acid synthesis in stringent strains of Escherichia coli. J. Mol.
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Biol. 31:145-148. 23. Neirlich, D. T. 1968. Amino acid control over RNA synthesis: a re-evaluation. Proc. Natl. Acad. Sci. U.S.A. 60:1345-1362. 24. Pato, M., nd K. von Meyenburg. 1970. Residual RNA synthesis in Eacherichia coli after inhibition ofinitiation of transcription by rifampicin. Cold Spring Harbor Symp. Quant. Biol. 36:497-504. 25. Sent, G., and S. Brenner. 1961. A genetic locus for the regulation of ribonucleic acid synthesis. Proc. Natl. Acad. Sci. U.S.A. 47:2006-2014. 26. Stephens, J., S. Artz, and B. Ame. 1976. Guanosine 5'-
diphosphate 3'-diphosphate (ppGpp): positive effector for histidine operon transcription and general signal for amino acid deficiency. Proc. Natl. Acad. Sci. U.S.A. 72:4389-4393. 27. Travers, A., M. Kamen, and M. Cashel. 1970. The in vitro synthesis of ribosomal RNA. Cold Spring Harbor Symp. Quant. Biol. 35:416418. 28. Vonder Haar, R., and E. Umbarger. 1974. Isoleucine and valine metabolism in Escherichia coli K-12: detection and memurement of ilv-specific messenger ribonucleic acid. J. Bacteriol. 120:687-606. 29. Yang, H., G. Zubay, E. Urm, G. Reeniss, and M. Cashel. 1974. Effects of guanosine tetraphosphate, guanosine pentaphosphate and Sy methylenyl guanosine pentaphosphate on gene expresion of Escherichia coli in vitro. Proc. Natl. Acad. Sci. U.S.A. 71:63-67.
JouItNAL oF BACT3i1OLOGY, Feb. 1977, p. 580-688 Copyright 0 1977 American Society for Microbiology
Influence of the Stringent Control System on the Transcription of Ribosomal Ribonucleic Acid and Ribosomal Protein Genes in Escherichia coli PATRICK P. DENNIS Department of Microbiology, University of British Columbia, Vancouver, British Columbia, Canada V6T 1 W5 Received for publication 30 August 1975
The fraction of the total ribonucleic acid (RNA) synthesis rate that is messenger RNA (mRNA) for ribosomal protein (r-protein) and ribosomal RNA (rRNA) has been estimated in valS(Ts) rel+ stringent and valS(Ts) relAl relaxed strains of Escherichia coli during a partial inhibition of valyl-transfer RNA aminoacylation. The partial inhibition was accomplished by shifting the strains from the permissive growth temperature of 29.50C to the semipermissive temperature of 35.50C. The RNA synthesized at the elevated temperature was pulse labeled with [3H]uracil. The fraction of the total incorporated 3H radioactivity in r-protein mRNA or in rRNA was estimated by specific hybridization to the transducing phages Aspcl, which carries about 15 r-protein genes, and Xilv5, which carries an rRNA transcription unit. The results clearly demonstrate that the rel gene influences the fraction of the total RNA synthesis rate that is rprotein mRNA and rRNA; in the rel+ strain these fractions are significantly decreased, and in the relAl strain they are significantly increased relative to control cultures. This indicates that the expression of the genes coding for the RNA and protein component of the ribosome are most likely regulated at the level of transcription. Furthermore, it appears that the distribution of functioning RNA polymerase between rRNA genes, r-protein genes, and other types of genes is influenced by the rel gene control system; presumably this influence is mediated through the unusual nucleotide guanosine tetraphosphate. In the bacterium Escherichia coli, the stringent control system regulates the production of the ribonucleic acid (RNA) and protein components of the ribosome (6, 8, 25). Amino acid or charged transfer RNA (tRNA) deprivation of rel+ stringent strains results in accumulation of guanosine tetraphosphate (ppGpp) and in reductions (i) in the rate ofaccumulation of stable RNA, (ii) in the amount of ribosomal protein (rprotein) messenger RNA (mRNA), and (iii) in the differential rate of r-protein synthesis (defined as r-protein synthesis rate/total protein synthesis rate). In contrast, amino acid deprivation of relA1 strains results (i) in a failure to accumulate ppGpp, (ii) in unabated accumulation of stable RNA, and (iii) in an elevation in the amount of r-protein mRNA and in the differential rate of r-protein synthesis (4, 6, 8, 14, 25). The precise mechanism whereby the stringent control system regulates RNA synthesis is the subject of controversy (11). Initially it was assumed that amino acid control over RNA synthesis was attributable to an arrest of initiation 580
that affected both stable RNA and mRNA transcription. It was subsequently shown that the synthesis of general and specific mRNA's (i.e., trp mRNA and lac mRNA) was essentially normal during amino acid deprivation in rel+ and relAl strains (13, 15, 19, 22, 23). Conversely, the accumulation of r-protein mRNA, like the accumulation of stable RNA, has recently been shown to be subject to stringent control (8). The influence of the amino acid control system on the accumulation of these RNAs could conceivably be mediated at the level of transcription as originally suggested or at the level of stability of the respective transcripts (10). In this paper, the effect of the stringent control system on the expression of rRNA and rprotein genes is examined by using RNA-deoxyribonucleic acid (DNA) hybridization (7, 9). The study uses isogenic rel+ and relAl strains carrying a temperature-sensitive mutation in the valyl-tRNA synthetase gene. A partial restriction of valyl-tRNA aminoacylation (and protein synthesis) results in elicitation of either the stringent or relaxed response and is
VOL. 129, 1977
TRANSCRIPTION OF rRNA AND r-PROTEIN GENES
achieved by shifting the respective rel+ and relAl cultures from a permissive growth temperature to a semipermissive growth temperature (6, 14). The rate of rRNA and r-protein accumulation and consequently ribosome biosynthesis is reduced in the rel+ strain at 35.50C relative to the control cultures; the rate of rRNA and r-protein accumulation and consequently ribosome biosynthesis is increased in the relAl strain relative to the control cultures. After these initial adjustments in the rates of ribosome biosynthesis, the temperature-sensitive strains maintain a more or less balanced condition of growth for some time at the semipermissive growth temperature of 35.50C, and protein synthesis continues at about 60 to 70% of the non-temperture-sensitive control rate (6). These conditions thus minimize any secondary and nonspecific effects on the expression of stable RNA and mRNA genes that might result from prolonged periods of severe amino acid deprivation. The transcriptional activity of rRNA and r-protein genes was estimated under these conditions by measuring the fraction of a pulse label that enters into rRNA and r-protein mRNA, respectively. (The transcriptional activity of a gene or group of genes is here defined and used to mean the fraction of the total RNA synthesis rate [nucleotides polymerized per unit time] that is transcribed from that gene or set of genes. For rRNA genes, it is [rate of rRNA synthesis/rate of total RNA synthesis]; for r-protein genes, it is [rate of r-protein mRNA synthesis/rate of total RNA synthesis].) The results clearly demonstrate that the transcriptional activity of rRNA and r-protein genes is reduced in the rel+ strain and increased in the relAl strain. It appears likely that the stringent control system influences directly the patterns of transcription of the genes coding for the RNA and protein components of the ribosome and consequently the global distribution of RNA polymerase between different classes of genes on the E. coli chromosome. MATERIALS AND METHODS Conditions of growth. The bacterial strains used were the parental strain NF314 (leu- valS+ rel+) and isogenic derivatives stringent NF536 [leu- valS(Ts) rel+] and relaxed NF537 [leu- valS(Ts) relA1I]. The strains were derived from E. coli B AS19 and were obtained from N. Fiil. Cultures were grown in minimal MOPS medium containing 0.2 mM phosphate (10yCi of 32p per ml) and supplemented with 0.2% glucose and 20 ,mg of leucine per ml (6). Growth was monitored as absorbance at 460 nm. The permissive growth temperature was 29.5°C, and the semipermissive temperature was 35.5°C. The experimental cultures were begun
581
by a 10-5 dilution of a fresh stationary-phase culture into 32P-labeled growth medium. When the cultures reached a density of 0.3, they were shifted from 29.5 to 35.5°C. After 15 min, [5-3H]uracil (25 Ci/mmol; 20 ,uCi/ml) was added. Incorporation was terminated after 1 min by pouring the cultures over ice, and RNA was extracted and purified as described (7). DNA-RNA hybridization. The 3H and 32p radioactivities in r-protein mRNA and in rRNA were assayed by hybridization to DNA from transducing phage Aspcl (17), carrying about 15 r-protein genes (obtained from M. Nomura), and Xilv5 (5), carrying rRNA genes (obtained from N. Fiil), respectively. The details of DNA preparation and the hybridization assay have been described (7, 9). Competitor RNA was obtained from strain NF314 grown in MOPS supplemented with 0.2% glucose and 0.4% vitamin-free Casamino Acids. Cultures were exposed to 50 ,ug of rifamycin per ml for 30 min before harvesting. This results in virtually complete decay of unstable RNA (24). At an input of 100 ,ug per hybridization assay, this rifamycin RNA was unable to compete r-protein mRNA. RNA concentrations were determined as absorbance at 260 nm (A260), using a Gilford spectrophotometer with a 1-cm light path. An A2680 of 1.0 is equivalent to about 50 ug of RNA per ml. Radioactivity was determined by spotting a measured portion of RNA solution onto a nitrocellulose filter and counting as described (7). The 32p specific activities of the RNAs were about 1.2 x 107 cpm/A260, and the 3H specific activities were: NF317 (valS+ rel+), 3.89 x 10 cpm/A260; NF536 [ualS(Ts) rel+], 8.79 x 105 cpm/A260; and NF537 [valS(Ts) relAl], 1.73 x 107 cpm/A2.. The difference in the incorporation of [3H]uracil during the 1-min pulse period reflects the influence of the stringent control system on the utilization of exogenous pyrimidine during amino acid deprivation rather than differences in the synthesis rate of RNA (12, 22). All data have been corrected for spillover of 32p into the 3H counting channel.
RESULTS Labeling of cellular RNA with [32P]orthophosphate and [3Hluracil. The effect of the stringent control system on the transcriptional activities of genes coding for RNA and protein components of the ribosome was examined by RNA-DNA hybridization. Cultures of the parental valS+ rel+, the stringent valS(Ts) rel+, and the relaxed valS(Ts) relAl strains were grown in medium containing [32P]orthophosphate to homogeneously label cellular nucleic acid. At zero time, the cultures were shifted from the permissive temperature of 29.50C to the semipermissive temperature of 35.50C. The temperature shift partially inhibited aminoacylation of valyl-tRNA and consequently resulted in a 30 to 40% reduction in the rate of protein synthesis in the valS(Ts) strains relative to the non-temperature-sensitive control strain (6); this resulted in the elicitation
582
DENNIS
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of the stringent or relaxed response. Nucleic acids synthesized at the elevated temperature were labeled after 15 min at 35.5°C by a brief 1-min exposure to [3H]uracil, and RNA was prepared. Nucleic acids synthesized in the 29.5°C control cultures (i.e., no temperature shift and therefore no inhibition of protein synthesis) were also labeled with [3H]uracil for 1 min, and RNA was prepared. The distribution of 3H and 32p radioactivities into the rRNA and r-protein mRNA in the respective RNA preparations was analyzed by hybridization to DNA from X transducing phages containing either rprotein genes from the "str-spc" region (17) or rRNA genes from the "ilv" region (5) of the bacterial chromosome. The amount of 32P radioactivity hybridizing to the respective DNAs gives the amount of r-protein mRNA or rRNA; the amount of 3H radioactivity gives an estimate of the transcriptional activities of r-protein genes and rRNA genes. It should be emphasized that each RNA preparation contained both 3H and 32p radioactivities. The hybridization of these radioactivities was determined in a single set of experiments but described separately; 32P hybridization data and the 3H data are presented below. Hybridization of 32P radioactivity. The hybridization of 32P-labeled RNA (prepared from
the three cultures that had been shifted to 35.5°C and pulse labeled with [3H]uracil) to Xtrk and Xspc1 DNA is illustrated (Fig. 1). Both Xtrk and Xspcl phages carry the aroE and trkA genes; in addition, the Xspcl phage carries about 15 r-protein genes. This represents about one-third to one-half of the total r-protein DNA on the bacterial chromosomes (7, 17). The fraction of the total 32p radioactivity that is r-protein mRNA and hybridizes to the Xspcl DNA is obtained from the slope of the Xspcl hybridization curve corrected for nonspecific hybridization to Xtrk DNA. It has been shown previously that X DNA and Xtrk DNA hybridize approximately equal amounts of input radioactivity; therefore, most or all of the radioactivity associated with Xtrk DNA is nonspecific (7, 8). The results of this experiment corroborate previous measurements (8) and indicate that r-protein mRNA hybridized to Xspcl DNA accounts for 0.175, 0.049, and 0.432% of the total RNA input radioactivity from the parental, the rel+, and the relAl strains, respectively (see legend of Table 1). Thus the amount of r-protein mRNA is subject to the influence of the stringent control system. Using RNA prepared from cultures grown at permissive temperature, the fraction of 32P radioactivity in r-protein mRNA was constant (Table 2; 8). Also note that the amount of
BiNFp536
A NF 314
x
C NF(537
Ea. Cl
2
2
o
so
o
0
s0
100
2-
30 o lo lo 20 o 3-0 1
150
200
0
s0
100
input RNA
150
200
0
s
o
s0
100
0
150
200
(microliters)
FIG. 1. Hyridization of [32P]RNA to Xtrk and Xspcl DNA. The RNA was prepared for hybridization from cultures that had been grown in 32P-labeled growth medium at 29.5°C, shifted to 35.5°C for 15 min, and pulse labeled with [3H]uracil for 1 min. Hybridization of 32p ratioactivity to DNA from Xtrk and Aspcl is illustrated in: (A) the parental strain; (B) the valS(Ts) rel+ strain; and (C) the valS(Ts) relAl strain. Specific hybridization of r-protein mRNA is obtained as the difference in radioactivity associated with Xspcl filters (0) and Xtrk filters (-). Radioactivity associated with Xtrk filters is more than 90% nonspecific (7, 8). The 32p specific activity of the input RNA was about 1.22 x 107 cpmlA260, and the input radioactivity (cpm) per 50 pi was: NF314, 5.82 x 105; NF536, 5.97 x 105; and NF537, 5.43 x 105. Each assay contained 50 to 200 pi of input RNA and six DNA filters (two containing 5 pg each of Xtrk DNA and four containing 5 pg each of Xspcl DNA) in a final volume of 2 ml of 2 x SSC (SSC = 0.15 M NaCl + 0.015 M sodium citrate). The average values from each assay are illustrated; the individual filters varied by less than +5% of the average values. The hybridization of the 3H radioactivity in the RNA preparation is illustrated in Fig. 4.
TRANSCRIPTION OF rRNA AND r-PROTEIN GENES
VOL. 129, 1977
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TABLE 1. Hybridization of [32P]RNA to Xspcl DNA and Ailv5 DNAa
Hybridization DNA Input radioactivity (cpm)
Strain
Hybridizedra-
~~~~(cpm)
H/I (%)
NF314 (vals+ rel+)
Aspcl Ailv5
5.82 x 105 3.10 x 103
1,016 1,256
0.175 (0.167)b 40.5 (42.0)
NF536 [valS(Ts) rel+]
Xspcl Xilv5
5.97 x 105 3.18 x 103
292 1,234
0.049 (0.056) 38.6 (38.2)
NF537 [valS(Ts) relAl]
Xspcl 5.43 x 105 2,348 0.432 (0.368) Ailv5 2.88 x 103 1,168 40.5 (37.6) a The data were obtained from the experiments illustrated in Fig. 1 and 2. The input radioactivity and the hybridized radioactivity are calculated at an input of 50 ,ul of RNA per assay. The hybridized radioactivity is four times greater than the values shown on the ordinates of Fig. 1 and 2 and accounts for the fact that each assay contained four specific DNA filters. The specific activity of 32P in the RNA preparations was about 1.22 x 107 cpm/A m. The ratio of radioactivity in specific hybrids to input radioactivity (H/I) is proportional to the fraction of the total input RNA that is homologous to the bacterial DNA carried on the Xspcl or Ailv5 DNA. The factor of proportionality is 1.33 and accounts for the fact that about 75% of the specific RNA enters into specific RNA-DNA hybrids at equilibrium under these assay conditions (7, 9). Thus 1.33 x 0.175% = 0.232% of the total input radioactivity is mRNA homologous to the 15 r-protein genes carried on the Aspcl DNA in the control NF314 culture at 35.5°C. This represents about one-third to one-half ofthe total r-protein mRNA. The RNA preparations contain about 20, 15, and 3 to 5% of the 32P radioactivity in DNA, tRNA, and mRNA, respectively. The hybridization efficiency of rRNA to Ailv5 DNA is again about 75% under these assay conditions. Therefore, at most about 50% of the input radioactivity should hybridize to Xilv5 DNA. b Values from a duplicate set of experiments. TABLE 2. Hybridization of [3HI32P]RNA labeled at the permissive temperature to Aspcl DNA and Xilv5
DNAa Percent radioactivity in specific hybrids (H/I) Strain
32P radioactiv- 3H radioactivity ity hybridized to DNA
Aspcl NF314 (valS+ rel+) 0.161 NF536 [valS(Ts) rel+] 0.159 NF537 [valS(Ts) 0.137 relA ]
Ailv5 40.0 40.5 35.7
hybridized to DNA
Aspcl 1.29 1.25 1.17
Ailv5 15.0 14.4 15.0
a The data were obtained from experiments similar to those described in detail in the legends to Fig. 1, 2, 4, and 5 and Tables 1 and 3. The RNA was prepared for hybridization from cultures grown in ['2P1 growth medium at 29.5°C and pulse labeled for 1 min with [3Mfuracil.
r-protein mRNA did not change appreciably after the temperature shift in the parental strain (Tables 1 and 2; 8). The specific hybridization of the 32P radioactivity to excess Xilv5 DNA indicated that at least 38 to 41% of the input radioactivity from each of the three respective RNA preparations was homologous to the bacterial substitution (Fig. 2 and Table 1). Similar results were obtained in control cultures that were not shifted to the elevated temperature (Table 2). The segment of bacterial DNA carried on the Xilv5 phage contains single copies of the 16S, 238, and 5S rRNA genes, and also a 4S gene coding
for tRNAlle and probably one additional tRNA gene (5, 21; unpublished data). About 95% of the 32P radioactivity hybridized to Xilv5 DNA could be competed by addition of 10 jig of competitor stable RNA (Fig. 3). Extrapolation to infinite competitor concentration indicated that virtually 100% of the 32P radioactivity could be displaced from hybrids. This means that virtually all of the 32p radioactivity hybridized to Xilv5 DNA was stable RNA and, as expected, the RNAs prepared from the three strains contained approximately equal proportions of rRNA. Hybridization of [3H]uracil pulse-labeled RNA. The RNA synthesized at the semipermissive temperature of 35.5°C was pulse labeled for 1 min with [3H]uracil. The fraction of the total 3H radioactivity in a given class of RNA thus reflects the transcriptional activity of the genes coding for that RNA relative to the total transcription occurring on the bacterial genome. The transcriptional activities of r-protein and rRNA genes were estimated by hybridization to Xspcl DNA and Xilv DNA, respectively (Fig. 4 and 5). From the results, it is clearly apparent that the stringent control mechanism influenced the transcriptional activities of these important genes coding for the RNA and protein components of the ribosome. Using RNA from the parental valS+ rel+ strain, 1.69 and 18.5% of the total 3H radioactivity hybridized specifically to Xspcl DNA and Xilv5 DNA, respectively (Table 3). In the
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DENNIS 0x C NF 537
10 .0
O-a
0,;
5
0
50
100
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200
0
100
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300
400
50
100
150
200
input RNA (microliters)
FIG. 2. Hybridization of [32P]RNA to X and Xilv5 DNA. The RNA was prepared for hybridization from cultures that had been grown in 32P-labeld growth medium at 29.5°C, shifted to 35.5°C, and pulse labeled for 1 min with [3H]uracil. Hybridization of 32p radioactivity to DNA from X and Xilv5 is illustrated in: (A) the parental strain; (B) the valS(Ts) rel+ strain; and (C) the valS(Ts) relAl strain. Specific hybridization of rRNA is obtained as the difference in radioactivity associated with Xilv5 filters (a) and X filters (a). The 32p specific activity of the input RNA was about 1.22 x 107 cpm/A260, and the input radioactivity (cpm) per 50 p1 was: NF314, 3.10 x 103; NF536, 3.18 x 103; and NF537, 2.88 x 103. Each assay contained 50 to 200 of input RNA and six DNA filters (two containing 5 pg each of XDNA and four containing 5 pg each of Xilv5 DNA) in a final volume of 2 ml of2 x SSC. The average values from each assay are illustrated; the individual filters varied by less than +5% of the average. The hybridization of the 3H radioactivity in the RNA preparation is illustrated in Fig. 5. A
;
NF 314
{B
C
NF 536
NF 537
20~
>Z u
1.o
micrograms of competitor RNA
FIG. 3. Hybridization of [32P/3H]RNA to Xilv5 DNA in the presence of nonradioactive stable RNA. The RNA was prepared for hybridization from cultures that had been grown in 32P-labeled growth medium at 29.5°C, shifted to 35.5°C for 15 min, and pulse labeled for 1 min with [3H]uracil. Competition of 32p (U) and 3H (0) radioactivity from hybridization with Xilv5 DNA by 0 to 10 pg of stable RNA is illustrated in; (A) the parental strain; (B) the valS(Ts) rel+ strain; and (C) the valS(Ts) relAl strain. Each assay contained three DNA filters (one containing 5 pg of X DNA and two containing 5 pg each of Xilv5 DNA), a constant amount of[32P13H]RNA, and 0 to 12 pg of nonradioactive competitor stable RNA. The input radioactivity (cpm) was: NF314,1.24 x 104 32P and 3.12 x 103 3H; NF536,2.54 x 104 32P and 1.83 x 103 3H; and NF537,1.15 x 104 32P and 1.64 x 104 3H. In the absence ofcompetitor, the radioactivity (cpm) hybridized specifically to the two Xilv5 DNA filters was: NF314, 4.57 x 103 32p and 5.32 x 102 3H; NF536, 8.87 x 103 32p and 8.8 x 10' 3H; and NF537, 4.19 x 103 32p and 4.50 x 103 3H. The zero competition values are the average ofthree identical assays that varied by less than ±+5% of the average. All other points are from single assays. The efficiency of hybridization of stable RNA in these assays is slightly less than in Fig. 2 and 5 because the amount ofspecific Xilv5 DNA was reduced to 10 pAg per assay. By replotting the data as the reciprocal of the percentage of radioactivity competed from hybrids versus reciprocal ofthe RNA competitor concentration and extrapolating the linear curve to infinite competitor, it becomes apparent that virtually 100% of the 32P radioactivity and 85 to 95% of the 3H radioactivity can be competed.
stringent valS(Ts) rel+ strain, the values were decreased to 0.81 and 7.01%, respectively. In the relaxed valS(Ts) relAl strain, the values were increased to 2.06 and 30.5%, respectively.
The distributions of 3H radioactivity in RNA prepared from 29.5°C control cultures were identical (Table 2). Thus these results clearly demonstrate that the stringent control system
TRANSCRIPTION OF rRNA AND r-PROTEIN GENES
VOL. 129, 1977 A NF 314
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2W
input RNA (microliters)
FIG. 4. Hybridization of [3H]RNA to Xtrk and Aspcl DNA. The RNA was prepared and hybridized to Xtrk and Xspcl DNA as described in the legend to Fig. 1. The 32p specific activity ofthe RNA was about 1.22 x 107 cpm/A260, and the 3H input radioactivity (cpm) per 50 1 was: NF314, 1.47 x 105; NF536, 4.30 X 104; and NF537, 7.71 x 105. Hybridization of the 3H radioactivity in the RNA prepared from the parental strain (A), the valS(Ts) rel+ strain (B), and the valS(Ts) reIAl strain (C) to Xtrk filters (U) and Aspcl filters (-) is illustrated. Hybridization of the 32p radioactivity in the input RNA to Atrk and Aspcl DNA is illustrated in Fig. 1. Further details are given in Table 3.
input RNA (microliters)
FIG. 5. Hybridization of [3H]RNA to A and Xilv5 DNA. The RNA was prepared and hybridized to A and Ailv5 DNA as described in the legend ofFig. 2. The 32P specific activity of the RNA was about 1.22 x 107 cpml A260, and the 3H input radioactivity (cpm) per 50 i1 was: NF314, 7.30 X 102; NF536, 228 x 102; and NF537, 4.10 x 103. Hybridization of 3H radioactivity in the RNA prepared from the parental strain (A), the valS(Ts) rel+ strain (B), and the valS(Ts) reLAU strain (C) to X filters (-) and Xilv5 filters (-) is illustrated. Hybridization of the 32P input radioactivity is illustrated in Fig. 2. Further details are given in Table 3.
influences the expression of rRNA and r-protein genes. Presumably this occurs at the level of transcription (see Discussion). Competition with nonradioactive stable RNA indicated that more than 80% of the 3H
radioactivity hybridized to Xilv5 DNA was rRNA (and a small amount of tRNA). The remaining 10 to 20% represented spacer sequences present in the 6,000-nucleotide transcript from rRNA genes that are degraded
586
J. BACTERIOL.
DENNIS
TABLE 3. Hybridization of 13H]RNA to Xspcl DNA and Xilv5 DNAa Strain
Hybridization DNA
Hybridized radioInput radioactivity activity (cpm) (cpm)
H/I (
NF314 (valS+ rel+)
Xspc1 Xilv5
1.47 x 105 7.80 x 102
2,488 144
1.69 (1.53)' 18.5 (18.2)
NF536 [valS(Ts) rel+]
Aspc1 Xilv5
4.3 x 104 2.28 x 102
384 16
0.81 (0.89) 7.01 (6.60)
2.06 (1.99) 15,900 7.71 x 105 Xspc1 4.10 x 103 30.5 (24.8) Xilv5 1,252 a The data were obtained from the experiments illustrated in Fig. 3 and 4. The input radioactivity (I) and the hybridized radioactivity (H) are calculated for an input of 50 ,ul of RNA per assay. The specific activities of [3H]RNA from the three strains were: NF314, 3.66 x 106 cpm/A2,0; NF536, 8.78 x 105 cpm/A260; and NF537, 1.74 x 107 cpm/A20. The specific activity of 32P in the three preparation was 1.22 x 107 cpm/A260. The differences in the 3H radioactivity incorporated during the 1-min pulse-labeling period illustrate the effect of the stringent control system on the uptake of exogenous pyrimidine during amino acid deprivation (11, 22). For further details see the legend of Table 1. b Values from a duplicate experiment.
NF537 [valS(Ts) relAl]
during maturation and assembly of ribosomal particles. In addition, about 1% or less of the noncompetable material was ilv mRNA (5, 28). DISCUSSION Turnover of [3HIRNA during the labeling period. During a 1-min pulse label, the average incorporated radioactive nucleotide is in RNA for considerably less than 30 s. This is because of the short lag (about 5 s) in the uptake of exogenous uracil from the growth medium and the dilution effect caused by the intracellular nucleotide pools. If the half-life of an RNA sequence is 1 min and decay is exponential, less than 25% of the incorporated nucleotides will be degraded by the end of the labeling period. The uptake lag, the pool dilution effects, and a longer half-life will reduce this percentage such that under normal growth conditions a negligible fraction of the pulselabeled RNA will be degraded. However, because of this small amount of turnover of labeled mRNA during the pulse period, the estimates will be slightly underestimated for the transcriptional activities of mRNA genes and slightly overestimated for stable RNA genes. Inhibition of protein synthesis could conceivably alter the half-life of unstable RNA sequences. For example, chloramphenicol treatment appears to stabilize mRNA (20). The experimental conditions used here, however, inhibit protein synthesis in the temperature-sensitive strains by only about 30% relative to the non-temperature-sensitive control strain. It is probable, therefore, that the stability of mRNA is not significantly altered. The stringent control system influences the expression of genes coding for the RNA and protein components of the ribosome (8). The
results summarized in Table 3 suggest that rRNA and r-protein genes are regulated at the level of transcription because the fraction of the 3H radioactivity entering into rRNA and rprotein mRNA in the rel+ strain was significantly less than in the control cultures; in this instance the amount of r-protein mRNA available for translation and the rate of accumulation of rRNA were reduced. In the relAl strain the opposite result was observed. The fraction of the 3H radioactivity entering into rRNA and r-protein mRNA was greater than in the control cultures, and the amount of r-protein mRNA available for translation and the rate of accumulation of stable RNA were elevated. Stabilization of mRNA by slight inhibition of protein synthesis would also contribute to the large accumulation of r-protein mRNA under these conditions. With regard to rRNA synthesis, the 3H radiaoctivity that cannot be competed from Xilv5 DNA hybrids using stable RNA represents, almost exclusively, the unstable spacer sequences from the 30S rRNA transcript (5, 9, 28). In all strains pulse labeled at 35.5°C, this material represents about 10% of the radioactivity hybridizing to Xilv5 DNA. This suggests that the maturation and stability of the 30S rRNA precursor in these strains are not significantly perturbed by the experimental conditions used. Stringent control and regulation of cell growth. The product of the rel gene is a protein, the stringent factor, which is associated with the bacterial ribosome (3). During limittion of aminoacylated tRNA, the stringent factor-ribosome complex produces ppGpp. This unusual nucleotide has been implicated both in vivo and in vitro as a negative effector in
VOL. 129, 1977
TRANSCRIPTION OF rRNA AND r-PROTEIN GENES
the control of transcription of rRNA genes (2, 14, 27) and r-protein genes (18; L. Lindahl and M. Nomura, unpublished data) and as a positive effector in the control of in vitro transcription of the trp and his operons (26, 29). Bertrand et al. (1) suggest that ppGpp might act at the attenuator site in the leader region of the tryptophan operon. The results summarized in Table 3 demonstrate a direct or indirect involvement of the rel gene product on the in vivo transcriptional activities of rRNA and r-protein genes. This involvement is presumably mediated by the nucleotide ppGpp at the level of transcription of rRNA and r-protein genes. In the valS(Ts) rel+ strain, ppGpp accumulates at 35.50C (6, 14) and transcription of rRNA and r-protein genes relative to total transcription is restricted; in the valS(Ts) relAl strain, ppGpp fails to accumulate and transcription of rRNA and r-protein genes is enhanced. In both strains the instantaneous rate of total RNA synthesis is about the same as in the parental control culture. [The relative instantaneous rates of total RNA synthesis in the three cultures can be roughly approximated from the relative rates of RNA accumulation and the fraction of the 3H radioactivity homologous to Xilv5 DNA. The relative rates of RNA accumulation in a similar experiment were: NF314, 1.40; NF536, 0.64; and NF537, 1.52 [6]. The estimates of the relative instantaneous rates of total RNA synthesis were calculated as follows: NF314, (1.40)(1/0.183) = 7.65; NF536 (0.64)(1/0.068) = 9.42; and NF537, (1.52)(1/ 0.276) = 5.51. These estimates are accurate only within a factor of two, but do indicate that the instantaneous rates of total RNA synthesis are approximately equal in the three respective cultures at 35.50C.] In total, these observations seem to point to the stringent control system as a major regulatory system in bacteria. The system is probably involved in the maintenance of a balanced rate of ribosome production in a given nutritional environment. The stringent factor protein serves as a sensor of the availability of high-energy phosphates and amino acids necessary to support the biosynthesis of proteins in that environment. A reduction in the charging levels of tRNA due to energy or amino acid limitation results in idling of ribosomes and accumulation of ppGpp. The nucleotide presumably has a negative effect on the transcription of genes coding for RNA and protein components of the ribosome and probably other factors required for protein synthesis (16; unpublished data). This allows the cell to conserve vital energy and to utilize limited
587
amino acids for the synthesis of necessary and important proteins. The nucleotide has a positive effect on the in vitro transcription of amino acid biosynthetic operons (26); translation in vivo of these RNAs into protein could potentially restore the proper levels of amino acids necessary for protein biosynthesis and exponential-phase growth. In addition, there are certainly other regulatory systems in the bacterium, some of which have not yet been fully characterized, that also influence the expression of these different classes of genes. ACKNOWLEDGMENTS Support for this work was obtained from the National Research Council of Canada. I thank Anita Quail for assistance in preparing the X transducing phages. LITERATURE CITED 1. Bertrand, K., L. Korn, F. Lee, T. Pratt, C. Squires, and C. Yanofsky. 1975. New features of the regulation of the tryptophan operon. Science 189:22-26. 2. Block, R. 1976. Synthesis of ribosomal RNA in a partially purified extract from Escherichia coli, p. 226240. In 0. Maaloe and N. Kjeldgaard (ed.), Alfred Benzon Symposium IX, Control of ribosome biosynthesis. Munksgaard, Copenhagen. 3. Block, R., and W. Haseltine. 1974. In vitro synthesis of ppGpp and pppGpp, p. 747-761. In M. Nomura, A. Tissieres, and P. Lengyel (ed.), Ribosomes. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 4. Cashel, M., and J. Gallent. 1969. Two compounds implicated in the function of the RC gene in Escherichia coli. Nature (London) 221:838-841. 5. Collins, J., N. Fiil, P. Jorgenaen, and J. Friesen. 1976. Gene cloning of Escherichia coli chromosomal genes important in the regulation of ribosomal RNA synthetases, p. 356-369. In 0. Maaloe and N. Kjeldgaard (ed.), Alfred Benzon Symposium IX, Control of ribosome biosynthesis. Munksgaard, Copenhagen. 6. Dennis, P., and M. Nomura. 1974. Stringent control of ribosomal protein gene expression in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 71:3819-3823. 7. Dennis, P., and M. Nomura. 1975. Regulation of the expression of ribosomal protein genes in Escherichia coli. J. Mol. Biol. 97:61-76. 8. Dennis, P., and M. Nomura. 1975. Stringent control of the transcriptional activities of ribosomal protein genes in E. coli. Nature (London) 225:460-465. 9. Dennis, P., and D. Nordan. 1976. Characterization of the hybridization between purified 168 and 23S ribosomal ribonucleic acid and ribosomal deoxyribonucleic acid from Escherichia coli. J. Bacteriol. 128:2834. 10. Donini, P. 1972. Turnover of ribosomal RNA during the stringent response in Escherichia coli. J. Mol. Biol. 72:553-569. 11. Edlin, G., and P. Broda. 1968. Physiology and genetics of the ribonucleic acid control locus in Escherichia coli. Bacteriol. Rev. 32:206-226. 12. Edlin, G., and J. Neuhard. 1967. Regulation of nucleotide triphosphate pools in Escherichia coli. J. Mol. Biol. 24:225-230. 13. Edlin, G., G. Stent, W. Baker, and C. Yanofsky. 1968. Synthesis of a specific messenger RNA during amino acid starvation of E. coli. J. Mol. Biol. 37:257-268. 14. Fill, N., K. Meyenburg, and J. Frieser. 1973. Accumulation and turnover of guanosine tetraphosphate in Escherichia coli. J. Mol. Biol. 71:769-783.
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DENNIS
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Biol. 31:145-148. 23. Neirlich, D. T. 1968. Amino acid control over RNA synthesis: a re-evaluation. Proc. Natl. Acad. Sci. U.S.A. 60:1345-1362. 24. Pato, M., nd K. von Meyenburg. 1970. Residual RNA synthesis in Eacherichia coli after inhibition ofinitiation of transcription by rifampicin. Cold Spring Harbor Symp. Quant. Biol. 36:497-504. 25. Sent, G., and S. Brenner. 1961. A genetic locus for the regulation of ribonucleic acid synthesis. Proc. Natl. Acad. Sci. U.S.A. 47:2006-2014. 26. Stephens, J., S. Artz, and B. Ame. 1976. Guanosine 5'-
diphosphate 3'-diphosphate (ppGpp): positive effector for histidine operon transcription and general signal for amino acid deficiency. Proc. Natl. Acad. Sci. U.S.A. 72:4389-4393. 27. Travers, A., M. Kamen, and M. Cashel. 1970. The in vitro synthesis of ribosomal RNA. Cold Spring Harbor Symp. Quant. Biol. 35:416418. 28. Vonder Haar, R., and E. Umbarger. 1974. Isoleucine and valine metabolism in Escherichia coli K-12: detection and memurement of ilv-specific messenger ribonucleic acid. J. Bacteriol. 120:687-606. 29. Yang, H., G. Zubay, E. Urm, G. Reeniss, and M. Cashel. 1974. Effects of guanosine tetraphosphate, guanosine pentaphosphate and Sy methylenyl guanosine pentaphosphate on gene expresion of Escherichia coli in vitro. Proc. Natl. Acad. Sci. U.S.A. 71:63-67.
