Vol. 130, No. 3 Printed in U.S.A.
JOURNAL OF BACTUIuOWGY, June 1977, p. 1109-1116 Copyright C 1977 American Society for Microbiology
Rate of Ribosomal Ribonucleic Acid Chain Elongation in Escherichia coli B/r During Chloramphenicol Treatment V. SHEN' AND H. BREMER* University of Texas at Dallas, Richardson, Texas 75080
Received for publication 25 January 1977
In Escherichia coli B/r growing in glucose-amino acids medium, the radioactive labeling of 5S ribosomal ribonucleic acid (rRNA) and transfer RNA (tRNA) was measured after the simultaneous addition to the bacteria of chloramphenicol (CAM) (100 ,ug/ml), rifampin (200 ,.g/ml), and radioactive uracil. Accumulation of 58 rRNA ceased 85 s after the addition of rifampin, independent of the presence or absence of CAM; this indicates that CAM did not affect the rRNA chain growth rate. Together with previous measurements of the synthesis of rRNA and messenger RNA under these conditions, the results imply that CAM caused a redistribution of RNA polymerase which greatly favored stable RNA synthesis (77 to 97% of total functioning RNA polymerase engaged in synthesis of rRNA and tRNA). Further, it is inferred that RNA polymerase molecules were activated that were inactive during exponential growth. The labeling of tRNA observed under these conditions suggests the existence of clusters of tRNA genes at the 3' end of long transcripts that resemble the rRNA precursor in length and response to CAM and may be parts of rRNA transcripts.
The synthesis of ribosomal ribonucleic acid (rRNA) and transfer RNA (tRNA) in bacteria can be greatly increased by certain inhibitors of protein synthesis such as chloramphenicol (CAM) (13, 14, 19, 27). Presumably this increase reflects a ppGpp-mediated increase in the affinity of the RNA polymerase for the stable RNA promoters (28-30). To find out to what extent an increase in the RNA chain elongation rate or in the polymerase activity (number of functioning RNA polymerase) contributes to the effect of CAM on rRNA synthesis, we have here measured the chain elongation rate of rRNA in the presence of CAM. From these and previous measurements on RNA synthesis obtained under comparable conditions (6, 27) one can then calculate the number of RNA polymerase molecules engaged in RNA synthesis before and after CAM addition. The results indicate that CAM does not affect rRNA chain elongation, but it appears to activate a fraction of RNA polymerase that is inactive during exponential growth. Such an activation had been previously suspected to occur (16), but its existence could not be proven (7). The mechanism of this control of RNA polymerase activity is not known. The method used here to determine rRNA chain growth was devised by Molin (20) and
involves use of the antibiotic rifampin. As an incidental result, this method shows a partial breakdown of rRNA synthesised in the presence of rifampin. Because of this effect, we have reevaluated some of the previous data on RNA synthesis from this laboratory, which were based on the assumption that rRNA made during rifampin run-out is stable and which are used here to evaluate the CAM experiments. Another incidental result obtained here concerns the synthesis of tRNA. Measurements of tRNA were included as a control to monitor the speed with which rifampin stops RNA chain initiation. Unexpectedly, tRNA synthesis was found to continue after rifampin addition at a reduced rate in a manner that suggests a clustering of several tRNA genes at the 3' end of long transcripts that might well be the rRNA precursors. rRNA precursors are known to contain a tRNA gene in the space between the 16S and 23S genes (15), but additional tRNA genes at the 3' end of the precursors have not been found yet.
I Present address: Department of Microbiology and Immunology, Washington University School of Medicine, St. Louis, MO 63110.
1109
MATERIALS AND METHODS Bacterial strain and growth conditions. Escherichia coli B/r (ATCC 12407) was grown at 37°C in minimal C medium (11) supplemented with 0.4% glucose and a mixture of 20 L-amino acids, each to a final concentration of 20 ,ug/ml. The experimental culture was inoculated with a fresh stationaryphase culture in the same medium (400-fold dilu-
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tion). Culture growth was followed by measuring the absorbance at 460 nm (Anao) (1-cm light path). Radioactive labeling. An exponential culture was labeled with [14C]uracil (0.25 gCi/ml, 62 mCi/ mmol), beginning at anAO of 0.1. All 14C label was chased into stable nucleic acids within two generations. At an A460 of 0.5 (zero time), a portion of the 14C culture was added to CAM (100 Sg/ml). A mixture of rifampin (final concentration, 200 ,ug/ml) and [3H]uracil (final concentration, 1.1 nmol/ml, 18 Ci/ mmol) was added to the untreated portion of the culture at t = 2.5 min and to the CAM-treated portion of the culture at t = 5 min. Samples (1 ml) were taken from both cultures at 10-s intervals during the first 2 min and 5 and 18 min after the addition of rifampin and [3H]uracil. The samples. were added to an equal volume of alcohol-phenol solution at 0°C (17). After the cells were collected by centrifugation, the bacteria were resuspended in 0.2 ml of 100°C electrophoresis sample solution [0.2 x tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetic acid-borate (TEB) buffer (25), 1% sodium dodecyl sulfate, 10% glycerol, 0.001% bromophenol blue], boiled for 45 s, and then stored at room temperature. Electrophoresis. A 10% polyacrylamide-bisacrylamide (19:1) slab gel (12 cm long, 1.5 mm thick) was used to separate the 5S rRNA and 4S tRNA. A small volume (75 ,u) of the lysate described above was used in each slot. Electrophoresis was at room temperature for 6 h at 10 mA in TEB buffer containing 0.1% sodium dodecyl sulfate. After electrophoresis the gels were dried on Whatman 3 MM filter paper and autoradiographed for 3 days on a sheet of Kodak PR Royal X-matic film. After development, the film was scanned on a Joyce-Loebl densitometer to locate the RNA bands. The gel (with the filter paper) was then cut into 2-mm slices. The RNA in the slices was hydrolyzed in 0.5 ml of 0.2 N NaOH overnight, and the radioactivity was counted after addition of 5 ml of a Biosolv-3-toluene mixture (Beckman) in a liquid scintillation spectrophotometer.
RESULTS The time required for the transcription of the main portion of the rRNA precursor can be measured by following the accumulation of 5S rRNA after inhibition of RNA chain initiation by rifampin (20). 58 rRNA is cotranscribed with 16S anad 23S rRNA (9, 10, 12, 15, 31). The end of the 5S section of the rRNA precursor is about 6,100 nucleotides distant from the rRNA promoter (10, 23, 31) so that, after rifampin addition, 5S rRNA is expected to accumulate at the pre-rifampin rate for the time it takes RNA polymerase molecules to transcribe 6,100 rRNA nucleotides; then further accumulation of 5S RNA should stop. In the experiment of Fig. la (closed circles) this time was 85 s; in Molin's experiments it was between 70 and 80 s (20). This time corresponds to a chain growth rate of approximately 75 nucleotides/s. The longer time in our experiment (85 s) might indicate a
slightly slower action of rifampin, since our strain was not rifampin permeable as was Molin's. If CAM is added to the culture 5 min before rifampin, the initial rate of accumulation of 5S RNA is twofold stimulated, as we have observed previously with this growth medium (glucose-amino acids) in the absence of rifampin (see 27), but the time it took to reach the maximum accumulation was not significantly altered (Fig. la, open circles). Thus, CAM did not affect the rRNA chain growth rate, and therefore the stimulation of rRNA and tRNA synthesis by CAM must involve an activation of RNA polymerase (see Discussion). Much of the 5S RNA synthesized in the presence of rifampin is unstable (Fig. lb), as was reported by Molin (20). Although this instability affects the kinetics of accumulation, it should not affect the time at which the break occurs in the accumulation curve due to the cessation of 5S RNA synthesis. The greater instability of rRNA synthesized in the presence of CAM (8, 14, 21, 27) shows up in the 5S curve immediately after labeling as a decrease in the rate of accumulation (Fig. la, open circles). The accumulation of 4S RNA after rifampin addition was immediately reduced to about one-third of the pre-refampin rate (Fig. la, triangular symbols; the increase in the isotope ratio is a measure for the increase in the specific radioactivity of RNA, which, without rifampin, is the same for all species of stable RNA; reference 32). Thus, rifampin stopped initiation of about two-thirds of the 4S RNA chains within 10 s, whereas the remaining onethird of the rate of 4S RNA synthesis stopped only after 80 to 100 s, again independent of the presence or absence of CAM. Most of this 4S RNA is assumed to be tRNA (see Discussion). This suggests that 20 to 30% of the tRNA synthesized in bacteria was transcribed at the end
+CAMp
20.
42
1.0
C,~~~~~~~-A
7%m Time after rifompicin (see)
Time after rifompicin (min )
FIG. 1. Labeling of 5S and 4S RNA in the presence of rifampin. Rifampin and [3H]uridine were added together at zero time (0, A); CAM was given 5 min earlier (O, A). For experimental details see the text. (a and b) Different time scales for the same experiment.
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VOL. 130, 1977
of long transcriptional units, which are similar to rRNA precursors in length and response to CAM (see Discussion); 30% is a maximum estimate since the slope of the tRNA curve is decreasing. A difficulty in the interpretation of the distributions of pulse-labeled RNA molecules arises from the contamination of 5S and 4S RNA species by other labeled nascent or unstable RNA molecules about 70 to 130 nucleotides long. In the data points of Fig. 1 this nascent RNA is included. We have redrawn the kinetics of Fig. 1 after subtraction of what we consider a maximum value for the background of radioactivity in nascent RNA, as indicated in the distributions of Fig. 2 (long dashes). The time of the break did not change by this procedure; however, the initial kinetic slopes were reduced, giving the impression of a 20- to 30-s labeling lag. Since this is unreasonably long in view of the fact that the lag in the labeling of total RNA under these conditions was only 5 to 10 s (independent of rifampin and CAM), we believe that the results are best described by the plot in Fig. 1, although fine details in these kinetics (e.g., the small break at 55 s in the 4S RNA kinetics) may not be significant.
DISCUSSION Control of the rRNA synthesis rate. The sudden increase in the rate of stable RNA synthesis after CAM or after a nutritional shift-up +CAM
32P
62"
might involve a redistribution of functioning RNA polymerase molecules over stable and messenger RNA (mRNA) genes or an activation of an "inactive reserve" of RNA polymerase (16). To find out to what extent redistribution or activation of RNA polymerase actually occurs, rRNA chain growth measurements such as in Fig. 1 must be combined with measurements of the synthesis rate of stable RNA and mRNA after a nutritional shift-up (7) and after CAM addition (27). Some of those previous measurements need a reevaluation due to the instability of rRNA synthesized in the presence of rifampin (20; Fig. 1) and due to the more detailed knowledge now available about the spacer regions in the rRNA precursors. In the following, we (i) discuss the implications of the instability of rRNA synthesized in the presence of rifampin for the previous measurements of RNA synthesis-related parameters, and (ii) estimate the distribution and functional activity of RNA polymerase under various growth conditions. (i) Instability of rRNA synthesized in the presence of rifampin: Implications for the interpretation of previous experiments on the control of RNA synthesis. The relative synthesis rate of mRNA and the chain elongation rate of rRNA were previously determined in this laboratory from the kinetics of RNA labeling in the presence of rifampin (1, 4-7). To evaluate those experiments, it was assumed that rRNA synthesized in the presence of rifampin is com121"
9
5
19.5
E a. 0 2-
z
z
.-I 3
12
-CAM I
4'~~~~33" ^
At
I-I
z A'~~~p 61" -4
W+,
A
9: I-
A
I-l_
120" |_ l_ | I |
1
5
|
- l_
|
I
~~~3
A
SLICE NUMBER
FIG. 2. Electrophoresis distributions of radioactive RNA for the experiment shown in Fig. 1. Symbols: (*) 3H label; (0) 14C label; (- - -) maximum background due to nascent RNA (see text); (-) ranges in which the radioactivities were summed for the formation of the 3HI14C ratio plotted in Fig. 1. At early times, 5S rRNA is in precursor form (10).
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SHEN AND BREMER
pletely stable; this is evidently not the case (20; Fig. lb). In the following, we shall discuss the possible errors in these estimates due to the partial instability of rRNA and their implications for the previous conclusion that there is no activation, but only a redistribution, of functioning RNA polymerase after a nutritional shift-up. Figure lb (closed circles) and Molin's data (20) indicate that about half of the 5S rRNA synthesized in the presence of rifampin decays within 15 min, whereas the other half is more stable. Most likely, the more stable portion of rRNA was synthesized early after the addition of rifampin to the culture and was stabilized by its incorporation into rather complete ribosomal particles, whereas the unstable portion was synthesized late after the addition ofrifampin and owes its instability to the lack of some ribosomal protein. This idea is consistent with our observations that stable rRNA can no longer be labeled 60 after rifampin addition (5), whereas rRNA labeled 2.0 min before rifampin addition is quite stable during 15 min in the presence of rifampin (32). In this case, the previous estimate of the synthesis rate of mRNA would still be correct (Fig. 3), but the rRNA chain growth rate would have been overestimated (Fig. 4). Both these implications agree with more direct measurements of the rate of mRNA synthesis by hybridization competition (27) and of the rRNA chain growth rate (20; Fig. 1). (If rRNA decayed at random with the same probability early or late after rifampin addition and with the same probability for all species of rRNA, then the synthesis rate of mRNA would have been overestimated by the rifampin method because part of the unstable RNA presumed to be mRNA would actually be rRNA, but the chain growth estimate should not be affected [see Discussion in reference 4].) We have now recalculated the distribution of RNA polymerase molecules over stable RNA and mRNA genes for E. coli B/r growing exponentially in three different media (succinate, glucose, and glucose plus 20 amino acids) and in the same media 5 min after the addition of CAM and after a nutritional shift-up on the following three assumptions. (i) The rRNA chain growth rate is 75 nucleotides/s, independent of the bacterial growth rate (20; Fig. la), rather than increasing with growth rate from 75 to 100 nucleotides/s (4), whereas the mRNA chain growth rate is 50 nucleotides/s (2; Discussion in reference 5). (ii) The rRNA precursor is 6,250 nucleotides long (23), of which about 5,100 nucleotides are incorporated into stable RNA (23S 3,200, 16S s
=
= 1,600, 5S = 120; 2 tRNA = 180; the number of tRNA genes per rRNA precursor is not exactly known [see below], but an error of 1 tRNA would make only a 1.5% difference). This means that the synthesis of total "stable" RNA, including spacer regions, is 1.2-fold greater than the observed accumulation of rRNA and tRNA, assuming that the total stable RNA in the cell consists of 85% rRNA and 15% tRNA (one-fifth of which may be drived from rRNA precursors). (iii) The fraction of the instantaneous rate of RNA synthesis that is stable RNA (p) or mRNA (1 - p) has been measured correctly by the rifampin method (4, 6) and by deoxyribonucleic acid-RNA hybridization (27); both methods gave identical values. However, if one uses mature rRNA for hybridization competition experiments, then one neglects the rRNA spacer material and counts it as mRNA; the same mistake is made with the rifampin method, which depends on the stability of rRNA and tRNA synthesized during the first 20 to 30 s after the addition of rifampin (Fig. 3) and which thus excludes the unstable spacers. Hence, these pvalues require a correction (factor 1.2 as above) if the spacer material is to be included (i.e., if stable RNA synthesis shall mean non-mRNA). The data derived from these assumptions are summarized in Table 1, which is discussed below. (ii) Distribution and function of RNA polymerase under various conditions of growth. According to Table 1, both CAM and a nutritional shift-up produce a redistribution, i.e., a shift of RNA polymerase from mRNA genes to stable RNA genes, and an activation of RNA polymerase: the redistribution is given by the increase in the parameter s (fraction of functioning RNA polymerase engaged in the synthesis of stable RNA); the activation is given by the increase in Np (number of functioning RNA polymerase molecules per genome, Table 1). Under either condition, the main effect is caused by the redistribution, as was concluded previously for the shift-up (7). For example, the shift from succinate- to glucose-amino acids medium causes an immediate 2.3-fold increase in s (from 0.35 to 0.81) and a 1.35-fold increase in Np (from 170 to 230 nascent RNA chains per genome). CAM causes an extreme reduction in the fraction of polymerase molecules transcribing mRNA genes; i.e., in CAM almost all (77 to 97%) functioning RNA polymerase transcribes stable RNA genes. Nevertheless, the CAM-induced increase in the rate of stable RNA synthesis cannot be completely accounted for by
VOL. 130, 1977
rRNA CHAIN ELONGATION AFTER CAM
0
-o
E
C.o a0 = 0.4
-
,
=
=
~0 o
b
0
00.4
O2
S_
_
.
1
Time after rifampicin(min) FIG. 3. Determination of the relative rate of synthesis ofmRNA [mI(m + s)] and ofstable RNA [s/(m + s)] from the kinetics of RNA labeling in the presence of rifampin (schematic example). (a-c) Evaluation under the assumption that all rRNA and tRNA synthesized in the presence of rifampin is stable. (dfJ Evaluation ifrRNA synthesized late after rifampin is unstable. (a) Observed kinetics of RNA labeling (5, 24). Radioactive uridine was given either at zero time, together with rifampin ( ), or 15 s after rifampin (-- -). Two early and two late samples (0) were taken (simultaneously) from both labeling tubes; the differences correspond to the label incorporated into total (m + s) and stable (s) RNA after 15s of labeling as indicated. The ratios m/(m + s) and si (m + s) correspond to the fractions of the instantaneous rate of RNA synthesis that is mRNA or stable RNA, respectively. (For further details, see ref. 4). (b, c) The kinetics shown in (a) were resolved into their two components, the labeling kinetics of stable rRNA and tRNA (b) and of unstable mRNA (c); the sum of the two corresponding curves in (b) and (c) is the curve in (a). The final levels of the curves in (b) correspond to the observed final levels in (a). (d) Comparison of the labeling of total RNA ( , same as top curve in a) and of stable RNA (--t-, ... same as top curves ind b and e). (e, The kinetics shown in (a) were resolved into their two components
1113
this redistribution, particularly the sevenfold increase that occurs when CAM is added to an exponential culture growing in succinate medium (Table 1: rs increases from 2.1 x 105 to 14.2 x 105 RNA nucleotides/min per genome). In this case, it is estimated that the number of growing RNA chains per genome doubles from 170 to 340; i.e., CAM produces a twofold "activation" of RNA polymerase. The lesser activation values (15 to 35%) estimated for CAM addition to glucose-minimal- or glucose-amino acids-grown bacteria, or for a nutritional shift-up, are marginally significant. The activation values depend on the fraction (p) of radioactively pulse-labeled RNA that is rRNA and tRNA, and on the stable RNA synthesis rate. These two parameters are measured with 10 and 5% accuracy, respectively (27); i.e., the main source of error comes from the determination of p, which produces a similar error in ip,. For glucose-amino acids-grown bacteria (Fig. 1), the minimum , value measured during exponential growth is 0.58 (27); the maximum ip8 value after CAM is obviously 1.0. Thus, CAM increases 'P, maximally 1.7fold. The observed increase in the stable RNA synthesis in this case varies between 1.8- and 2.0-fold (27), which is still greater than the maximum increase in 4u., suggesting that some activation of RNA polymerase does in fact occur. The distribution of RNA polymerase over stable RNA and mRNA genes is assumed to be regulated by the intracellular concentration of guanosine tetraphosphate (ppGpp), which affects the affinity of RNA polymerase for the rRNA promoters (28, 30). Both CAM and a shift-up reduce the synthesis of ppGpp. In exponentially growing E. coli bacteria more than 50% of the total RNA polymerase is not functioning at any time (3, 18). It was not known whether this nonfunctioning RNA polymerase can be activated. The estimates of Table 1 (increase in N,) suggest that such an activation occurs, although in most instances only to a modest extent. Initiator fmet tRNAfIet and elongation factor TuTs, which have been reported to stimulate RNA polymerase activity in vitro (26, 29), might be responsible for the in vivo activation of RNA polymerase inferred here. as in (b) and (c) but under the assumption that part of the rRNA is unstable (see above and text). The graph shows that this assumption does not affect the value of the relative synthesis rate of mRNA [differences (m + s) and (s)].
1114
SHEN AND BREMER I
I
J. BACTERIOL.
I
I
60
80
7
6
r
~5
\
23s
0
;~~~ 16s
o
20
\
\ 40
Time after rifampicin (sec FIG. 4. Determination of the synthesis time of rRNA from the radioactive labeling of 23S and 16S rRNA after rifampin addition (schematic example). Rifampin was given at zero time; radioactive uridine was given at the times indicated on the abscissa. The radioactivity in 23S and 16S molecules was determined 15 min later, when all unstable RNA (mRNA and rRNA spacer material) had decayed. After rifampin addition, only nascent RNA chains were labeled; the later after rifampin the labeling began, the fewer molecules were labeled, hence the decreasing curves (for details of the analysis and theoretical expectation, see ref. 5). Symbols: (---) Theoretical expectation if (i) the specific radioactivity of precursors is immediately constant (see below); (ii) the rRNA chain elongation rate is 75 nucleotidesls; (iii) the 3' ends of the (mature) 16S and 23S portions are 1,800 and 5,600 nucleotides distant from the rRNA promoter (i.e., the times at which no more label will enter 16S and 23S molecules are 24 and 75 s, respectively). The horizontal distance between the 16S and 23S curve (+-) is 51 s, corresponding to a chain growth rate of (5,600 minus 1,800)151 = 75 nucleotidesis. If the specific radioactivity of the precursor pool would increase with labeling time, both curves would be shifted to the left (i.e., actually lowered) without affecting the horizontal distance (5). ( ) Observed curves for glucose-amino acids medium (5). The observed horizontal distance was only 32 s, suggesting a much higher chain elongation rate (the estimate was 106 nucleotidesls, based on the assumption that the 3' ends of the 16S and 23S portions of the rRNA precursor were 3,000 nucleotides apart, rather than 3,800). (....) Expected curve if all rRNA synthesized later than 55 s were unstable and all rRNA synthesized earlier were stable. The observed curves suggest that (i) very little of the rRNA synthesized from 0 to 25 s after rifampin
Synthesis of tRNA after rifampin addition. The 4S material labeled in the presence of rifampin (Fig. 1) is assumed to be tRNA because of its stability (Fig. lb) and because its electrophoresis distribution coincides with that of tRNA (Fig. 2). There is no other RNA known to exist with these properties. However, early after rifampin addition this material mightbe contaminated by intermediates of rRNA maturation, which might be the cause of the small break in the 4S curves (Fig. la) around 55 s. The synthesis of a substantial amount of tRNA after rifampin addition has also been observed previously (24). It indicates the existence of tRNA genes at the end of long transcriptional units, in fact, as long as rRNA transcriptional units. Further, these transcriptional units must be transcribed about as frequently as rRNA genes, and their transcription must increase after CAM addition to the same extent as rRNA. Judging from the slope of the curves in Fig. 1, this tRNA accounts for 20 to 30% of the total tRNA synthesis. Since there are 10 tRNA molecules synthesized for every ribosome, the results would obtain if two tRNA genes were located at the 3' end of the rRNA transcriptional units. Synthesis of tRNA from genes located in the spacer between 16 and 23S rRNA should stop about 30 s after rifampin addition; thus, this tRNA cannot account for the bulk of the tRNA observed. Currently, there is no indication for additional tRNA genes at the 3' ends of the rRNA precursors (31; D. Schlessinger, personal communication). However, since no other class of long transcript is made with sufficient frequency, at least three speculative possibilities remain: (i) some tRNA promoters are relatively rifampin resistant (this possibility is unlikely since it implies that the rifampin-resistant promoters become rifampin sensitive precisely at 75 to 80 s after rifampin addition, independent of the rifampin concentration used); or (ii) the full rRNA transcripts are even longer in vivo than the 30S pre-rRNA, and fragiments containing tRNA are processed off during transcription by a hypothetical enzyme still active in the mutant that permitted the discovery of the 30S transcript. (iii) The results could also be explained by the existence of long clusters of tRNA genes at the 3' end of less frequently is degraded (16S RNA; parallel slopes of the observed and expected 23S curve in this range); (ii) all (23S) rRNA synthesized after 55 s is degraded; (iii) between 25 and 55 s, an increasing fraction of 23S RNA is degraded (changing vertical distance between the 23S curves indicated by and . ).
rRNA CHAIN ELONGATION AFTER CAM
VOL. 130, 1977
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TABLE 1. Effect of growth medium and CAM on the distribution and activity of RNA polymerase Growth medium
Parameter"
Units (symbol)
Growth rate*
Doublings/h (j)
Stable RNA synthesis rate*
Nucleotides x 10-l/ min per genome
Glucose + Succinate
Glucose
0.67
1.36
From ref. 6, 7, and 27; the values in ref. 6 and 7 were multiplied by 1.2 to include rRNA spacer material (see text)
(r.) Exponential growth
2.1 14.2
5' after CAM 5' shift-upb
Relative rate of stable RNA synthesis*
RNA polymerase synthesizing stable RNA
Exponential growth
c
0.81 0.98 0.81 = p/[p + (1 - p)c,Icm] (See ref. 5); c, = stable RNA
chain growth rate = 75 nu0.26 0.77
0.52 0.87
0.65 0.97 0.65
Nascent RNA chains/genome
(Np)
cleotides/s (ref. 20); cm
170 340 (100)C
340 390 (15)
=
mRNA chain growth rate = 50 nucleotides/s (ref. 2, 5)
Np
5' shift-up" b
0.63 0.91
Fraction of total functioning RNA polymerase (%')
5' after CAM a
21 39 6.7
From ref. 6, 7, and 27; the values in ref. 6 and 7 were multiplied by 1.2 to include rRNA spacer material (see text) 0.35 0.84
Exponential growth 5' after CAM 5' shift-upb Number of functioning RNA polymerase
8.2 15.6
Fraction of stable/ total RNA synthesis (p)
Exponential growth 5' after CAM 5' shift-upb
Equation or reference
amino acids 2.14
=
rJ(cs s.)
670 840 (25 230 (35)
Parameters marked with asterisks were measured; the others were calculated. Shift-up from succinate-minimal to glucose-amino acids medium; observed parameters from ref. 7. Numbers in parentheses give the percent increase (activation of RNA polymerase).
transcribed (non-rRNA) precursors coregulated with rRNA. In this case the clustering of many tRNA genes compensates for the infrequent transcription. ACKNOWLEDGMENTS This work was supported by Public Health Service grant 15412 from the National Institute of General Medical Sciences.
We thank D. Schlessinger for his critical reading of this manuscript.
LITERATURE CITED 1. Bremer, H., J. Hymes, and P. Dennis. 1974. Ribosomal RNA chain growth rate and RNA labeling patterns in Escherichia coli B/r. J. Theor. Biol. 45:379-404. 2. Bremer, H., and D. Yuan. 1968. RNA chain growth rate in Escherichia coli. J. Mol. Biol. 38:163-180. 3. Dalbow, D. 1973. Synthesis of RNA polymerase in Escherichia coli B/r growing at different rates. J. Mol. Biol. 75:181-184. 4. Dennis, P., and H. Bremer. 1973. A method for determination of the synthesis rate of stable and unstable RNA in Escherichia coli. Anal. Biochem. 56:489-501. 5. Dennis, P., and H. Bremer. 1973. Regulation of RNA
synthesis in E. coli B/r: an analysis of shift-up. I. Ribosomal RNA chain growth rate. J. Mol. Biol. 75:145-159. 6. Dennis, P., and H. Bremer. 1974. Macromolecular composition during steady-state growth of Escherichia coli B/r. J. Bacteriol. 119:270-281. 7. Dennis, P., and H. Bremer. 1974. Regulation of RNA synthesis in E. coli B/r: an analysis of a shift-up. VII. Stable RNA synthesis rate and ribosomal RNA chain growth rate following a shift-up. J. Mol. Biol. 89:233239. 8. Dubin, D. T., and A. T. Elkort. 1965. A direct demonstration of the metabolic turnover of chloramphenicol RNA. Biochim. Biophys. Acta 103:355-358. 9. Ginsburg, D., and J. A. Steitz. 1975. The 30S ribosomal precursor RNA from E. coli. A primary transcript containing 23S, 16S, and 5S sequences. J. Biol. Chem. 250:5647-5654. 10. Hayes, F., 0. M. Vasseur, N. Nikolaev, D. Schiessinger, J. Sriwidada, A. Krol, and C. Branlant. 1975. Structure of a 30S pre-ribosomal RNA of E. coli. FEBS Lett. 56:85-91. 11. Helmstetter, C. E. 1967. Rate of DNA synthesis during the division cycle of E. coli B/r. J. Mol. Biol. 24:417427. 12. Jordan, B. R., J. Feunteun, and R. Monier. 1970. Identification of a 5S rRNA precursor in exponentially
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SHEN AND BREMER
growing E. coli cell. J. Mol. Biol. 50:605-615. 13. Kurland, C. G., and 0. Maaloe. 1962. Regulation of ribosomal and transfer RNA synthesis. J. Mol. Biol. 4:193-210. 14. Lazzarini, R. %A., and E. Santangelo. 1968. Effect of chloramphenicol on the synthesis and stability of ribonucleic acid in Bacillus subtilis. J. Bacteriol. 95:1212-1220. 15. Lund, E., J. E. Dahlberg, L. Lindahl, S. R. Jaskunas, P. Dennis, and M. Nomura. 1976. Transfer RNA genes between 16S and 23S rRNA genes in rRNA transcription units of E. coli. Cell 1:165-177. 16. Maaloe, 0. 1969. An analysis of bacterial growth. Dev. Biol. Symp. 3:33-58. 17. Manor, H., D. Goodman, and G. S. Stent. 1969. RNA chain growth rate in E. coli. J. Mol. Biol. 39:1-23. 18. Matzura, H., B. Hansen, and J. Zeuthen. 1973. Biosynthesis of the and 8' subunits of RNA polymerase in Escherichia coli. J. Mol. Biol. 74:9-20. 19. Midgeley, J. E. M., and W. J. H. Gray. 1971. The control of RNA synthesis in bacteria. The synthesis and stability of RNA in chloramphenicol-inhibited cultures of E. coli. Biochem. J. 122:149-159. 20. Molin, S. 1976. Control of ribosome synthesis, p. 333341. In N. 0. Kjeldgaard and 0. Maaloe (ed.), Alfred Benzon Symposium IX, Munksgaard. Academic Press Inc., New York. 21. Neidhardt, C. F., and F. Gros. 1957. Metabolic instability of the RNA synthesized by E. coli in the presence of chloromycetin. Biochim. Biophys. Acta 25:513-520. 22. Nierlich, D. P. 1972. Regulation of RNA synthesis in growing bacterial cells. II. Control over the composition of the newly made RNA. J. Mol. Biol. 72:765-777.
J. BACTERIOL. 23. Nikolaev, N., D. Schiessinger, and P. K. Wellauer. 1974. 30S preribosomal RNA of Escherichia coli and products of cleavage by RNase III: length and molecular weight. J. Mol. Biol. 86:741-747. 24. Pato, M., and K. von Meyenburg. 1970. Residual RNA synthesis in Escherichia coli after inhibition of initiation of transcription by rifampicin. Cold Spring Harbor Symp. Quant. Biol. 35:497-504. 25. Peacock, A. C., and C. W. Dingman. 1967. Resolution of multiple RNA species by polyacrylamide gel electrophoresis. Biochemistry 6:1818-1827. 26. Pongs, O., and N. Ulbrich. 1976. Specific binding of formylated initiator tRNA to E. coli RNA polymerase. Proc. Natl. Acad. Sci. U.S.A. 73:3064-3067. 27. Shen, V., and H. Bremer. 1976. Chloramphenicol-induced changes in the synthesis of ribosomal, transfer, and messenger ribonucleic acids in Escherichia coli B/r. J. Bacteriol. 130:1098-1108. 28. Travers, A. 1976. Modulation of RNA polymerase specificity by ppGpp. Mol. Gen. Genet. 147:225-232. 29. Travers, A. 1976. RNA polymerase specificity and the control of growth. Nature (London) 263:641-646. 30. van Ooyen, A. J. J., M. Gruber, and P. Jorgenson. 1976. The mechanism of action of ppGpp on rRNA synthesis in vitro. Cell 8:123-128. 31. Wu, M., and N. Davidson. 1975. Use of gene 32 protein staining of SS polynucleotides for gene mapping by electron microscopy: application to the 080 d3 ilv su+7 system. Proc. Natl. Acad. Sci. U.S.A. 72:4506-4510. 32. Yuan, D., and V. Shen. 1975. Stability of ribosomal and transfer ribonucleic acid in Escherichia coli B/r after treatment with ethylenedinitrilotetraacetic acid and rifampin. J. Bacteriol. 122:425-431.
JOURNAL OF BACTUIuOWGY, June 1977, p. 1109-1116 Copyright C 1977 American Society for Microbiology
Rate of Ribosomal Ribonucleic Acid Chain Elongation in Escherichia coli B/r During Chloramphenicol Treatment V. SHEN' AND H. BREMER* University of Texas at Dallas, Richardson, Texas 75080
Received for publication 25 January 1977
In Escherichia coli B/r growing in glucose-amino acids medium, the radioactive labeling of 5S ribosomal ribonucleic acid (rRNA) and transfer RNA (tRNA) was measured after the simultaneous addition to the bacteria of chloramphenicol (CAM) (100 ,ug/ml), rifampin (200 ,.g/ml), and radioactive uracil. Accumulation of 58 rRNA ceased 85 s after the addition of rifampin, independent of the presence or absence of CAM; this indicates that CAM did not affect the rRNA chain growth rate. Together with previous measurements of the synthesis of rRNA and messenger RNA under these conditions, the results imply that CAM caused a redistribution of RNA polymerase which greatly favored stable RNA synthesis (77 to 97% of total functioning RNA polymerase engaged in synthesis of rRNA and tRNA). Further, it is inferred that RNA polymerase molecules were activated that were inactive during exponential growth. The labeling of tRNA observed under these conditions suggests the existence of clusters of tRNA genes at the 3' end of long transcripts that resemble the rRNA precursor in length and response to CAM and may be parts of rRNA transcripts.
The synthesis of ribosomal ribonucleic acid (rRNA) and transfer RNA (tRNA) in bacteria can be greatly increased by certain inhibitors of protein synthesis such as chloramphenicol (CAM) (13, 14, 19, 27). Presumably this increase reflects a ppGpp-mediated increase in the affinity of the RNA polymerase for the stable RNA promoters (28-30). To find out to what extent an increase in the RNA chain elongation rate or in the polymerase activity (number of functioning RNA polymerase) contributes to the effect of CAM on rRNA synthesis, we have here measured the chain elongation rate of rRNA in the presence of CAM. From these and previous measurements on RNA synthesis obtained under comparable conditions (6, 27) one can then calculate the number of RNA polymerase molecules engaged in RNA synthesis before and after CAM addition. The results indicate that CAM does not affect rRNA chain elongation, but it appears to activate a fraction of RNA polymerase that is inactive during exponential growth. Such an activation had been previously suspected to occur (16), but its existence could not be proven (7). The mechanism of this control of RNA polymerase activity is not known. The method used here to determine rRNA chain growth was devised by Molin (20) and
involves use of the antibiotic rifampin. As an incidental result, this method shows a partial breakdown of rRNA synthesised in the presence of rifampin. Because of this effect, we have reevaluated some of the previous data on RNA synthesis from this laboratory, which were based on the assumption that rRNA made during rifampin run-out is stable and which are used here to evaluate the CAM experiments. Another incidental result obtained here concerns the synthesis of tRNA. Measurements of tRNA were included as a control to monitor the speed with which rifampin stops RNA chain initiation. Unexpectedly, tRNA synthesis was found to continue after rifampin addition at a reduced rate in a manner that suggests a clustering of several tRNA genes at the 3' end of long transcripts that might well be the rRNA precursors. rRNA precursors are known to contain a tRNA gene in the space between the 16S and 23S genes (15), but additional tRNA genes at the 3' end of the precursors have not been found yet.
I Present address: Department of Microbiology and Immunology, Washington University School of Medicine, St. Louis, MO 63110.
1109
MATERIALS AND METHODS Bacterial strain and growth conditions. Escherichia coli B/r (ATCC 12407) was grown at 37°C in minimal C medium (11) supplemented with 0.4% glucose and a mixture of 20 L-amino acids, each to a final concentration of 20 ,ug/ml. The experimental culture was inoculated with a fresh stationaryphase culture in the same medium (400-fold dilu-
1110
J. BACTERIOL.
SHEN AND BREMER
tion). Culture growth was followed by measuring the absorbance at 460 nm (Anao) (1-cm light path). Radioactive labeling. An exponential culture was labeled with [14C]uracil (0.25 gCi/ml, 62 mCi/ mmol), beginning at anAO of 0.1. All 14C label was chased into stable nucleic acids within two generations. At an A460 of 0.5 (zero time), a portion of the 14C culture was added to CAM (100 Sg/ml). A mixture of rifampin (final concentration, 200 ,ug/ml) and [3H]uracil (final concentration, 1.1 nmol/ml, 18 Ci/ mmol) was added to the untreated portion of the culture at t = 2.5 min and to the CAM-treated portion of the culture at t = 5 min. Samples (1 ml) were taken from both cultures at 10-s intervals during the first 2 min and 5 and 18 min after the addition of rifampin and [3H]uracil. The samples. were added to an equal volume of alcohol-phenol solution at 0°C (17). After the cells were collected by centrifugation, the bacteria were resuspended in 0.2 ml of 100°C electrophoresis sample solution [0.2 x tris(hydroxymethyl)aminomethane-ethylenediaminetetraacetic acid-borate (TEB) buffer (25), 1% sodium dodecyl sulfate, 10% glycerol, 0.001% bromophenol blue], boiled for 45 s, and then stored at room temperature. Electrophoresis. A 10% polyacrylamide-bisacrylamide (19:1) slab gel (12 cm long, 1.5 mm thick) was used to separate the 5S rRNA and 4S tRNA. A small volume (75 ,u) of the lysate described above was used in each slot. Electrophoresis was at room temperature for 6 h at 10 mA in TEB buffer containing 0.1% sodium dodecyl sulfate. After electrophoresis the gels were dried on Whatman 3 MM filter paper and autoradiographed for 3 days on a sheet of Kodak PR Royal X-matic film. After development, the film was scanned on a Joyce-Loebl densitometer to locate the RNA bands. The gel (with the filter paper) was then cut into 2-mm slices. The RNA in the slices was hydrolyzed in 0.5 ml of 0.2 N NaOH overnight, and the radioactivity was counted after addition of 5 ml of a Biosolv-3-toluene mixture (Beckman) in a liquid scintillation spectrophotometer.
RESULTS The time required for the transcription of the main portion of the rRNA precursor can be measured by following the accumulation of 5S rRNA after inhibition of RNA chain initiation by rifampin (20). 58 rRNA is cotranscribed with 16S anad 23S rRNA (9, 10, 12, 15, 31). The end of the 5S section of the rRNA precursor is about 6,100 nucleotides distant from the rRNA promoter (10, 23, 31) so that, after rifampin addition, 5S rRNA is expected to accumulate at the pre-rifampin rate for the time it takes RNA polymerase molecules to transcribe 6,100 rRNA nucleotides; then further accumulation of 5S RNA should stop. In the experiment of Fig. la (closed circles) this time was 85 s; in Molin's experiments it was between 70 and 80 s (20). This time corresponds to a chain growth rate of approximately 75 nucleotides/s. The longer time in our experiment (85 s) might indicate a
slightly slower action of rifampin, since our strain was not rifampin permeable as was Molin's. If CAM is added to the culture 5 min before rifampin, the initial rate of accumulation of 5S RNA is twofold stimulated, as we have observed previously with this growth medium (glucose-amino acids) in the absence of rifampin (see 27), but the time it took to reach the maximum accumulation was not significantly altered (Fig. la, open circles). Thus, CAM did not affect the rRNA chain growth rate, and therefore the stimulation of rRNA and tRNA synthesis by CAM must involve an activation of RNA polymerase (see Discussion). Much of the 5S RNA synthesized in the presence of rifampin is unstable (Fig. lb), as was reported by Molin (20). Although this instability affects the kinetics of accumulation, it should not affect the time at which the break occurs in the accumulation curve due to the cessation of 5S RNA synthesis. The greater instability of rRNA synthesized in the presence of CAM (8, 14, 21, 27) shows up in the 5S curve immediately after labeling as a decrease in the rate of accumulation (Fig. la, open circles). The accumulation of 4S RNA after rifampin addition was immediately reduced to about one-third of the pre-refampin rate (Fig. la, triangular symbols; the increase in the isotope ratio is a measure for the increase in the specific radioactivity of RNA, which, without rifampin, is the same for all species of stable RNA; reference 32). Thus, rifampin stopped initiation of about two-thirds of the 4S RNA chains within 10 s, whereas the remaining onethird of the rate of 4S RNA synthesis stopped only after 80 to 100 s, again independent of the presence or absence of CAM. Most of this 4S RNA is assumed to be tRNA (see Discussion). This suggests that 20 to 30% of the tRNA synthesized in bacteria was transcribed at the end
+CAMp
20.
42
1.0
C,~~~~~~~-A
7%m Time after rifompicin (see)
Time after rifompicin (min )
FIG. 1. Labeling of 5S and 4S RNA in the presence of rifampin. Rifampin and [3H]uridine were added together at zero time (0, A); CAM was given 5 min earlier (O, A). For experimental details see the text. (a and b) Different time scales for the same experiment.
1111
rRNA CHAIN ELONGATION AFTER CAM
VOL. 130, 1977
of long transcriptional units, which are similar to rRNA precursors in length and response to CAM (see Discussion); 30% is a maximum estimate since the slope of the tRNA curve is decreasing. A difficulty in the interpretation of the distributions of pulse-labeled RNA molecules arises from the contamination of 5S and 4S RNA species by other labeled nascent or unstable RNA molecules about 70 to 130 nucleotides long. In the data points of Fig. 1 this nascent RNA is included. We have redrawn the kinetics of Fig. 1 after subtraction of what we consider a maximum value for the background of radioactivity in nascent RNA, as indicated in the distributions of Fig. 2 (long dashes). The time of the break did not change by this procedure; however, the initial kinetic slopes were reduced, giving the impression of a 20- to 30-s labeling lag. Since this is unreasonably long in view of the fact that the lag in the labeling of total RNA under these conditions was only 5 to 10 s (independent of rifampin and CAM), we believe that the results are best described by the plot in Fig. 1, although fine details in these kinetics (e.g., the small break at 55 s in the 4S RNA kinetics) may not be significant.
DISCUSSION Control of the rRNA synthesis rate. The sudden increase in the rate of stable RNA synthesis after CAM or after a nutritional shift-up +CAM
32P
62"
might involve a redistribution of functioning RNA polymerase molecules over stable and messenger RNA (mRNA) genes or an activation of an "inactive reserve" of RNA polymerase (16). To find out to what extent redistribution or activation of RNA polymerase actually occurs, rRNA chain growth measurements such as in Fig. 1 must be combined with measurements of the synthesis rate of stable RNA and mRNA after a nutritional shift-up (7) and after CAM addition (27). Some of those previous measurements need a reevaluation due to the instability of rRNA synthesized in the presence of rifampin (20; Fig. 1) and due to the more detailed knowledge now available about the spacer regions in the rRNA precursors. In the following, we (i) discuss the implications of the instability of rRNA synthesized in the presence of rifampin for the previous measurements of RNA synthesis-related parameters, and (ii) estimate the distribution and functional activity of RNA polymerase under various growth conditions. (i) Instability of rRNA synthesized in the presence of rifampin: Implications for the interpretation of previous experiments on the control of RNA synthesis. The relative synthesis rate of mRNA and the chain elongation rate of rRNA were previously determined in this laboratory from the kinetics of RNA labeling in the presence of rifampin (1, 4-7). To evaluate those experiments, it was assumed that rRNA synthesized in the presence of rifampin is com121"
9
5
19.5
E a. 0 2-
z
z
.-I 3
12
-CAM I
4'~~~~33" ^
At
I-I
z A'~~~p 61" -4
W+,
A
9: I-
A
I-l_
120" |_ l_ | I |
1
5
|
- l_
|
I
~~~3
A
SLICE NUMBER
FIG. 2. Electrophoresis distributions of radioactive RNA for the experiment shown in Fig. 1. Symbols: (*) 3H label; (0) 14C label; (- - -) maximum background due to nascent RNA (see text); (-) ranges in which the radioactivities were summed for the formation of the 3HI14C ratio plotted in Fig. 1. At early times, 5S rRNA is in precursor form (10).
1112
J. BACTERIOL.
SHEN AND BREMER
pletely stable; this is evidently not the case (20; Fig. lb). In the following, we shall discuss the possible errors in these estimates due to the partial instability of rRNA and their implications for the previous conclusion that there is no activation, but only a redistribution, of functioning RNA polymerase after a nutritional shift-up. Figure lb (closed circles) and Molin's data (20) indicate that about half of the 5S rRNA synthesized in the presence of rifampin decays within 15 min, whereas the other half is more stable. Most likely, the more stable portion of rRNA was synthesized early after the addition of rifampin to the culture and was stabilized by its incorporation into rather complete ribosomal particles, whereas the unstable portion was synthesized late after the addition ofrifampin and owes its instability to the lack of some ribosomal protein. This idea is consistent with our observations that stable rRNA can no longer be labeled 60 after rifampin addition (5), whereas rRNA labeled 2.0 min before rifampin addition is quite stable during 15 min in the presence of rifampin (32). In this case, the previous estimate of the synthesis rate of mRNA would still be correct (Fig. 3), but the rRNA chain growth rate would have been overestimated (Fig. 4). Both these implications agree with more direct measurements of the rate of mRNA synthesis by hybridization competition (27) and of the rRNA chain growth rate (20; Fig. 1). (If rRNA decayed at random with the same probability early or late after rifampin addition and with the same probability for all species of rRNA, then the synthesis rate of mRNA would have been overestimated by the rifampin method because part of the unstable RNA presumed to be mRNA would actually be rRNA, but the chain growth estimate should not be affected [see Discussion in reference 4].) We have now recalculated the distribution of RNA polymerase molecules over stable RNA and mRNA genes for E. coli B/r growing exponentially in three different media (succinate, glucose, and glucose plus 20 amino acids) and in the same media 5 min after the addition of CAM and after a nutritional shift-up on the following three assumptions. (i) The rRNA chain growth rate is 75 nucleotides/s, independent of the bacterial growth rate (20; Fig. la), rather than increasing with growth rate from 75 to 100 nucleotides/s (4), whereas the mRNA chain growth rate is 50 nucleotides/s (2; Discussion in reference 5). (ii) The rRNA precursor is 6,250 nucleotides long (23), of which about 5,100 nucleotides are incorporated into stable RNA (23S 3,200, 16S s
=
= 1,600, 5S = 120; 2 tRNA = 180; the number of tRNA genes per rRNA precursor is not exactly known [see below], but an error of 1 tRNA would make only a 1.5% difference). This means that the synthesis of total "stable" RNA, including spacer regions, is 1.2-fold greater than the observed accumulation of rRNA and tRNA, assuming that the total stable RNA in the cell consists of 85% rRNA and 15% tRNA (one-fifth of which may be drived from rRNA precursors). (iii) The fraction of the instantaneous rate of RNA synthesis that is stable RNA (p) or mRNA (1 - p) has been measured correctly by the rifampin method (4, 6) and by deoxyribonucleic acid-RNA hybridization (27); both methods gave identical values. However, if one uses mature rRNA for hybridization competition experiments, then one neglects the rRNA spacer material and counts it as mRNA; the same mistake is made with the rifampin method, which depends on the stability of rRNA and tRNA synthesized during the first 20 to 30 s after the addition of rifampin (Fig. 3) and which thus excludes the unstable spacers. Hence, these pvalues require a correction (factor 1.2 as above) if the spacer material is to be included (i.e., if stable RNA synthesis shall mean non-mRNA). The data derived from these assumptions are summarized in Table 1, which is discussed below. (ii) Distribution and function of RNA polymerase under various conditions of growth. According to Table 1, both CAM and a nutritional shift-up produce a redistribution, i.e., a shift of RNA polymerase from mRNA genes to stable RNA genes, and an activation of RNA polymerase: the redistribution is given by the increase in the parameter s (fraction of functioning RNA polymerase engaged in the synthesis of stable RNA); the activation is given by the increase in Np (number of functioning RNA polymerase molecules per genome, Table 1). Under either condition, the main effect is caused by the redistribution, as was concluded previously for the shift-up (7). For example, the shift from succinate- to glucose-amino acids medium causes an immediate 2.3-fold increase in s (from 0.35 to 0.81) and a 1.35-fold increase in Np (from 170 to 230 nascent RNA chains per genome). CAM causes an extreme reduction in the fraction of polymerase molecules transcribing mRNA genes; i.e., in CAM almost all (77 to 97%) functioning RNA polymerase transcribes stable RNA genes. Nevertheless, the CAM-induced increase in the rate of stable RNA synthesis cannot be completely accounted for by
VOL. 130, 1977
rRNA CHAIN ELONGATION AFTER CAM
0
-o
E
C.o a0 = 0.4
-
,
=
=
~0 o
b
0
00.4
O2
S_
_
.
1
Time after rifampicin(min) FIG. 3. Determination of the relative rate of synthesis ofmRNA [mI(m + s)] and ofstable RNA [s/(m + s)] from the kinetics of RNA labeling in the presence of rifampin (schematic example). (a-c) Evaluation under the assumption that all rRNA and tRNA synthesized in the presence of rifampin is stable. (dfJ Evaluation ifrRNA synthesized late after rifampin is unstable. (a) Observed kinetics of RNA labeling (5, 24). Radioactive uridine was given either at zero time, together with rifampin ( ), or 15 s after rifampin (-- -). Two early and two late samples (0) were taken (simultaneously) from both labeling tubes; the differences correspond to the label incorporated into total (m + s) and stable (s) RNA after 15s of labeling as indicated. The ratios m/(m + s) and si (m + s) correspond to the fractions of the instantaneous rate of RNA synthesis that is mRNA or stable RNA, respectively. (For further details, see ref. 4). (b, c) The kinetics shown in (a) were resolved into their two components, the labeling kinetics of stable rRNA and tRNA (b) and of unstable mRNA (c); the sum of the two corresponding curves in (b) and (c) is the curve in (a). The final levels of the curves in (b) correspond to the observed final levels in (a). (d) Comparison of the labeling of total RNA ( , same as top curve in a) and of stable RNA (--t-, ... same as top curves ind b and e). (e, The kinetics shown in (a) were resolved into their two components
1113
this redistribution, particularly the sevenfold increase that occurs when CAM is added to an exponential culture growing in succinate medium (Table 1: rs increases from 2.1 x 105 to 14.2 x 105 RNA nucleotides/min per genome). In this case, it is estimated that the number of growing RNA chains per genome doubles from 170 to 340; i.e., CAM produces a twofold "activation" of RNA polymerase. The lesser activation values (15 to 35%) estimated for CAM addition to glucose-minimal- or glucose-amino acids-grown bacteria, or for a nutritional shift-up, are marginally significant. The activation values depend on the fraction (p) of radioactively pulse-labeled RNA that is rRNA and tRNA, and on the stable RNA synthesis rate. These two parameters are measured with 10 and 5% accuracy, respectively (27); i.e., the main source of error comes from the determination of p, which produces a similar error in ip,. For glucose-amino acids-grown bacteria (Fig. 1), the minimum , value measured during exponential growth is 0.58 (27); the maximum ip8 value after CAM is obviously 1.0. Thus, CAM increases 'P, maximally 1.7fold. The observed increase in the stable RNA synthesis in this case varies between 1.8- and 2.0-fold (27), which is still greater than the maximum increase in 4u., suggesting that some activation of RNA polymerase does in fact occur. The distribution of RNA polymerase over stable RNA and mRNA genes is assumed to be regulated by the intracellular concentration of guanosine tetraphosphate (ppGpp), which affects the affinity of RNA polymerase for the rRNA promoters (28, 30). Both CAM and a shift-up reduce the synthesis of ppGpp. In exponentially growing E. coli bacteria more than 50% of the total RNA polymerase is not functioning at any time (3, 18). It was not known whether this nonfunctioning RNA polymerase can be activated. The estimates of Table 1 (increase in N,) suggest that such an activation occurs, although in most instances only to a modest extent. Initiator fmet tRNAfIet and elongation factor TuTs, which have been reported to stimulate RNA polymerase activity in vitro (26, 29), might be responsible for the in vivo activation of RNA polymerase inferred here. as in (b) and (c) but under the assumption that part of the rRNA is unstable (see above and text). The graph shows that this assumption does not affect the value of the relative synthesis rate of mRNA [differences (m + s) and (s)].
1114
SHEN AND BREMER I
I
J. BACTERIOL.
I
I
60
80
7
6
r
~5
\
23s
0
;~~~ 16s
o
20
\
\ 40
Time after rifampicin (sec FIG. 4. Determination of the synthesis time of rRNA from the radioactive labeling of 23S and 16S rRNA after rifampin addition (schematic example). Rifampin was given at zero time; radioactive uridine was given at the times indicated on the abscissa. The radioactivity in 23S and 16S molecules was determined 15 min later, when all unstable RNA (mRNA and rRNA spacer material) had decayed. After rifampin addition, only nascent RNA chains were labeled; the later after rifampin the labeling began, the fewer molecules were labeled, hence the decreasing curves (for details of the analysis and theoretical expectation, see ref. 5). Symbols: (---) Theoretical expectation if (i) the specific radioactivity of precursors is immediately constant (see below); (ii) the rRNA chain elongation rate is 75 nucleotidesls; (iii) the 3' ends of the (mature) 16S and 23S portions are 1,800 and 5,600 nucleotides distant from the rRNA promoter (i.e., the times at which no more label will enter 16S and 23S molecules are 24 and 75 s, respectively). The horizontal distance between the 16S and 23S curve (+-) is 51 s, corresponding to a chain growth rate of (5,600 minus 1,800)151 = 75 nucleotidesis. If the specific radioactivity of the precursor pool would increase with labeling time, both curves would be shifted to the left (i.e., actually lowered) without affecting the horizontal distance (5). ( ) Observed curves for glucose-amino acids medium (5). The observed horizontal distance was only 32 s, suggesting a much higher chain elongation rate (the estimate was 106 nucleotidesls, based on the assumption that the 3' ends of the 16S and 23S portions of the rRNA precursor were 3,000 nucleotides apart, rather than 3,800). (....) Expected curve if all rRNA synthesized later than 55 s were unstable and all rRNA synthesized earlier were stable. The observed curves suggest that (i) very little of the rRNA synthesized from 0 to 25 s after rifampin
Synthesis of tRNA after rifampin addition. The 4S material labeled in the presence of rifampin (Fig. 1) is assumed to be tRNA because of its stability (Fig. lb) and because its electrophoresis distribution coincides with that of tRNA (Fig. 2). There is no other RNA known to exist with these properties. However, early after rifampin addition this material mightbe contaminated by intermediates of rRNA maturation, which might be the cause of the small break in the 4S curves (Fig. la) around 55 s. The synthesis of a substantial amount of tRNA after rifampin addition has also been observed previously (24). It indicates the existence of tRNA genes at the end of long transcriptional units, in fact, as long as rRNA transcriptional units. Further, these transcriptional units must be transcribed about as frequently as rRNA genes, and their transcription must increase after CAM addition to the same extent as rRNA. Judging from the slope of the curves in Fig. 1, this tRNA accounts for 20 to 30% of the total tRNA synthesis. Since there are 10 tRNA molecules synthesized for every ribosome, the results would obtain if two tRNA genes were located at the 3' end of the rRNA transcriptional units. Synthesis of tRNA from genes located in the spacer between 16 and 23S rRNA should stop about 30 s after rifampin addition; thus, this tRNA cannot account for the bulk of the tRNA observed. Currently, there is no indication for additional tRNA genes at the 3' ends of the rRNA precursors (31; D. Schlessinger, personal communication). However, since no other class of long transcript is made with sufficient frequency, at least three speculative possibilities remain: (i) some tRNA promoters are relatively rifampin resistant (this possibility is unlikely since it implies that the rifampin-resistant promoters become rifampin sensitive precisely at 75 to 80 s after rifampin addition, independent of the rifampin concentration used); or (ii) the full rRNA transcripts are even longer in vivo than the 30S pre-rRNA, and fragiments containing tRNA are processed off during transcription by a hypothetical enzyme still active in the mutant that permitted the discovery of the 30S transcript. (iii) The results could also be explained by the existence of long clusters of tRNA genes at the 3' end of less frequently is degraded (16S RNA; parallel slopes of the observed and expected 23S curve in this range); (ii) all (23S) rRNA synthesized after 55 s is degraded; (iii) between 25 and 55 s, an increasing fraction of 23S RNA is degraded (changing vertical distance between the 23S curves indicated by and . ).
rRNA CHAIN ELONGATION AFTER CAM
VOL. 130, 1977
1115
TABLE 1. Effect of growth medium and CAM on the distribution and activity of RNA polymerase Growth medium
Parameter"
Units (symbol)
Growth rate*
Doublings/h (j)
Stable RNA synthesis rate*
Nucleotides x 10-l/ min per genome
Glucose + Succinate
Glucose
0.67
1.36
From ref. 6, 7, and 27; the values in ref. 6 and 7 were multiplied by 1.2 to include rRNA spacer material (see text)
(r.) Exponential growth
2.1 14.2
5' after CAM 5' shift-upb
Relative rate of stable RNA synthesis*
RNA polymerase synthesizing stable RNA
Exponential growth
c
0.81 0.98 0.81 = p/[p + (1 - p)c,Icm] (See ref. 5); c, = stable RNA
chain growth rate = 75 nu0.26 0.77
0.52 0.87
0.65 0.97 0.65
Nascent RNA chains/genome
(Np)
cleotides/s (ref. 20); cm
170 340 (100)C
340 390 (15)
=
mRNA chain growth rate = 50 nucleotides/s (ref. 2, 5)
Np
5' shift-up" b
0.63 0.91
Fraction of total functioning RNA polymerase (%')
5' after CAM a
21 39 6.7
From ref. 6, 7, and 27; the values in ref. 6 and 7 were multiplied by 1.2 to include rRNA spacer material (see text) 0.35 0.84
Exponential growth 5' after CAM 5' shift-upb Number of functioning RNA polymerase
8.2 15.6
Fraction of stable/ total RNA synthesis (p)
Exponential growth 5' after CAM 5' shift-upb
Equation or reference
amino acids 2.14
=
rJ(cs s.)
670 840 (25 230 (35)
Parameters marked with asterisks were measured; the others were calculated. Shift-up from succinate-minimal to glucose-amino acids medium; observed parameters from ref. 7. Numbers in parentheses give the percent increase (activation of RNA polymerase).
transcribed (non-rRNA) precursors coregulated with rRNA. In this case the clustering of many tRNA genes compensates for the infrequent transcription. ACKNOWLEDGMENTS This work was supported by Public Health Service grant 15412 from the National Institute of General Medical Sciences.
We thank D. Schlessinger for his critical reading of this manuscript.
LITERATURE CITED 1. Bremer, H., J. Hymes, and P. Dennis. 1974. Ribosomal RNA chain growth rate and RNA labeling patterns in Escherichia coli B/r. J. Theor. Biol. 45:379-404. 2. Bremer, H., and D. Yuan. 1968. RNA chain growth rate in Escherichia coli. J. Mol. Biol. 38:163-180. 3. Dalbow, D. 1973. Synthesis of RNA polymerase in Escherichia coli B/r growing at different rates. J. Mol. Biol. 75:181-184. 4. Dennis, P., and H. Bremer. 1973. A method for determination of the synthesis rate of stable and unstable RNA in Escherichia coli. Anal. Biochem. 56:489-501. 5. Dennis, P., and H. Bremer. 1973. Regulation of RNA
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