INTRODUCTION

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Escherichia coli cells will grow over a temperature range of about 40°C, and remarkably, the cell growth rate increases in response to increasing temperature like a simple chemical reaction in a central normal range of its growth temperatures (20 to 37°C). At both high and low temperatures, this relationship breaks down. It is thought that at low temperature, protein initiation becomes rate limiting for growth (3, 6), but it is not known what is rate limiting at high temperature. We have examined one important aspect of cell physiology, translation, as a step toward understanding the effect of temperature on cell growth.

It is known from studies of cell growth in different media that E. coli tightly controls the levels of the protein synthesizing machinery. At different growth rates at the same temperature, the rate at which ribosomes are working (peptide chain elongation rate) is fairly constant, but the number of ribosomes per cell increases markedly with increasing growth rate (15, 21). Thus, at high growth rates many ribosomes are working whereas at lower growth rates fewer ribosomes are working, but the ribosomes in both cells (growing at different rates) are working at approximately the same rate. It should be noted that there is also an increase in peptide chain elongation rate at high growth rates which may have important regulatory functions, but the change in protein synthetic capacity is primarily due to alterations in the levels of ribosomes (21). Thus, we know that cells change the number of ribosomes per cell to increase their growth rate in response to richer medium, but how do cells regulate the protein synthetic machinery in response to temperature changes? We can answer this question by determining the number of ribosomes per cell (and the fraction actually engaged in protein synthesis) and the peptide chain elongation rate; together these factors determine the protein synthetic capacity.

Previous work in this lab and others has shown that the levels of ribosomal protein and RNA are constant between 23 and 42°C in a given medium. Herendeen et al. (12) have used two-dimensional gel electrophoresis to measure the steady-state levels of ribosomal proteins in cells grown at various temperatures in glucose-rich medium. Their results show that there is only small variation in the levels of individual ribosomal proteins in cells growing in this range and that as a group they neither increase nor decrease with increasing temperature. In addition, Ryals et al. (28) have shown that the level of rRNA is constant over this temperature range.

We have determined the polypeptide chain elongation rate throughout the range of E. coli growth temperatures as well as after a shift to high temperature. In addition, we have confirmed that the level of ribosomal components is constant between 37 and 42°C and have examined the polysome profiles of cells growing at 15, 37, and 42°C. The results show that peptide chain elongation rate increases as a function of temperature. In the normal temperature range, the rate of peptide chain elongation matches the growth rate, but at both high and low temperatures there appears to be an excess of protein synthetic machinery. At low temperature this may indicate a defect in initiation of translation, but at high temperature there is not an excessive pool of nontranslating ribosomes. This indicates that protein degradation occurs at a much higher rate at the higher temperature.

	MATERIALS AND METHODS

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Strains and growth conditions. E. coli W3110 (prototrophic) was used in all experiments. Cells were grown in MOPS (3-[N-morpholine]propanesulfonic acid) medium (20) supplemented with 0.4% glucose, 20 amino acids, vitamins, and bases as previously described (36). Medium for amino acid labeling experiments was the same except leucine and methionine concentrations were lower (80 and 20 µM, respectively, instead of 800 and 200 µM) and uridine was omitted when labeling was with radioactive uridine. Cells were grown aerobically in a rotary water bath, and cell growth was followed in a Zeiss spectrophotometer at 420 nm. Cell growth was followed for at least six generations of steady-state growth before any experiments were initiated. Temperature shifts were initiated by diluting the culture growing at the preshift temperature into a flask containing 1/2 volume of culture medium preincubated at the postshift temperature.

Peptide elongation rate measured by -galactosidase and alpha fragment induction. One way to determine the peptide chain elongation rate is to measure the time it takes to make -galactosidase enzyme after induction with isopropyl--D-thiogalactopyranoside (IPTG). The time when the first -galactosidase molecule is made can be extrapolated from the graph, and since we know that -galactosidase contains 1,024 amino acids, we can calculate the number of amino acids incorporated per second. An example is shown in Fig. 1A. The square root of -galactosidase activity (minus background) initially gives a linear graph which then curves downward as mRNA breakdown becomes significant (4, 29). An example of this is shown in Fig. 1B; only the linear portion of the curve is shown. Thus, the peptide chain elongation rate can be estimated by measuring the time it takes to make the first -galactosidase molecule after induction of the lac operon. This is the time needed for induction and initiation of the gene as well as translation of -galactosidase.

View larger version (15K): [in this window] [in a new window]   FIG. 1.   Determination of polypeptide chain elongation rate by measurement of -galactosidase induction. -Galactosidase production was induced in strain W3110 at 37°C growing in glucose-rich MOPS by the addition of IPTG at time zero. (A) Enzyme activity; (B) square root of enzyme activity (E) minus background activity (Eo) plotted against time of induction. Note that some points are missing from the early portion of the graph since E  Eo was less than or equal to zero.

Peptide elongation rate measured by pulse-labeling experiments. In a second method to measure the elongation rate (2, 7), cells are pulsed with a radioactive amino acid ([³H]leucine) for a short time (5 to 15 s) and then chased with an unlabeled amino acid. The pulse is so short that only a fraction of each polypeptide being translated at the time is labeled. During the chase, samples are taken periodically to measure radioactivity in completed protein. Radioactivity is chased into completed protein until the polypeptide initiated during the pulse (and thus with only amino acids near its N terminus labeled) is finally completed and no more radioactivity is chased into the completed protein. The time when this occurs is the translation time of the protein. Radioactivity in completed protein is followed by separating proteins by two-dimensional gel electrophoresis and determining the amount of radioactivity in individual protein spots. A control culture labeled with another isotope ([³⁵S]methionine) is mixed with the samples to control for sampling error.

Two-dimensional gel electrophoresis and measurement of radioactivity in protein spots. Two-dimensional gel electrophoresis was performed as previously described (1, 23, 35). Extracts were made, and half was placed on each of two gels. These duplicate gels were stained with Coomassie blue and dried, and autoradiograms were prepared. Spots were excised and prepared for quantification as described previously (24). Trichloroacetic acid precipitation was done to obtain the ³H/³⁵S ratio in each sample extract.

Calculation of radioactivity for pulse-chase experiments. The radioactivity in completed protein is expressed as ³H/³⁵S in the spot divided by ³H/³⁵S in total protein (trichloroacetic acid precipitates). In addition, a correction for the amino acid composition of each protein was made by multiplying the radioactivity measurement by the leucine/methionine ratio in total protein divided by the leucine/methionine ratio in the protein assayed.

RNA/protein ratios. RNA/protein ratios were calculated from colorimetric assays of RNA (orcinol method) and protein (Lowry assay). The assays were performed essentially as described elsewhere (5, 8). Five-milliliter samples of cultures growing at the appropriate temperature were taken at a number of different cell densities (OD₄₂₀ = 0.2 to 1.0) and added to 0.4 ml of ice-cold 70% perchloric acid. After incubation for 30 min on ice, the samples were centrifuged for 10 min at 12,000 × g. The precipitates were washed in 2.5 ml of 0.5 M perchloric acid and repelleted. These pellets were resuspended in 0.25 ml of water, and portions were used for protein assay and RNA assay.

Polysome isolation. Polysomes were isolated as described previously (27, 31, 32) from cells which had been labeled with radioactive uridine. Cultures were labeled for one generation with either 0.25 µCi of [¹⁴C]uridine (100 µCi/mmol; ICN) or 2.5 µCi of [³H]uridine (60 Ci/mmol; ICN) per ml and then subjected to a one-generation chase with unlabeled uridine (2 mg/ml).

	RESULTS

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Peptide chain elongation rate as determined by -galactosidase induction time. The most obvious reason for cell growth rate to increase in response to temperature is that the rate of polypeptide elongation may increase as a function of temperature, identically to the growth rate. We first wanted to determine if this was true within the normal temperature range (25 to 37°C) and then see what happens outside the normal range (i.e., at 42°C and at 10 to 20°C).

View larger version (13K): [in this window] [in a new window]   FIG. 2.   (A) Measurements of peptide chain elongation rate by the -galactosidase induction method. Peptide chain elongation rate (closed circles) was calculated from the -galactosidase induction time at the indicated temperatures. Growth rate (open circles) was determined by optical density measurements done on the same cultures. The average of data from several experiments is plotted with the standard error indicated. Points with no error bars had less than 2% standard error. aa, amino acids. (B) Peptide chain elongation rate relative to growth rate at various temperatures. Elongation rate as determined by -galactosidase induction time at each temperature is plotted versus the specific growth rate at the same temperature. The error bars represent 1 standard error. Points with no error bars had less than 2% standard error.

View larger version (17K): [in this window] [in a new window]   FIG. 3.   Peptide elongation rate at low temperature. Peptide chain elongation rate (closed circles) was calculated from the -galactosidase induction time at the indicated temperatures. Growth rate (open circles) was determined by optical density measurements done on the same cultures. The average from several experiments is plotted. The maximum standard error for any elongation rate value was 7%, and the maximum error for the growth rate was 2%. aa, amino acids.

Peptide chain elongation rate as determined by labeling kinetics. Since the -galactosidase induction method measures the translation time of a single protein, it may not be representative of the majority of proteins. Therefore, the pulse-chase method (see Materials and Methods) was used to measure the completion time of proteins at 37 and 42°C. The results of these studies confirm that the polypeptide chain elongation rate is higher at the higher temperature, although there is little difference in growth rate (Fig. 4). The elongation rates of RNA polymerase beta subunit are 24 amino acids/s at 42°C and 19 amino acids/s at 37°C. The elongation rate from the -galactosidase experiments was lower, but the difference between the two temperatures was the same (approximately 30%). For the pulse-chase experiments, we routinely examined five or six proteins (-galactosidase, RNA polymerase beta subunit, DnaK, ValS, polynucleotide phosphorylase, and pyruvate dehydrogenase), but since translation is so fast at 42°C, only the results of the largest protein examined, RNA polymerase beta subunit, were consistently usable. The other proteins gave results which either confirm the RNA polymerase results or were merely consistent with those results qualitatively. Thus, at high temperature, there appears to be an excess of protein synthetic capacity in that the cells growing at 37 and 42°C contain the same number of ribosomes and the growth rates are the same, but there is a 30% increase in polypeptide chain elongation rate at 42°C. One possibility is that a fraction of ribosomes are inactive. Similarly, pulse-chase experiments were used to confirm the -galactosidase induction results at 15°C, demonstrating that elongation rate is not limiting for growth at 15°C (data not shown).

View larger version (15K): [in this window] [in a new window]   FIG. 4.   Results of pulse-chase experiments at 37 and 42°C. Radioactivity recovered from a single protein spot (RNA polymerase beta subunit) is plotted versus time of chase. The data from three experiments at 37°C (open symbols) and two at 42°C (closed symbols) are shown. Radioactivity is expressed as described in Materials and Methods. Each point represents the average from values obtained from duplicate gels. The translation time at each temperature is indicated by an arrow. The standard errors of the calculated translation times are 2% at 37°C and 3% at 42°C.

Ribosome concentration. Previous studies have shown that the levels of ribosomal protein (12) and rRNA (28) are constant both within the normal temperature range and at high temperature. To confirm that the levels of ribosomes are constant between 37 and 42°C in our standard E. coli strain and our medium, the ratio of RNA to protein was measured in cells growing at each temperature. This ratio has been shown to reflect accurately the levels of ribosomes in cells when used to measure the levels in cells growing in different media (e.g., reference 21). These measurements showed no significant difference in the RNA/protein ratio between cells growing at 42°C (0.98 ± 0.04 [standard error]; n = 12) and those growing at 37°C (1.00 ± 0.02; n = 14). Therefore, the measurements made previously are confirmed, and we can conclude that cells growing at 37 and 42°C contain the same amount of ribosomal material.

View larger version (21K): [in this window] [in a new window]   FIG. 5.   (A) Polysome profiles from cells grown at 37 or 42°C. Cells were grown for several generations in the presence of either [14C]uridine or [3H]uridine at 37 or 42°C. Cells were mixed and polysomes were separated on sucrose gradients as described in Materials and Methods. The data were normalized such that the total radioactivity isolated in fractions 16 to 127 was the same for the two isotopes. Shown is the actual 3H counts/minute in each fraction and the normalized 14C values. Subunits and monosomes are centered as follows: 30S subunits at approximately fraction 20, 50S subunits at fraction 29, and 70S monosomes at fraction 40. (B) Polysome profiles from cells grown at 37 or 15°C. Cells grown in the presence of radioactive uridine at either 37 or 15°C were mixed, and polysomes were separated on a sucrose gradient. The data are normalized such that the total radioactivity in fractions 5 to 126 was the same for both isotopes. Shown is the actual 14C counts/minute and normalized 3H values. Subunits and monosomes are centered as: 30S subunits at fraction 20, 50S subunits at fraction 27, and 70S monosomes at fraction 38.

Temperature shifts. It is of interest now to ask what happens after a shift to higher temperature. We expect one of two results: either the polypeptide chain elongation rate increases immediately upon a shift to 42°C, presumably as a passive response to the increased temperature, or the cell needs to actively increase the elongation rate, a process which could require time.

View larger version (11K): [in this window] [in a new window]   FIG. 6.   Peptide chain elongation rate after a shift to high temperature, determined by the pulse-labeling method. Cells growing at 37°C were shifted to 42°C, and the pulse was initiated 15 s later. Labeling was terminated after 15 s, and samples were taken for two-dimensional gel electrophoresis. Shown is the radioactivity isolated from a single protein spot (RNA polymerase beta subunit) versus time of chase. Radioactivity is expressed as described in Materials and Methods. The translation time is 55 s (indicated by the arrow), and the calculated elongation rate is 24 amino acids/s. The standard error of the calculated translation time is approximately 3%.

	DISCUSSION

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The results presented have shown that within the normal temperature range, the rate of polypeptide chain elongation increases, allowing faster growth without the need for increased synthesis of ribosomes. Thus, these results fit with our current understanding of the control of protein synthesis in E. coli.

We have found that cells growing at low temperature contain a large pool of ribosomal subunits (Fig. 5B). It is quite unusual to find a circumstance where a wild-type cell (growing in steady state) has a large pool of nontranslating ribosomes. The obvious question is why doesn't the cell regulate its level of rRNA and protein such that it simply meets its needs at this temperature as it does in the normal temperature range? Cells growing at low temperature seem to require a higher concentration of ribosomes for protein synthesis. Perhaps this requirement is necessary to overcome the block in protein initiation as has been postulated (3, 6, 13), or possibly cells maintain a pool of ribosomes to meet future demands (14). Under most other conditions, E. coli cells maintain only the level of ribosomes per unit of cell mass required for protein synthesis. This seems sensible, since it would be very wasteful to maintain an excess of ribosomes. These extra ribosomes by definition do not contribute to cell growth. There are at least two situations in which it may make sense to have excess ribosomes. The first is in starvation or very slow growth. It is known that the relationship between the number of ribosomes per unit of mass and growth rate breaks down at very low growth rates (21). One reasonable model to explain this is that cells are "waiting for better days" (14). Slowly growing or starving cells which maintain a relatively high level of ribosomes would be able to respond rapidly to an increase in the nutritional richness of their medium. Applying the same logic to cells growing at low temperature, it is possible that these cells too are waiting for better days, i.e., a higher growth temperature.

When one considers our measurements of the fraction of functioning ribosomes (polysomes) at low temperature, our measurements of protein synthesis fit quite well with the observed growth rate. At 13.5°C, the levels of ribosomal proteins decrease to approximately 65% of the level at 37°C (12). The number of functional ribosomes (polysomes) is 83% of the 37°C value (Fig. 5B) and the peptide elongation rate is 11.6% of the rate at 37°C (Fig. 3). Using the 13.5°C value for the levels of ribosomal proteins (which should be close to the 15°C value), the protein synthetic capacity (number of ribosomes times the fraction working as polysomes times the elongation rate) at 15°C is 6.3% of the capacity at 37°C. This is quite similar to the difference in growth rate: cells at 15°C grow at 5.5% of the rate at 37°C.

At 42°C, where the specific growth rate is only about 2.5% higher than that at 37°C, we have shown that the number of functioning ribosomes per unit of cell mass is approximately the same as at 37°C. Second, polypeptide chain elongation rate is not rate limiting for growth; it increases in proportion to temperature even at high temperature when growth rate is no longer increasing. RNA elongation rate is assumed to increase also, since in the -galactosidase induction experiments the results are dependent on both translation and transcription rates. Also, the elongation rate of stable RNA has been shown to increase as a function of temperature and is proportional to growth rate (28). To summarize, the calculated protein synthetic capacity (defined above) at 42°C is 1.32 ± 0.05 (standard error calculated by using the Gaussian approximation formulae) relative to the value at 37°C (1.0 ± 0.05). Thus, the results obtained at high temperature are surprising since they appear to violate our current models of the regulation of protein synthesis. It is possible that, as discussed above for cells grown at low temperature, cells growing at high temperature maintain a pool of ribosomes in anticipation of future demands. However, in this case we would expect to find a pool of nontranslating ribosomes in these cells (as at low temperature), which we do not. Thus, this explanation tells us nothing about the nature of the defect in cells growing at 42°C.

A second model to explain why cells growing at high temperature appear to have an excess of protein synthetic activity involves protein degradation. It is possible that all of these ribosomes are indeed functioning and producing protein but that the rate of protein degradation is greatly increased. This model is very attractive in light of what we know about temperature upshifts and the heat shock response as discussed below. A problem with this model is that measurements of protein degradation at various temperatures have not shown an increase above the normal temperature effect (25, 26). It is possible, however, that degradation of newly synthesized protein can occur at rates high enough to account for the discrepancy. Most measurements of protein degradation have been done such that degradation only of long-lived proteins has been examined and show that 1 to 2% of cellular protein is degraded per generation (18). It is much more difficult to measure short-term degradation. Two studies have estimated the degradation of newly synthesized protein. First, Pine (26) showed that a minimum of 5% of the labeled amino acid incorporated in a 5-s pulse was released during the next 45 s. This rapid degradation was followed by a lower rate of about 2% per generation. A second series of experiments obtained similar results (37) showing that degradation of newly synthesized protein is significant and distinct from degradation of stable proteins. Furthermore, as seen in Fig. 4, we consistently find a decrease in the radioactivity in completed protein at 42°C, but not at lower temperatures, after the initial break in the curve, indicating that newly synthesized proteins are indeed degraded at a higher rate at high temperature than at normal temperatures. Thus, the rate of degradation of newly synthesized protein may be quite high (18). If, for some reason, polypeptides cannot fold as quickly at 42°C, newly synthesized protein would be more susceptible to proteolysis.

We also show in this work that peptide elongation rate increases immediately upon an upshift in temperature. Temperature upshifts induce the heat shock response, which results in the induction of 20 or so proteins, many of which are chaperones or proteases (34; reviewed in references 11 and 22). The response is mediated by the induction of the sigma factor ³². Do the observations about peptide elongation rate fit into our current thinking about the induction mechanism and function of the heat shock response?

The model of heat shock induction that results from the observations of ³² induction and heat shock protein function can be summarized as follows (reviewed in reference 11). Upon an increase in temperature, the heat shock chaperones are increasingly bound to unfolded protein and are thus titrated away from ³². This results in an increase in the stability of ³² and its rate of synthesis (30, 33). The increase in ³² levels is sufficient to induce the synthesis of the heat shock proteins, including the chaperones. When the levels of chaperones are sufficient to bind all of the cell's unfolded protein, they can bind ³² and decrease its level.

The model assumes that the concentration of unfolded protein is increased to induce a heat shock response. The source of unfolded protein probably differs in different inducing conditions, but a major source upon heat treatment is likely to be newly synthesized protein. As shown in this report, the rate of synthesis of nascent polypeptides increases immediately upon heat shock, and this may be the source of unfolded protein. To our knowledge there is no direct evidence that proteins synthesized before a temperature shift to 42°C actually unfold at the new temperature; thus, it seems likely that an imbalance between the rate of protein translation and the rate of growth triggers the heat shock response by causing a transient accumulation of newly translated, unfolded protein.

Implied in this model is that the rate of chaperone function does not increase in parallel to growth rate with increasing temperature or that the need for chaperone function increases faster than growth rate. Higher levels of the chaperones are required at higher temperatures. This could be for a number of reasons: the mechanism of chaperone function may not be a simple catalytic reaction, protein folding may be slowed by increasing temperature, or more proteins may require chaperones to help them fold at high temperature. A series of experiments by Horowitz and coworkers begins to address this issue (16, 17). They have studied the folding of rhodanese in vitro at different temperatures. Rhodanese will fold significantly at low temperature (10°C) without the presence of chaperones, but at a higher temperature (37°C), the protein precipitates. Addition of the chaperone GroEL/GroES enables the protein to fold at 37°C, with no precipitation. Interestingly, addition of the chaperone at low temperature inhibits protein folding. This system is very similar to what is observed in vivo. Chaperone synthesis (and all heat shock protein synthesis) is inhibited at low temperature but is clearly required at high temperature. Heat shock mutants of either individual chaperones or ³² form large protein-rich inclusion bodies at high temperature (reference 10 and unpublished results).

Thus, the increased rate of peptide chain elongation upon a heat shock fits this heat shock induction model quite well and in fact is a necessary part of the model. The increase in the rate of protein elongation is the trigger that generates the signal for the heat shock response.