De novo Synthesis and Assembly of rRNA into Ribosomal Subunits during Cold Acclimation in Escherichia coli

    De novo synthesis and assembly of rRNA into ribosomal subunits during cold acclimation in Escherichia coli Lolita Piersimoni, Mara Giangrossi, Paolo Marchi, Anna Brandi, Claudio O. Gualerzi, Cynthia L. Pon PII: DOI: Reference:

S0022-2836(16)00156-X doi: 10.1016/j.jmb.2016.02.026 YJMBI 65012

To appear in:

Journal of Molecular Biology

Received date: Revised date: Accepted date:

5 January 2016 25 February 2016 26 February 2016

Please cite this article as: Piersimoni, L., Giangrossi, M., Marchi, P., Brandi, A., Gualerzi, C.O. & Pon, C.L., De novo synthesis and assembly of rRNA into ribosomal subunits during cold acclimation in Escherichia coli, Journal of Molecular Biology (2016), doi: 10.1016/j.jmb.2016.02.026

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ACCEPTED MANUSCRIPT De novo synthesis and assembly of rRNA into ribosomal subunits during cold acclimation in Escherichia coli

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Lolita Piersimoni1, Mara Giangrossi2, Paolo Marchi, Anna Brandi2, Claudio O. Gualerzi and Cynthia L. Pon

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Present address: 1 Department of Biological Chemistry University of Michigan Ann Arbor, MI 48109

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Correspondence to Claudio O. Gualerzi: Laboratory of Genetics Dept. of Biosciences and Biotechnology University of Camerino, 62032 Camerino, Italy e-mail [email protected]

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Laboratory of Genetics, Department of Biosciences and Biotechnology, University of Camerino, 62032 Camerino, Italy

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School of Biosciences and Veterinary Medicine University of Camerino 62032 Camerino, Italy

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Running title: rRNA synthesis after cold stress

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ABSTRACT

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During the cold adaptation that follows a cold-stress, bacterial cells undergo many

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physiological changes and extensive reprogramming of their gene expression pattern. Bulk

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gene expression is drastically reduced, while a set of cold shock genes is selectively and transiently expressed. The initial stage of cold acclimation is characterized by the establishment of a stoichiometric imbalance of the translation initiation factors

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(IFs)/ribosomes ratio that contributes to the preferential translation of cold shock

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transcripts. Whereas de novo synthesis of the IFs following cold stress has been documented, nothing was known concerning activity of the rrn operons during the cold acclimation period. In this work we focus on the expression of the rrn operons and the fate

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of rRNA after temperature downshift. We demonstrate that in Escherichia coli, rRNA synthesis does not stop during the cold acclimation phase but continues with a greater

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contribution of the P2 compared to the P1 promoter and all seven rrn operons are active although their expression levels change with respect to pre-stress conditions. Eight hours

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after the 37°  10°C temperature downshift, the newly transcribed rRNA represents up to 20% of total rRNA and is preferentially found in the polysomes. However, with respect to the de novo synthesis of the IFs, both rRNA transcription and maturation are slowed down drastically by cold stress, thereby accounting in part for the IFs/ribosomes stoichiometric imbalance. Overall, our data indicate that new ribosomes, possibly suitable to function at low temperature, are slowly assembled during cold acclimation.

Keywords cold-shock; rrn operons; rRNA synthesis; rRNA maturation; ribosome assembly

Introduction

ACCEPTED MANUSCRIPT In all living organisms ribosome biogenesis is an essential process which accounts for a significant fraction of the cell‘s energy budget, at least in rapidly growing bacteria.

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Synthesis and assembly of an intact ribosome entails a number of complex and regulated

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functions such as rDNA transcription, rRNA processing and folding, the coordinated

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synthesis of ribosomal proteins and their stepwise binding to the nascent rRNAs, while the RNA and proteins undergo various types of post-transcriptional and post-translational modifications, respectively [1-8]. To meet its changing demand for protein synthesis as a

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function of its metabolic state, the bacterial cell adjusts the number of ribosomes in

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proportion to its growth rate [9-13] and variations of the level of ribosomes in the cell are geared to variations in the levels of the three initiation factors (IFs) IF1, IF2 and IF3. These factors are present in the cell in a sub-stoichiometric amount with respect to the ribosomes

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at a level (~ 0.15) which reflects the abundance of the “native” 30S ribosomal subunits, namely the subunits which, having a copy of each IF bound, are amenable to initiate

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protein synthesis [14].

However, in cells undergoing cold-adaptation after a cold-stress the de novo

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synthesis of IFs [15,16] produces an up to at least 3-fold increase of the IFs/ribosome ratio, thereby violating the dogma of its rigorous constancy. In turn, it has been shown that this IFs/ribosomes imbalance plays an important role in the establishment of a translational bias, whereby cold-shock mRNAs are preferentially translated whereas translation of noncold-shock mRNAs is inhibited [17-19]. Whether a blockage of ribosome synthesis and assembly may also contribute to the cold-stress-induced imbalance of the IFs/ribosomes stoichiometric ratio remained an open question that the present study sought to answer by investigating the transcriptional activity of the rrn operons and promoters after a temperature downshift and analyzing the fate of the pre-existing and de novo synthesized rRNA during cold adaptation.

ACCEPTED MANUSCRIPT RESULTS rRNA synthesis does not stop during cold acclimation

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To test whether rRNA synthesis occurs after temperature downshift, primer

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extension assays were performed on the RNA extracted from control cells (i.e.

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immediately before cold stress) and from cells taken at different times during cold adaptation following a 37°  10°C temperature downshift. For these experiments we used a universal primer (primer a) complementary to a sequence present in all rRNA operons

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and corresponding to the region upstream of the 5' end of 17S precursor of 16S rRNA (Fig.

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1a). Thus, the products of the primer extensions obtained with this primer correspond exclusively to the RNase III unprocessed transcripts originating from either P1 or P2

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promoter. As seen in Fig. 1b, the transcripts originating from P1 give rise to two primer

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extension products, both larger than those of P2, as expected; the upper band corresponds to transcripts originating from P1 of rrnA, rrnB, rrnC and rrnG and the lower

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band to those of rrnD, rrnE and rrnH that are shorter by about 8-9 bases [20]. The transcripts from the P2 promoters yield multiple (five) primer extension products that can

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be attributed to sequence heterogeneity near the transcription initiation sites [21]. As seen in Fig. 1b,c, 16S rRNA precursors are detected in all cell samples, albeit in different amounts. Transcripts originating from both P1 and P2 were detected in the RNA extracted at different times after cold-shock, whereas the transcripts of control cells originated mainly from P1. Quantification of these data indicates that the levels of unprocessed transcripts from both P1 and P2 increase during cold adaptation, reaching a maximum between 2 and 3 hours after the stress. Furthermore, the increase is substantially higher for the P2 than for the P1 transcript so that the P2/P1 transcript ratio increases during the first 2 hours of cold-adaptation (Fig 1d). At the later stages of cold adaptation as well as in cold-adapted cells, the level of all precursors diminishes but the level of P2 transcripts remains higher than P1 transcripts.

ACCEPTED MANUSCRIPT These results leave open the question of whether the observed increase of the primer extension products is due to de novo rRNA synthesis after cold-shock and/or to the

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accumulation of unprocessed intermediates, possibly caused by a reduction of the

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efficiency by which the rRNA precursors are processed at low temperature. Indeed, the

III and/or on the structure of its RNA substrate.

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low temperature could have a direct effect on the activity of the processing enzyme RNase

To answer these questions, primer extension analyses were performed on total

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RNA extracted from cells in which de novo rRNA synthesis was inhibited by rifampicin

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treatment. As seen in Fig. 2a, b, the level of unprocessed transcripts originating from both P1 and P2 promoters is drastically reduced after exposure to the antibiotic and the rate of reduction depends upon the phase of cold adaptation. On the other hand, when the same

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RNA samples were subjected to primer extension from primer b, complementary to an internal region of 16S rRNA (Fig. 1a), the level of processed 16S rRNA was found to be

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approximately the same before and after various times of cold-shock (Fig. S1). This indicates that rifampicin affects the levels of unprocessed, neo-synthesized rRNA, but

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does not cause major variations in the level of the bulk rRNA. Thus, the presence of the rRNA precursors can be taken as evidence that de novo rRNA synthesis continues during the cold adaptation phase that follows cold stress.

Maturation of rRNA precursors slows down during cold adaptation From the rates of disappearance of the rRNA precursors after rifampicin treatment (Fig. 2a), it was possible to estimate the half-lives of the precursors as a function of the time elapsed after cold stress (Fig. 2b). These data indicate that during the initial phases of cold acclimation, rRNA maturation is slowed down considerably. The half-lives of the transcripts originating from P1 and P2 increase during cold acclimation from 20-22 min after 30 min to about 30 min after 2.5 - 4.0 hours of cold-shock and overall are

ACCEPTED MANUSCRIPT approximately two orders of magnitude longer than before the stress (t 1/2 ~18 sec at 37°C, Fig. S2). Furthermore, after cold stress the half-lives of the transcripts originating from P1

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and P2 promoters are essentially the same. This finding is important insofar as it indicates

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that the observed increase of the P2/P1 transcript ratio (Fig.1d) is not due to a differential

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stability of the precursors but instead to a preferential transcriptional activity from the P2 promoter during cold adaptation.

The effect of cold adaptation on rRNA maturation was further studied by quantifying

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the amount of 17S and 16S rRNA present in the cells before and after temperature

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downshift. For this purpose total rRNA extracted from cells labeled with [32P]orthophosphate at 37°C prior to the stress or immediately after a temperature downshift and then maintained at either 37°C or 10°C for various times was subjected to agarose gel

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electrophoresis to separate 16S and 17S rRNA (Fig.3a). As seen from these autoradiograms, during cold acclimation the amount of 17S precursor is greater than (up to

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2 hrs) or equivalent to (after 2-3 hrs) that of 16S rRNA and represents a substantial proportion of the total radioactive transcript. By contrast, in the cells kept at 37°C the 17S

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rRNA initially present is rapidly converted to mature 16S rRNA that is essentially the only small subunit RNA species present after 15 min. In the same autoradiograms the timedependent accumulation of 23S rRNA can also be observed, the process being substantially slower under cold adaptation conditions than at 37°C (see also below). To determine the rate at which 17S rRNA matures into 16S, cells were subjected to pulses with radioactive orthophosphate (for 2 min at 37°C in one case and for 30 min after cold-shock in another case) followed by a chase with an excess of non-radioactive orthophosphate and addition of rifampicin to block de novo transcription. The total radioactive RNA extracted from these cells was subjected to electrophoresis and the amount of radioactivity present in the 17S rRNA band was determined as a function of time elapsed after rifampicin addition. From the data obtained (Fig. 3b), it was possible to

ACCEPTED MANUSCRIPT estimate that the t1/2 of 17S rRNA increases from  0.3 min at 37°C to  60 min after coldshock at 10°C; this corresponds to a  200-fold reduction of the maturation rate.

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Maturation of the 17S rRNA precursor involves cleavages at both 3' and 5' ends of

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the transcript. To analyze whether cold stress affects the maturation at both ends of the

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17S precursor, non-radioactive RNA extracted at different times during cold acclimation was subjected to Northern blotting [22] and hybridization with three radioactively labeled probes, one (equivalent to primer b in Fig.1a) complementary to an internal region of

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mature 16S rRNA that served as control and the other two complementary to the 5’ and 3’

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ends of the 17S rRNA precursor (probes c and d in Fig.1a, respectively). The level of the rRNA unprocessed at either the 5’ or 3’ end was determined by quantifying the

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radioactivity associated with the electrophoretically resolved 17S rRNA band. As seen

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from Fig.3c, within the first 6 hours of cold adaptation the amount of rRNA unprocessed at either end increases at essentially the same rate up to 4-5 fold with respect to the pre-

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stress situation. After 6 hours the amount of unprocessed rRNA starts to decline. These data allow for the conclusion that not only the initial cleavage by RNase III but also the

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overall 17S rRNA maturation is slowed down as a result of the cold stress.

Time course of 16S and 23S rRNA synthesis during cold acclimation After the demonstration that de novo rRNA synthesis occurs after cold-shock, the next experiment was designed to study the time course of its synthesis. For this purpose, the radioactivity associated with the electrophoretically resolved rRNA bands in samples taken at different times during cold acclimation from cells labeled with [32P] orthophosphate at the onset of the stress was quantified (Fig. 4a). As seen from the figure, the bands corresponding to full length 16S and 23S rRNA are very faint up to 60-90 min after the stress and their intensities increase linearly for at least 8 hours (Fig. 4b). Accumulation of full length radioactive 16S rRNA is faster than 23S rRNA so that the 23S/16S rRNA

ACCEPTED MANUSCRIPT stoichiometric ratio is < 1 for at least 5 hours. However, after longer times this ratio increases and becomes ~1.25 after 8 hours, still far away from the expected value of ~2

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(Fig. 4c). Although the observed time-dependent incorporation of radioactive phosphate

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into complete rRNA molecules could result from the completion of the synthesis of

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molecules whose transcription had already started at the time of the stress and/or from de novo started syntheses, these findings provide an additional indication that rRNA transcription continues during cold acclimation, but several hours are needed before the

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synthesis is completed.

Determination of the amount of newly synthesized rRNA An important question concerns the quantitative aspect of the de novo rRNA

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transcription occurring during cold acclimation. To determine the actual amount of the newly synthesized rRNA with respect to the total cellular rRNA, the cells were exposed to

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[32P]-phosphate at different times after cold stress and lysed; after removal of the membranes and of all acid insoluble radioactivity, the amount of free phosphate and its

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specific radioactivity were determined as described in Materials and Methods. After some initial oscillation, the phosphate specific radioactivity was found to remain essentially constant starting from approximately 60 min after the cold-shock. From the comparison of the specific radioactivities of the free phosphate and of the 16S and 23S rRNA present in the cells, it was possible to determine the percentage of newly synthesized rRNA with respect to total rRNA and to estimate that from 2 to 8 hours after the stress the fraction of newly synthesized rRNA increases from ~ 3% to ~21% of total rRNA (Table 1). Since overall the total amount of cellular RNA superficially appears unaffected by the cold stress, the finding that a fairly large percentage of rRNA is synthesized de novo during cold acclimation implies that part of the pre-existing rRNA becomes metabolically unstable after the cold stress. For this reason and also because several types of stress

ACCEPTED MANUSCRIPT were found to cause degradation of the rRNA present in partially assembled, misassembled or idle ribosomes [23, 24], a possible effect of cold stress on rRNA stability

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was investigated.

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rRNA stability after cold stress

To determine the rRNA half-lives during cold-adaptation, completely and homogeneously labeled rRNA was obtained in cells exposed to an extended radioactive

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pulse and subsequently treated with rifampicin and with an excess of non-radioactive

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phosphate. The radioactivity associated with the electrophoretically resolved 16S and 23S rRNA bands was then quantified. About 35 % of the 23S and 45% of the 16S rRNA synthesized before the stress is lost during the first 30 min following cold-shock, whereas

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the remaining rRNA is quite stable for at least 8 hours (Fig. 5a). A similar loss of rRNA has been previously observed as a result of a drastic decrease in the growth rate induced by

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an energy source downshift [23, 24]. Since rifampicin inhibits initiation but not elongation of transcription, the slight increase in the amount of both rRNAs that follows the initial rapid

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drop (Fig. 5a) can be attributed to the completion with non-radioactive precursors of incomplete radioactive molecules whose synthesis had begun at the time of the chase and rifampicin addition. The rRNA synthesized at the onset of cold-shock (i.e. within the initial 30 min) remains fairly stable for about 1-1.5 hours before declining to about 50% of its initial value (Fig. 5b) whereas the rRNAs synthesized at the end of the cold acclimation phase (i.e. between 4 and 4.5 hours after the stress) remain fairly stable (Fig. 5c).

Activity of individual rrn operons under cold-acclimation conditions Seven constructs were prepared to determine whether cold stress might have different or selective effects on the activity of the individual rrn operons present in E. coli [20]. Each construct, a derivative of pKK322, contained the cat reporter gene under the

ACCEPTED MANUSCRIPT dual transcriptional control of the core elements of the P1 and P2 promoters of each of the seven rrn operons. These constructs (schematically illustrated in Fig. S3) were used to

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investigate the intrinsic activity of the core promoters in combination with the regulatory

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regions present upstream of the individual operons; these include the UAS sequences as

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well as the FIS and H-NS binding sites [25]. On the other hand, to avoid complications related to phenomena not having a direct bearing on the activity of the promoters, the nutlike sequence involved in transcriptional anti-termination occurring downstream of P2 [26]

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was not included in the constructs. E. coli cells transformed with individual constructs were

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grown at 37°C in either rich (LB) or poor (M9) medium and subjected to cold stress at 10°C for two hours. The steady state levels of the cat transcripts originating from both P1 and P2 promoters of the individual rrn operons present in the total RNA extracted from these

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cells were quantified by slot-blot analysis using a radioactive oligonucleotide complementary to the cat reporter gene. The levels of the RNAs detected in this way

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depend upon the ratio of the rates at which they are synthesized and degraded. However, because the RNAs produced from the constructs are the same in all cases, the rates of

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degradation under a given growth condition can be taken as being identical, regardless of the promoter from which the transcripts originate. Thus, the RNA levels determined by slot blot analysis reflect directly the rates of transcription from the individual rrn operons and the RNA levels reflect the promoter strengths before and after cold stress. In cells growing in LB broth at 37°C the transcripts derived from rrnA, rrnB, rrnE and rrnH are more abundant than those originating from rrnC, rrnD and rrnG (Fig. 6a, c), whereas in poor medium the transcripts deriving from the P1 and/or P2 promoter(s) of rrnG are the most abundant (Fig. 6b, d). Under these growth conditions, also the transcripts originating from rrnA and rrnB, although much less abundant than those of rrnG, are slightly more abundant than those originating from the other operons (Fig. 6b, d). The situation changes after two hours of cold-shock. In LB, the transcripts originating from

ACCEPTED MANUSCRIPT rrnA and rrnB are the most abundant while those originating from the other operons can be ranked in the order: rrnH > rrnG > rrnE > rrnD > rrnC. In M9 medium, the activity of the

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promoters can be ranked in the order rrnD ~ rrnG > rrnA > rrnB > rrnH > rrnE > rrnC.

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The present evidence that the activity of the rrn promoters is influenced by the

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growth conditions of the cells also during cold acclimation agrees with and extends earlier findings obtained with cells growing at the optimal temperature [21, 27]. The above results reflect the levels of the transcripts made by the combined activities of the P1 and P2 core

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promoters of the individual rrn operons, thereby providing insight into their relative

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strengths as well as of the likely effects of transcriptional factors that target the promoterupstream regions.

From the ratios of the transcript levels before and after two hours of cold stress (Fig.

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6c), it is possible to determine that in rich medium rrnA, rrnB and rrnG are the operons displaying the greatest responsiveness to cold stress (ca. 6 to 8-fold increase). Operons

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rrnD and rrnH display an intermediate responsiveness whereas the remaining operons are the least responsive. The situation is quite different in poor medium; after the stress the

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level of the rrnD transcript increases almost 4-fold whereas that of rrnG becomes lower (Fig. 6d). All the other operons, with the exception of rrnA, do not to respond to the stress. Additional comments concerning these results are presented in the Discussion section.

Differential use of P1 and P2 promoters before and after cold-shock To determine which of the seven P2 promoters are responsible for the overall higher activity of the P2 promoters with respect to the P1 promoters observed during cold acclimation (Fig. 1b, c), constructs containing the cat reporter gene under the control of either the P1 or P2 promoter derived from each rrn operon were prepared (see Supplementary Material). The upstream region is present in the constructs containing the P1 but not in those

ACCEPTED MANUSCRIPT containing the P2 promoters and none of the constructs contains the entire transcriptional anti-termination sequence. Following growth at 37°C in rich medium (LB), the cells

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transformed with these constructs were subjected to cold-shock and the steady state level

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of cat RNA transcript analyzed by slot blot assays as described above. The control cells

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(growth at 37°C) contain transcripts originating from all seven P1 promoters with the highest level being observed with rrnH and rrnE promoters (Fig. 7a, c) whereas transcripts originating from P2 promoters are hardly detectable (Fig. 7b, d). The picture changes after

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cold-shock; all seven rrn P2 promoters are activated by the stress with those of rrnB, rrnG

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and rrnH being the most affected followed by rrnA and rrnD (Fig. 7b, d). On the other hand, the RNA transcribed from the P1 promoters of rrnC, rrnD and rrnA increases >2.5fold whereas the activity of the other promoters is either hardly affected (rrnB and rrnE) or

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even reduced (rrnG and rrnH) (Fig. 7a, c). In conclusion, the above results indicate that the P1 and P2 promoters display a different behavior vis-à-vis the cold stress and confirm the

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premise that the P1 promoters are mainly used at 37°C whereas all P2 promoters are preferentially activated during the early phases of cold acclimation. Additional comments

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concerning these results are presented in the Discussion section.

Nature and fate of the neo-synthesized rRNA The nature and fate of the rRNAs synthesized after cold stress were investigated by pulse chase experiments. For this purpose, 30 minutes after cold-shock (at 10°C), the cells were given a pulse of [32P] orthophosphate followed 30 min later by a chase with an excess of non-radioactive phosphate. A sample of the cell culture was withdrawn immediately after the chase and samples were taken after further incubations for 60, 180 and 420 min; these times correspond to cell exposure to cold-adaptation conditions for a total of 1, 2, 4 and 8 hours. The extracts obtained from these cells were then subjected to sucrose density gradient centrifugation under conditions optimized for the analysis of

ACCEPTED MANUSCRIPT dissociated ribosomal subunits (Fig. 8a) while samples of total RNA were analyzed by agarose gel electrophoresis (Fig. 8b). The sucrose density gradient profiles reveal well

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resolved, almost perfectly symmetrical A260 peaks of 30S and 50S ribosomal subunits in

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the expected ~1:2 stoichiometric ratio whereas the radioactive RNA appears rather

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heterogeneous and does not co-sediment with the ribosomal subunits for at least 3 hours after the labeling (or 4 hours after the cold stress). Only 7 hours after the labeling (8 hours after the cold-shock) there is a fairly good correspondence between the UV absorbance of

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the ribosomal subunits and the rRNA radioactivity. However, the ratio of the radioactivity

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corresponding to 23S and 16S rRNA is still 1:1.5, somewhat lower than the expected ~1:2 ratio.

Electrophoretic analysis of the labeled RNA present in the cell extracts (Fig. 8b)

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reveals that there is hardly any full-length radioactive rRNA synthesized one hour after the cold-shock. Trace amounts of 16S rRNA become visible two hours after the stress

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whereas 23S rRNA does not seem to be present yet; only 3-4 hours after the stress an intense band of radioactive 16S rRNA and a detectable band of 23S rRNA can be

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observed. Subsequently, the intensities of 16S RNA and, in particular, of 23S RNA bands continue to increase even 12 hrs after the stress indicating that during cold adaptation the synthesis of complete rRNA molecules is a very slow process. If we disregard the possibility that a fraction of the rRNA molecules might undergo nucleolytic degradation before their transcription is complete, the time required to reach the plateaus should roughly correspond to the time required for the completion of the synthesis of those molecules that had been labeled near their 5’ end. If this assumption is correct, the time required to synthesize a complete 16S rRNA would be between 5 and 6 hours whereas the synthesis of the twice as large 23S rRNA would require a 2-2.5-times longer time. From these rough calculations, it would seem that cold-shock at 10°C slows down the elongation rate of rRNA transcription approximately 600-fold.

ACCEPTED MANUSCRIPT The fate of the 16S rRNA synthesized before cold stress and during cold adaptation was investigated in a series of experiments in which 70S ribosomes and polysomes

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isolated from [32P]-labeled cells at different times after the stress were analyzed for their

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radioactive rRNA content. As seen from the figures, increasing amounts of 16S rRNA

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synthesized at 37°C immediately prior to the stress accumulate in the 70S ribosome for at least 6 hours after the stress (Fig. 9a). The same rRNA is also found in the polysomal fraction; however, after an initial increase during the first 1-2 hours of cold adaptation, its

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level declines and the polysomes start accumulating increasing amounts of the 16S rRNA

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synthesized 30 and 60 min after cold-shock (Fig. 9b). Indeed, 3 and 6 hours after the stress the amount of this newly synthesized RNA present in the polysomal fraction is larger compared to that synthesized prior to the cold-shock (Fig. 9b). Overall, these results

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indicate that within the first two hours of cold adaptation the 16S rRNA synthesized prior to the cold stress is essentially the only radioactive 16S rRNA contained in both 70S

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monomers and polysomes, whereas at later stages of cold acclimation the 16S rRNA synthesized after the stress is preferentially included in the active ribosome fraction

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represented by the polysomes, unlike that synthesized before the stress that ends up preferentially in (presumably idle) 70S ribosomes (Fig. 9a, b)

Discussion During the period of cold adaptation that follows a cold stress, the bacterial cells undergo many physiological changes and extensive reprogramming of their gene expression pattern. A peculiar feature of this phase is the establishment of a cold-shock translational bias, whereby a small number of "cold-shock" transcripts is selectively translated whereas translation of bulk, non-cold-shock transcripts is either drastically reduced or abolished [15-19, 28-35].

ACCEPTED MANUSCRIPT Aside from the cis-acting elements present in the paradigm cold-shock cspA mRNA [35], an important role in the establishment of the translational bias is played by the three

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initiation factors (IFs) whose levels increase with respect to the ribosomes, giving rise to a

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characteristic imbalance of their otherwise constant level with respect to the ribosomes

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[14]. Whereas it has been shown that cold-shock-induced expression of the genes encoding these factors contributes to this imbalance by increasing the value of the numerator of the IFs/ribosomes ratio [15,16], nothing was known about the fate of rRNA

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transcription and ribosome assembly following a cold stress. Thus, the aim of this study

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was to fill this gap in our understanding of the global response of the bacterial cell to a cold stress and determine whether a change of the denominator might also contribute to the imbalance.

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The present data demonstrate not only that rRNA transcription does not stop after cold stress but also that rRNA molecules are synthesized de novo and eventually

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assembled into the active polysomal fraction of the cell. However, the rate of rRNA transcription becomes very slow during cold adaptation and several hours are necessary

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before this occurs; it can be surmised from the estimated times required to complete the synthesis of 16S and 23S rRNA molecules that the rate of transcription at 10°C is 2-3 orders of magnitude slower than before the stress. In addition, also the maturation at both 5’ and 3’ ends of the 17S rRNA precursor slows down so that the rate of 16S rRNA formation is reduced by more or less the same extent. The slow rate of 17S rRNA maturation could stem from a reduced activity of RNase III, the endonuclease responsible for the initial cleavages of the precursor rRNAs and/or from a cold-induced modification of the secondary structures of its substrate RNA, constituted by a partially double stranded stretch of RNA [36]. Furthermore, the rate of rRNA maturation seems to decrease mainly when the cell undergoing cold adaptation accumulates several cold-shock helicases and RNA chaperones that could potentially disrupt/alter the RNase III binding sites [17, 34, 37].

ACCEPTED MANUSCRIPT Finally, the cold-shock-induction of the evolutionarily conserved protein YmdB that interacts with the catalytic region of RNase III and affects its RNA cleavage capacity [38]

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could also contribute to the reduced activity of RNase III. Additional cleavages at both 5’

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and 3’ ends of the 17S rRNA are also required to produce mature 16S rRNA and they may

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be retarded as well during cold adaptation by the fact that these enzymes require rather mature ribonucleoprotein complexes as substrates [39].

Thus, overall this study demonstrates that whereas during cold acclimation initiation

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factors continue to accumulate [15,16], transcription, maturation and assembly of rRNA

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into complete subunits are significantly retarded so that the amount of ribosomes is comparatively reduced thereby contributing to the IFs/ribosome imbalance. In this study we have also compared in vivo the activity of the promoters of the

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seven individual rrn operons before and after cold stress in rich (LB) and in minimal medium. It should be recalled, in this connection, that E. coli cells growing in rich medium

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synthesize proteins more actively and divide much faster than those growing in minimal medium [40] in addition to having a fundamentally different metabolism and different levels

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of factors that influence positively or negatively transcription (see below). Accordingly, these cells contain a larger number of ribosomes and other components of the translational apparatus [41] and the genes encoding these components are expressed at a significantly higher level [40], consistent with the known axiom that their regulation is growth rate-dependent. In particular, several studies have indicated that whereas the P1 promoters are controlled by the growth rate, transcription from the P2 promoters remains nearly constant [42,43]. However, these conclusions were drawn from determination of enzyme expression that depends upon both transcription and translation. For this reason we measured promoter activities by semi-quantitative slot blot analysis of the transcript of the cat reporter gene controlled by the P1 and P2 promoters. If P1 activities are under growth rate control, unlike those of P2, transcription of the different rrn operons at 37°C in

ACCEPTED MANUSCRIPT LB should be primarily due to the P1 promoters; on the other hand, both in minimal medium and after cold shock, two situations that drastically reduce growth rate, the

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transcriptional activity should be accounted for primarily by the P2 promoters.

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In agreement with this premise, our results show that in LB the level of transcription

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of the individual operons at 37°C displays the largest differences with the corresponding levels observed after cold shock (gray bars in the histogram of Fig. 6 c) whereas in minimal medium the amounts of transcripts of the individual operons are rather similar at

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37°C and after cold shock (gray bars in the histogram of Fig. 6 d). The finding that at 37°C

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in rich medium the rrnG operon is the least active whereas it is very active in minimal medium both before and after cold stress is also consistent with the above interpretation. Analysis of the activity of the individual P1 and P2 promoters of the seven rrn

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operons reveals a wide variability of the activity levels of the P1 promoters both before and after cold stress (red and blue bars in the histogram of Fig. 7c) but that there is no major

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alteration of their relative activity levels pre and post stress (gray bars in the histogram of Fig. 7c). This finding suggests that the seven P1 promoters are differently affected by the

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intracellular conditions but respond in similar ways to the stress. On the contrary, all the P2 promoters display similar levels of activity at 37°C (red bars in the histogram of Fig. 7d) whereas they respond to different extent to the cold stress (blue and gray bars in the histogram of Fig. 7d). In conclusion, this study demonstrates that the de novo rRNA transcription during cold adaptation involves primarily the P2 promoters of all seven rrn operons albeit to different extents. Some considerations could be made in an attempt to rationalize the different activities of the various promoters that we have detected before and after cold stress. The structures of the P1 promoter cores are very similar in all rrn operons whereas the P1-upstream regions differ considerably. In fact, all upstream regions contain intrinsic

ACCEPTED MANUSCRIPT curvatures centered at approximately the same distance from the P1 promoter, but differ for the degree of intrinsic curvature and for the affinities displayed for nucleoid-associated

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proteins such as Fis, H-NS, and Lrp with which they form complexes having different

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structures and topologies [25]. The intrinsic curvature is highest for the rrnB and rrnD and

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less pronounced for rrnA and rrnE operons [44]. In addition to being under the antagonistic control of transcriptional regulators Fis and H-NS [25, 45-47], Fis and Lrp [44], the individual P1 are differently affected by the concentrations of ppGpp, DksA and the NTPs

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[48].

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On the other hand, whereas a direct effect of Fis and ppGpp on the activity of the P2 promoters is hardly observable, indirect effects on their activity are likely to occur. Under optimal growth conditions the individual promoters are engaged in a genome-wide

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competition for a limited amount of RNA polymerase (RNAP) and are not saturated with RNAP so that the free RNAP concentration contributes to affect the rrn promoter activities

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[49, 50]. It is likely that after cold stress the level of free RNAP increases as a consequence of the overall reduced transcriptional activity and that the P2 promoters may

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benefit from this new situation. Furthermore, a reduced activity of P1, in addition to making available a larger number of free RNAP molecules, would reduce the probability that the P2 promoters are occluded by a transcription elongation complex originating at the upstream promoters [51, 52]. As a result of all these functional interplays, under normal growth conditions, the rRNA is transcribed to different extents from the promoters of the different rrn operons and the individual promoters do not respond in the same way to environmental cues [53]. Here we have shown that the individual rrn promoters indeed respond in different ways to cold stress in different growth media. However, to find a rational explanation for these effects is very difficult, and beyond the scope of this study, because cold stress causes a large number of partially defined physiological variations that may influence the

ACCEPTED MANUSCRIPT transcriptional activity of each promoter. Nevertheless, at least a few considerations could be made. After cold stress, the intracellular levels of Fis, H-NS and ppGpp change

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drastically; the transcriptional activator Fis and the repressor ppGpp virtually disappear,

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whereas the level of the repressor H-NS more than doubles [22, 29, 30, 54, 55]. These

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changes are expected to influence directly the activity of the P1 promoters and indirectly that of the P2 promoters.

Furthermore, although no information is available as to possible cold-stress-induced

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changes of the cellular nucleotide triphosphate pool, it is noteworthy that rrnD, the only

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operon beginning with guanine instead of adenine, is the most cold-shock responsive operon in minimal medium.

Analysis of the fate of the rRNA transcribed de novo after cold shock indicates that,

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as the cold adaptation phase progresses, the new rRNA molecules tend to be preferentially included in the polysomes that likely represent the active ribosomal fraction.

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At the same time a large proportion of the rRNA synthesized before the stress becomes metabolically unstable, while the rest is preferentially found in presumably idle 70S

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monomers [52, 56]. We have estimated that the maximum amount of the rRNA synthesized de novo during cold adaptation corresponds to approximately 20% of the total rRNA of the cell, whereas almost half of the rRNA synthesized before the stress is degraded during the initial phases of cold adaptation. This situtation is similar to that observed in cells undergoing a drastic growth rate decrease induced by an energy downshift [23, 24]. By a differential regulation of the individual rrn operons [25] the cell could modulate the type of rRNA synthesized in response to an environmental change. The minor sequence differences of the rrn operons, together with rRNA and r-protein modifications, may contribute to give rise to a ribosome heterogeneity that has been suggested to represent a new level of translational regulation [57].

ACCEPTED MANUSCRIPT Since the sequence of the rRNA synthesized from a given operon before and after the cold stress should be identical, to find the rationale for the replacement of the old rRNA

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by the new is a challenging task. The small structural heterogeneities within the sequences

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of the different rrn operons [58] and the cold stress-induced change in their levels of

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expression may contribute to the formation of specialized ribosomes adapted to function at low temperatures. Moreover, since the rRNA is extensively modified post-transcriptionally [59] and the modified nucleotides are clustered within functionally conserved regions with

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at least some of these modifications playing a role in the translation initiation stage [60], it

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is possible that the modifications of the pre-stress rRNA are not suitable to support protein synthesis during cold adaptation and in cold adapted cells and/or that the newly synthesized rRNA may be necessary to start a de novo assembly of ribosomal subunits to

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replace subunits that might have been irreversibly inactivated by the stress.

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MATERIALS AND METHODS

Cell growth, cold stress induction and preparation of cell extracts

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Escherichia coli MRE600 cells were grown at 37°C in low phosphate medium (100 mM Tris-HCl, pH7.7, 0.5% glucose, 0.5% peptone, 10mM NH4Cl, 0.7 mM NaNO3, 1mM Na2SO4, 0.5 mM MgSO4∙7H2O, 0.05 mM MnCl2∙4H2O, 0.02 mM FeSO4∙7H2O). When A600 = 0.3 or 0.6 was reached the culture was transferred to 10°C and the cells collected by centrifugation at 5K rpm for 3 min (GSA rotor Sorvall); the pellet was washed with saline solution (0.9% NaCl) and then pelleted by 2 min centrifugation at 10K rpm (Eppendorf 5417R centrifuge); the RNA was then obtained from the pellet by phenol extraction [22]. In the case of radioactively labeled cells, the pellets were instead resuspended in Buffer1 (10 mM TrisHCl pH 7.6; 10 mM MgAcetate; 60 mM NH4Cl) containing 0.4 M NaCl and ruptured in a Misonix3000 sonicator with three cycles of 20 sec 6 W pulse in ice followed

ACCEPTED MANUSCRIPT each time by a 20 sec pause and finally centrifuged 20 min at 14K rpm at 4°C in an

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Eppendorf table centrifuge to clarify the crude extract from the pelleted cell debris.

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In vivo [32P] RNA labeling

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Cells grown to A600 = 0.3 to 0.6 as described above were labeled by addition of 1 μCi/ml [32P]-Orthophosphate (PerkinElmer) either at 37°C or after cold stress at the time and for the duration indicated in each case.

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Pulse-labeling experiments

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As indicated in “Results”, in some experiments the pulse with radioactive phosphate was followed by a chase with a 1000-fold excess of non-radioactive orthophosphoric acid. For each experiment, time and length of the pulse-chase are indicated in the text. In the cases

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indicated, rifampicin (250 μg/ml) was offered at the time of the chase. Preparation of radioactive “standard” rRNA

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Fully labeled total RNA was obtained by incubating 300 ml of E. coli MRE600 culture with 1mM [32P]-Orthophosphate (PerkinElmer) during growth at 37°C from A 600=0.2 to 0.6. The

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cells were harvested, sonicated, centrifuged and the S30 was loaded on a 10%-40% sucrose gradient to prepare ribosomal subunits as described below. The pelleted subunits were resuspended in Buffer 1 and subjected to two phenol and two chloroform (1:1 = v:v) extractions to remove proteins.

Inorganic phosphate determination Cell pellets from 40 ml of culture grown and cold-shocked as described above were resuspended in 400 μl of Buffer 1 and ruptured by sonication as described. The extracts were clarified by centrifugation at 4°C for 20 min at 14K rpm and 95 μl Buffer 1 were added to 5 μl of the crude extracts; 10 μl of TCA (100%) were then added and the sample left for 10 min on ice before centrifugation for 15 min at 14K rpm (5417R Eppendorf

ACCEPTED MANUSCRIPT centrifuge). The supernatant was adjusted to pH 6 by addition of 10 N NaOH and the sample volume brought to 1 ml with Buffer 1 before addition of 30 μl of reaction mixture

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(3.6 M sulfuric acid; 70 mM ammonium molybdate; 20 mM potassium antimony (III) oxide

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tartrate hemihydrate) and 30 μl of 0.4 M ascorbic acid. After 5 min incubation at room

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temperature, the A882 of the sample was determined and the concentration of the inorganic phosphate determined by reference to a standard curve (from 0.001 to 1 μg) of KH2PO4

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(Sigma) in Buffer 1.

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Preparation of ribosomal subunits and polysomes

Cells obtained from 100 ml of culture were collected by centrifugation and the resulting pellets were resuspended in 0.5 ml of low-salt buffer (Buffer 2) (10mM Tris-HCl, pH7.6; 1

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mM MgAc; 60 mM NH4Cl) and subjected to sonication on ice. After centrifugation (20 min at 14K rpm at 4°C) to remove cell debris, the resulting extracts were subjected to

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centrifugation (16 h at 24K rpm in a SW41 Beckman rotor at 4°C) through a 10-40% sucrose density gradient in Buffer 2. The gradient fractions containing the ribosomal

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subunits, as judged from their A260, were pooled, the subunits recovered by centrifugation (2 h at 80K rpm in the Sorvall M120 Discovery ultracentrifuge) and finally resuspended in 200 μl of Buffer 1. For polysomes preparation the cells were subjected to the freeze–thaw and lysozyme method followed by sucrose gradient centrifugation [61] whereby 30S and 50S subunits, 70S monosomes and polysomes were separated. The different ribosomal species were individually pooled, pelleted by centrifugation (1 h at 120K rpm in the Sorvall M120 Discovery ultracentrifuge) and finally resuspended in Buffer 1. Sucrose density gradients Gradients were loaded with 10 to 15 A260 of cell crude extract and subjected to ultracentrifugation (SW 28 rotor, 20000rpm for 18 hrs). The gradients were fractionated using a peristaltic pump and the A260 of each fraction was determined using a SHIMADZU

ACCEPTED MANUSCRIPT UV-1601 spectrophotometer, while the radioactivity present in each fraction was

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determined in a WALLAC MicroBeta instrument.

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RNA analyses

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Total RNA was obtained by hot phenol extraction [22] starting from 15 ml cell cultures grown and cold-stressed as described above. The RNA was used for quantitative measurements, primer extension and other RNA analyses.

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Primer extension analyses

Primer extensions were carried out on 5 μg of total RNA at 65°C for 45 min with 1U of

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PNG retrotranscriptase according to the procedure described [16] using 5 pmol 32Pradiolabeled oligonucleotide “a” (5’-GATTGTCTGATAAATTGTT-3’). The reactions were

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stopped by ethanol precipitation, the samples dissolved in running buffer, loaded onto a 8M urea gel and subjected to electrophoretic analysis. The radioactive cDNA

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corresponding to P1 and P2 transcripts was quantified in the Molecular Imager (Bio-Rad FX) using Quantity One® software.

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Radioactivity quantification

For the quantification of 23S, 17S and 16S rRNAs, 2 μg or 5 μg of labeled total RNA were subjected to electrophoresis on 1.2% agarose gels. After electrophoresis, the gels were placed on Hybond membrane (Amersham) and then heat-dried under vacuum. The radioactivity in the individual rRNA species present in each lane of the dried gels were quantified in the Molecular Imager (Bio-Rad FX) using Quantity One® software and the amounts normalized with respect to the total ethidium bromide-stained RNA. To obtain an absolute quantification of 16S and 23S RNAs, increasing amounts of labeled “standard” rRNA, purified from subunits (see above), were loaded together with total RNA samples. Unlabeled RNA extracted according to the hot phenol protocol [22] was subjected to slot blot hybridization analyses as described [62] but for the use of SSC instead of SSPE . The

ACCEPTED MANUSCRIPT slot blots were probed with a 32P labeled oligonucleotide (5’AGTTTGCTCATGGAAAACGGTGTAACAAGG-3’) specific for cat gene. Total RNA

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samples were also used for Northern blot analysis by hybridization with 5′-end-labeled

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oligonucleotides “b” (5’- TGTGTTAGGCCTGCCGCCAG-3’), “c” (5’-

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CTTGCGACGTTAAGAATCCG-3’) and “d” (5’- AGWACGCTTCTTTAAGG-3’) (Fig1a) as described in [22]. The samples were normalized for the rRNA content. After hybridization,

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analyzed using Quantity One® software.

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the radioactivity on the filters was determined in a Molecular Imager (Bio-Rad FX) and

Figure legends

Fig. 1. Activity of rrn P1 & P2 promoters after cold-shock

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(a) Schematic representation of an E. coli rrn operon indicating the positions of the core elements (-35 and -10) and the transcriptional start point (+1) of the P1 and P2 promoters.

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The arrows pointing left (a and b) and the bars (c and d) below the 17S rRNA precursor indicate the position where the primers used in primer extension analysis and the probes

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used in Northern blotting anneal with the transcript (see Material and Methods). (b) Primer extension analysis of total RNA extracted from E. coli cells before (lanes 37°C) and after the times (indicated above each lane) following a (37°C  10°C) cold-stress. The primer used for these extensions is indicated as “a” in panel (a). The transcription products originating from P1 and P2 promoters are indicated on the left side. The asterisk indicates premature arrests of the transcriptase likely due to the presence of several Gs and Cs. The lanes indicated GACT are from sequencing reactions. (c) Levels of radioactivity, expressed as arbitrary units (a.u.) of the bands corresponding to the transcripts of rrn P1 (gray bars) and P2 (black bars) promoters are plotted as a function of the time elapsed after cold-stress. The bands at the level of the asterisk were not considered so that the activity of the P1 promoters is likely to be slightly underestimated.

ACCEPTED MANUSCRIPT (d) P2/P1 transcription level ratios to quantify the differential use of the two promoters as a function of the time of cold adaptation. Values shown in (c) and (d) are the results of at

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least three primer extension experiments.

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Fig. 2 De novo transcription of rRNA during cold acclimation

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(a) Primer extension analysis of the transcripts originating from P1 and P2 after treatment with rifampicin. The primer extension analysis was carried out as indicated in Fig. 1b on total RNA extracted from cells treated with rifampicin at different time after cold stress and

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withdrawn at 30 min intervals. The times of the first and last sampling after addition of

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rifampicin are indicated above the lanes; (b) stability (t1/2) of the RNase III-unprocessed transcripts originating from P1 () and P2 () promoters as determined from their rate of disappearance as a function of the time elapsed after rifampicin treatment of cells at

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different stages of cold acclimation. The transcript levels were obtained by quantification of primer extension products (panel a).

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Fig. 3. Effect of cold stress on rRNA maturation (a) Electrophoretic separation of rRNA purified from [32P]- orthophosphate labeled cells.

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Left panel: cells in exponential phase (A600 = 0.6) were labeled at 37°C and further incubated at 37°C for the indicated times. Right panel: cells labeled at the onset of the 37°C to 10°C downshift and incubated at 10° C for the indicated times. Separation of 16S and 17S RNA was achieved by performing extended electrophoretic runs. The lane St contains radioactive 16S rRNA. The arrows on the left side of the panel indicate the positions to which 23S, 17S, and 16S rRNA migrate. (b) The level (percent) of radioactively labeled 17S rRNA precursor is plotted as a function of the time elapsed after the chase with non-radioactive orthophosphate and rifampicin addition taking the prerifampicin level as 100 percent. RNA extracted from cells labeled and incubated at 37°C () and labeled at the onset of cold stress and incubated at 10°C (). (c) Rate of accumulation of 17S rRNA unprocessed at the 5’ end () and 3’ end (▲) as a function of

ACCEPTED MANUSCRIPT the time elapsed after cold stress; the increase of precursors relative to their steady state levels at time zero taken as 1 is indicated in the ordinate.

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Fig. 4 rRNA synthesis after cold stress

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(a) Autoradiogram of an agarose gel (1.2%) electrophoresis of total RNA (2 μg/lane)

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extracted at the indicated times after cold stress from [32P]-labeled cells. The positions of 23S and 16S rRNA migration are indicated by the arrows; (b) levels of radioactivity expressed in arbitrary units (a.u.) quantified for each electrophoretically resolved 16S ()

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indicated times elapsed after cold stress.

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and 23S () rRNA band; (c) ratio of the 23S/16S rRNA radioactivity as a function of the

Fig. 5 Determination of rRNA stability during cold adaptation Residual (percent) radioactively labeled 16S (gray tracings) and 23S (black tracings)

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rRNAs after a chase with an excess of non-radioactive phosphate and the addition of rifampicin (250 µg/ml) to cells pulsed (a) 2 min at 37°C before the cold stress and (b) in the

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early (0-30 min) and (c) late (4-4.5 h) cold acclimation phase. After the pulse chase and rifampicin addition, the cells were incubated under cold stress conditions (10°C) for the

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times indicated in the abscissae. Fig. 6 Effect of cold-stress on transcriptional activity of individual rrn operons Slot blot analysis of the steady state levels of cat mRNA transcribed under the control of P1 + P2 promoters of the indicated rrn operons before (37°C) and 2 hours after cold stress (37°C 10°C) as indicated above the lanes in cells growing in LB (a) and M9 (b) medium. The radioactivity levels determined for each slot of panels (a) and (b) are presented in the histograms (c) and (d), respectively. Red bars and blue bars represent the radioactivity levels recorded for non-stressed and cold-stressed cells, respectively. The ratios of the radioactivity levels for each rrn operon after cold stress vs. before cold stress are indicated by the gray bars (ordinate on the right). The histograms of panels (c) and (d) do not present error bars because these slot blot experiments are not amenable for statistical

ACCEPTED MANUSCRIPT analysis. However, similar experiments were repeated two times and yielded results consistent with those presented here.

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Fig. 7 Effect of cold-stress on transcriptional activity of individual rrn promoters

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Slot blot analysis of the steady state levels of cat mRNA transcribed under the control of

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P1 (panels a and c) and P2 (panels b and d) promoters of the indicated rrn operons before (37°C) and after (37°C 10°C) cold stress and corresponding radioactivity levels of each slot. The values recorded for non-stressed and cold-stressed cells are indicated by red

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and blue bars, respectively. Ratios of the levels in cold-stressed and non-stressed cells of

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the cat reporter gene transcript from P1(c) and P2 (d) are indicated by the gray bars. The histograms of panels (c) and (d) do not present error bars because these slot blot

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experiments are not amenable for statistical analysis. However, similar experiments were

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repeated two times and yielded results consistent with those presented here. Fig. 8 Analysis of newly synthesized rRNA after cold stress.

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(a) Sucrose density gradient (10-30%) analysis of the S30 fractions from cells subjected to pulse-chase with [32P] orthophosphate between 30 and 60 min after cold-shock and

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collected at the times of cold stress indicated to the left of the A 260 (black) and radioactivity (red) profiles; (b) autoradiography of the electrophoretic analysis on agarose (1%) gel of total radioactive RNA extracted with hot phenol from the same pulse-chased cells analyzed in (a). The positions of 16S and 23S rRNA, determined by appropriate markers, are indicated by arrows. The times at which the cell extracts were prepared following the pulse-chase between 30 and 60 min after cold-shock are: lanes 1 through 6 = 1, 2, 3, 4, 8 and 12 hours. Fig. 9 Fate of 16S rRNA synthesized before and after cold stress. [32P]- labeled 16S rRNA was synthesized in cells during a pulse-chase at 37°C for 2 min just before T-downshift to 10°C (red bars), or during the first 30 min (blue bars) and between 30 and 60 min (green bars) following the T-downshift to 10°C. Samples of these

ACCEPTED MANUSCRIPT cells were withdrawn at the times of cold adaptation indicated in the abscissae and analyzed for their content of electrophoretically resolved labeled 16S rRNA with respect to

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the total 16S rRNA present in the ribosomal fractions. The percent of newly synthesized

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16S rRNA thus determined in (a) 70S monomers and (b) the polysomal fraction are

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reported in the ordinates of the histograms.

Acknowledgments

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This work was initially supported by two Italian MIUR grants (FIRB 2001 and PRIN 2005)

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to CLP. We gratefully acknowledge the kind support offered to us by colleagues and friends throughout the world who made it possible to complete this work with their generous gifts of the necessary materials and chemicals. The help of Dr. S. Stella and Mr.

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D. Palombi in some early phases of this work is also gratefully acknowledged. REFERENCES

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1. Nierhaus, K.H. (1991). The assembly of prokaryotic ribosome. Biochimie 73: 739-755. 2. Nierhaus, K.H. (2004). Ribosomal assembly- Assembly of the prokaryotic ribosome. In Protein Synthesis and Ribosome Structure. (eds. K.H. Nierhaus and D.N. Wilson), pp. 85105. Wiley-Vch Verlag, Weinheim. 3. Williamson, J.R. (2003). After the ribosome structures: how are the subunits assembled? RNA 9: 165-167. 4. Anton, B.P., Saleh L., Benner, J.S., Raleigh, E.A., Kasif, S. & Roberts, R.J. (2008). RimO, a MiaB-like enzyme, methylthiolates the universally conserved Asp88 residue of ribosomal protein S12 in Escherichia coli. Proc. Natl. Acad. Sci. U.S.A. 105: 1826–1831. 5. Xu, Z., O'Farrell, H.C., Rife, J.P. & Culver, G.M. (2008). A conserved rRNA methyltransferase regulates ribosome biogenesis. Nat. Struct. Mol. Biol. 15: 534-536. 6. Desai, P.M., Culver, G.M. & Rife, J.P. (2011). Site-directed mutants of 16S rRNA reveal important RNA domains for KsgA function and 30S subunit assembly. Biochemistry 50: 854-863. 7. Nesterchuk, M.V., Sergiev, P.V. & Dontsova, O.A. (2011). Posttranslational modifications of ribosomal proteins in Escherichia coli. Acta Naturae 3: 22-33. 8. Shajani, Z., Sykes, M.T. & Williamson, J.R. (2011). Assembly of bacterial ribosomes. Annu. Rev. Biochem. 80: 501-526. 9. Kiss, A., Sain, B. & Venetianer, P. (1977). The number of rRNA genes in Escherichia coli. FEBS Lett. 79: 77–79. 10. Sarmientos, P. & Cashel, M. (1983). Carbon starvation and growth-rate dependent regulation of the Escherichia coli ribosomal RNA promoters: differential control of dual promoters. Proc. Natl. Acad. Sci. U.S.A. 80: 7010–7013.

ACCEPTED MANUSCRIPT

AC

CE P

TE

D

MA

NU

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T

11. Gourse, R.L., deBoer, H.A. & Nomura, M. (1986). DNA determinants of rRNA synthesis in E. coli: growth rate dependent regulation, feedback inhibition, upstream activation, antitermination. Cell 44: 197–205. 12. Gourse, R.L., Gaal, T., Bartlett, M.S., Appleman, J.A. & Ross, W. (1996). RNA transcription and growth rate-dependent regulation of ribosomes synthesis in Escherichia coli. Annu. Rev. Microbiol. 50: 645-677. 13. Bremer, H. & Dennis, P.P. (1987). Modulation of chemical composition and other parameters of the cell by growth rate. In Escherichia coli and Salmonella typhimurium: Cellular and Molecular Biology, Vol. 2 (eds. F.C. Neidhardt, J.L. Ingraham, K.B., Low, B. Magasanik, M. Schaechter, H.E. Umbarger), pp. 1527–1542. American Society for Microbiology, Washington, DC. 14. Howe, J.G. & Hershey J.W. (1983). Initiation factor and ribosome levels are coordinately controlled in Escherichia coli growing at different rates. J. Biol. Chem. 258: 1954–1959. 15. Giangrossi, M., Brandi, A., Giuliodori, A.M., Gualerzi, C.O. & Pon, C.L. (2007). Coldshock-induced de novo transcription and translation of infA and role of IF1 during cold adaptation. Mol. Microbiol. 64: 807-821. 16. Giuliodori, A.M., Giangrossi, M., Brandi, A., Gualerzi, C.O. & Pon, C.L. (2007). Coldstress-induced de novo expression of infC and role of IF3 in cold-shock translational bias. RNA 13: 1355-1365. 17. Gualerzi, C.O., Giuliodori, A.M. & Pon, C.L. (2003). Transcriptional and posttranscriptional control of cold shock genes. J. Mol. Biol. 331: 527-539. 18. Giuliodori, A.M., Brandi, A., Gualerzi, C.O. & Pon, C.L. (2004). Preferential translation of cold-shock mRNAs during cold adaptation. RNA 10: 265-276. 19. Gualerzi, C.O., Giuliodori, A.M., Brandi, A., Di Pietro, F., Piersimoni, L., Fabbretti, A. & Pon, C.L. (2011). Translation initiation at the root of the cold-shock translational bias. In Ribosomes: Structure, Function, and Dynamics (eds. MV Rodnina, W Wintermeyer, R Green), pp. 143-154. Springer Verlag, New York. 20. Lindahl, L. & Zengel J.M. (1986). Ribosomal genes in Escherichia coli. Ann. Rev. Genet. 20: 297-326. 21. Condon, C., Philips, J., Fu, Z.H., Squires, C. & Squires, C.L. (1992). Comparison of the expression of the seven ribosomal RNA operons in Escherichia coli. EMBO J. 11: 41754185. 22. Brandi, A., Spurio, R., Gualerzi, C.O. & Pon, C.L. (1999). Massive presence of the Escherichia coli 'major cold-shock protein' CspA under non-stress conditions. EMBO J. 18: 1653-1659. 23. Johnsen, K., Molin, S., Karlström, O. & Maaloe O. (1977). Control of protein synthesis in Escherichia coli: analysis of an energy source shift-down. J. Bacteriol. 131: 18-29. 24. Molin, S., Von Meyenburg, K., Maaloe, O., Hansen, M.T. & Pato, M.L. (1977). Control of ribosome synthesis in Escherichia coli: analysis of an energy source shift-down. J. Bacteriol. 131: 7-17. 25. Hillebrand, A., Wurm, R., Menzel, A. & Agent, R. (2005). The seven E. coli ribosomal RNA operon upstream regulatory regions differ in structure and transcription factor binding efficiencies. Biol. Chem. 386: 523-534. 26. Li, S.C., Squires, C.L. & Squires, C. (1984). Antitermination of E. coli rRNA transcription is caused by a control region segment containing lambda nut-like sequences. Cell 38: 851–860. 27. Condon, C., French, S., Squires, C. & Squires, C.L. (1993). Depletion of functional RNA operons in Escherichia coli causes increased expression of the remaining intact copies. EMBO J. 12: 4305-4315.

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28. Jones, P.G., VanBogelen, R. A. & Neidhardt, F.C. (1987). Induction of proteins in response to low temperature in Escherichia coli. J. Bacteriol. 169: 2092-2095. 29. Jones , P.G, Cashel, M., Glaser, G. & Neidhardt, F.C. (1992). Function of a relaxedlike state following temperature downshifts in Escherichia coli. J. Bacteriol. 174: 39033914. 30. La Teana, A., Brandi, A., Falconi, M., Spurio, R., Pon, C.L. & Gualerzi, C.O. (1991). Identification of a cold shock transcriptional enhancer of the Escherichia coli gene encoding nucleoid protein H-NS. Proc. Natl. Acad. Sci. U.S.A. 88: 10907-10911. 31. Brandi, A., Pietroni, P., Gualerzi, C.O, Pon, C.L. (1996). Post-transcriptional regulation of CspA expression in Escherichia coli. Mol. Microbiol. 19: 231-240. 32. Goldenberg, D., Azar, I., Oppenheim , A.B., Brandi , A., Pon, C.L. & Gualerzi, C.O. (1997). Role of Escherichia coli cspA promoter sequences and translational apparatus adaptation in the cold shock response. Mol. Gen. Genet. 256: 282-290. 33. Weber, M.H., Klein, W., Müller, L., Niess, U.M. & Marahiel, M.A. (2001). Role of the Bacillus subtilis fatty acid desaturase in membrane adaptation during cold shock. Mol. Microbiol. 39: 1321-1329. 34. Weber, M.H. & Marahiel, M.A. (2003). Bacterial cold shock responses. Sci. Prog. 86: 9-75. 35. Giuliodori, A.M., Di Pietro, F., Marzi, S., Masquida, B., Wagner, R., Romby, P., Gualerzi, C.O. & Pon, C.L. (2010). The cspA mRNA is a thermosensor that modulates translation of the cold-shock protein CspA. Mol. Cell. 37: 21-33. 36. Drider, D. & Condon, C. (2004). The continuing story of endoribonuclease III. J. Mol. Microbiol. Biotechnol. 8: 195-200. 37. Yamanaka, K. (1999). Cold shock response in Escherichia coli. J. Mol. Microbiol. Biotechnol. 1: 193-202. 38. Kim, K.S., Manasherob, R. & Cohen, S.N. (2008). YmdB: a stress-responsive ribonuclease-binding regulator of E. coli RNase III activity. Genes Dev. 22: 3497-3508. 39. Deutscher, M.P. (2009). Maturation and degradation of ribosomal RNA in bacteria. Progr. Mol. Biol. Trans. Sci. 85: 369-391. 40. Tao. H., Bausch C., Richmond, C., Blattner, F.R. & Conway T. (1999). Functional genomics: expression analysis of Escherichia coli growing on minimal and rich media. J. Bact. 181, 6425–6440. 41. Keener, J. & M. Nomura. (1996). Regulation of ribosome synthesis, p. 1417–1431. In F. C. Neidhardt, R. Curtiss III, J. L. Ingraham, E.C.C. Lin, K.B. Low, B. Magasanik, W.S. Reznikoff, M. Riley, M. Schaechter, & H.E. Umbarger (ed.), Escherichia coli and Salmonella: cellular and molecular biology, 2nd ed. ASM Press, Washington, D.C. 42. Gourse, R. L., de Boer, H.A. & Nomura, M. (1986) DNA determinants of rRNA synthesis in E. coli: growth rate dependent regulation, feedback inhibition, upstream activation, antitermination. Cell 44:197–205. 43. Josaitis, C.A., Gaal, T. & Gourse, R.L. (1995). Stringent control and growth-ratedependent control have nonidentical promoter sequence requirements. Proc. Natl. Acad. Sci. USA 92:1117–1121. 44. Pul, U., Lux, B., Wurm, R. & Wagner, R. (2008). Effect of upstream curvature and transcription factors H-NS and LRP on the efficiency of Escherichia coli rRNA promoters P1 and P2 - a phasing analysis. Microbiology 154: 2546-2558. 45. Falconi, M., Higgins, N.P., Spurio, R., Pon, C.L. & Gualerzi, C.O. (1993). Expression of the gene encoding the major bacterial nucleoid protein H-NS is subject to transcriptional auto-repression. Mol. Microbiol. 10: 273-282. 46. Falconi, M., Brandi, A., La Teana, A., Gualerzi, C.O. & Pon, C.L. (1996). Antagonistic involvement of FIS and H-NS proteins in the transcriptional control of hns expression. Mol. Microbiol. 19: 965-975.

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47. Afflerbach, H., Schroder, O. & Wagner, R. (1998). Effects of the Escherichia coli DNAbinding protein H-NS on rRNA synthesis in vivo. Mol. Microbiol. 28: 641–653 48. Kolmsee, T., Delic, D., Agyenim, T., Calles, C. & Wagner, R. (2011). Differential stringent control of Escherichia coli rRNA promoters: effects of ppGpp, DksA and the initiating nucleotides. Microbiology 157: 2871-2879. 49. Dennis, P.P., Ehrenberg, M. & Bremer, H. (2004). Control of rRNA synthesis in Escherichia coli: a systems biology approach. Microbiol. Mol .Biol. Rev. 68: 639-668. 50. Jin, D.J., Cagliero, C. & Zhou, Y.N. (2012). Growth rate regulation in Escherichia coli. FEMS Microbiol. Rev. 36: 269-287. 51. Zhang, X. & Bremer, H. (1996) Effects of Fis on ribosome synthesis and activity and on rRNA promoter activities in Escherichia coli. J. Mol. Biol. 259: 27-40. 52. Zhang, X., Dennis, P., Ehrenberg, M. & Bremer, H. (2002). Kinetic properties of rrn promoters in Escherichia coli. Biochimie 84: 981-996. 53. Liebig, B. & Wagner, R. (1995). Effects of different growth conditions on the in vivo activity of the tandem Escherichia coli ribosomal RNA promoters P1 and P2. Mol. Gen. Genet. 249: 328-335. 54. Pao, C.C. & Dyess, B.T. (1981). Stringent control of RNA synthesis in the absence of guanosine 5'-diphosphate-3'-diphosphate. J. Biol. Chem. 256: 2252-2257. 55. Mackow, E.R. & Chang, F.N. (1983). Correlation between RNA synthesis and ppGpp content in Escherichia coli during temperature shifts. Mol. Gen. Genet. 192: 5-9. 56. Vila-Sanjurjo, A., Schuwirth, B.S., Hau, C.W. & Cate, J.H. (2004). Structural basis for the control of translation initiation during stress. Nat. Struct. Mol. Biol. 11: 1054-1059. 57. Byrgazov, K., Vesper, O. & Moll, I. (2013). Ribosome heterogeneity: another level of complexity in bacterial translation regulation. Curr. Opin. Microbiol. 16: 133-139. 58. Wagner, R. (2009). Translational components in prokaryotes: genetics and regulation of ribosomes. In Encyclopedia of Life Sciences (ELS). John Wiley & Sons, Ltd: Chichester. 59. Limbach, P.A., Crain, P.F. & McCloskey, J.A. (1994). Summary: the modified nucleosides of RNA. Nucleic Acids Res. 22: 2183-96 60. Das, G., Thotala, D.K., Kapoor, S., Karunanithi, S., Thakur, S.S., Singh, N.S. & Varshney, U. (2008). Role of 16S rRNA methylations in translation initiation in Escherichia coli. EMBO J. 27: 840-851. 61. Ron, E.Z., Kohler, R.E. & Davis, B.D. (1966). Increased stability of polysomes in an Escherichia coli mutant with relaxed control of RNA synthesis. Proc. Natl. Acad. Sci. U.S.A. 56: 471–475. 62. Marchi, P., Longhi, V., Zangrossi, S., Gaetani, E., Briani, F. & Dehò, G. (2007). Autogenous regulation of Escherichia coli polynucleotide phosphorylase during cold acclimation by transcription termination and antitermination. Mol. Gen. Genomics 278: 7584.

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rRNA transcription and assembly were analyzed during cold adaptation.

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 after cold-shock 16S and 23S rRNA transcription and maturation continue at slower rate

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 rRNA molecules are synthesized de novo from all rrn operons of E. coli, mainly from P2 promoters rRNA newly transcribed during cold acclimation is preferentially found in polysomes.

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Ribosomes are slowly assembled during cold adaptation

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