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Department of Molecular Microbiology, John Innes Centre, Norwich, UK
Correspondence
R. Gary Sawers
sawers{at}staff.uni-marburg.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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1.2 kb. All eight transcripts have the same 3' end. The 5' ends of three of the transcripts, termed 6, 6a and 7, are located upstream of the operon. The promoters generating transcripts 6 and 7 are anaerobically regulated by FNR and ArcAP, while promoter 6a is constitutively active. The 5' ends of the other five transcripts are all located within the operon. Most of the 5' ends of these operon-internal transcripts result from RNA polymerase-dependent processing of the three longer primary transcripts, 6, 6a and 7. Here, it is demonstrated that subsequent to, and distinct from, these processing events, post-transcriptional modification of these transcripts also occurs through the action of the endoribonuclease RNase E. Transcripts 6 and 7 exhibit differential stability with half-lives of 1 and 5 min, respectively. Transcript 7, which has the longer half-life, is the longest transcript of the operon and has a 340 base untranslated leader. Two of the operon-internal transcripts, 4 and 5, also have comparatively short half-lives in the wild-type, which are significantly increased in a mutant with impaired RNase E activity. A precursor-product relationship is observed between the longer transcripts 3–7 and transcripts 1 and 2. The 5' ends of transcripts 1 and 2 are closest to the pflB gene and have half-lives of approximately 7–8 min. The consequence of this regulation is an accumulation of full-length pflB transcript and comparably low levels of dicistronic transcript. This ensures different levels of synthesis of the formate transporter FocA and pyruvate formate-lyase during anaerobic growth, while maintaining coordinate regulation. Transcript analysis throughout the growth phase revealed that maximal anaerobic expression of the focA-pflB operon was restricted to exponentially growing cells. Expression of transcript 7 peaked in early to mid-exponential phase, while the levels of transcript 6 steadily accumulated toward the late-exponential phase of growth. Taken together, these findings indicate that although subject to common positive control by ArcAP and FNR, the transcripts generated by promoters 6 and 7 are subject to differential temporal and post-transcriptional regulation.
Present address: Max-Planck-Institut für Terrestrische Mikrobiologie, Karl-von-Frisch-Straße, D-35043 Marburg, Germany
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Coburn & Mackie, 1999). Since the mean half-life of mRNA species in bacteria like E. coli lies between 2 and 4 min, control of mRNA turnover and degradation play key roles in influencing gene expression (Kennell, 2002; Kushner, 2002; Li & Altman, 2004; Ow et al., 2002). In particular, differential endoribonucleolytic processing and stability of polycistronic transcripts allow modulation of the cellular concentrations of gene products while maintaining coordination of product synthesis (Coburn & Mackie, 1999; Higgins & Smith, 1986; Kennell, 2002; Kushner, 2002; Newbury et al., 1987). Usually these processing reactions form part of the general turnover cycle of mRNAs and regulation is a consequence of whether particular portions of transcripts are more or less susceptible to further degradation by exoribonucleases.
There are three major characterized cytoplasmic endoribonucleases in Escherichia coli, and these include the enzymes RNase III, RNase E and RNase G. While RNase III plays a major role in processing specific transcripts, its role in mRNA turnover is limited (Aristarkhov et al., 1996; Kushner, 2002). On the other hand, RNase E and its paralogue RNase G have major roles in mRNA turnover (Li et al., 1999; Tock et al., 2000; Umitsuki et al., 2001; Wachi et al., 1997) and both enzymes are conserved in a large number of prokaryotes (Kushner, 2002). The activity of RNases E and G is markedly affected by the presence of a 5'-end monophosphate on transcripts, and these transcripts are cleaved at a distant site (Bouvet & Belasco, 1992; Coburn & Mackie, 1999; Mackie, 1998; Tock et al., 2000). The recent structural determination of the catalytic domain of RNase E (Callaghan et al., 2005) has provided important insights into how the enzyme catalyses cleavage at a distance. RNase E is also associated through its C-terminal domain with a large protein complex called the ‘RNA degradosome’ (Carpousis, 2002); however, there is still considerable debate as to what role the ‘degradosome’ plays in mRNA turnover (Ow et al., 2000; Kennell, 2002; Kushner, 2002). RNase E initiates turnover of many mRNA species through selective processing at specific sites. The findings of this study show that RNase E is involved in initial events during the turnover of the anaerobically inducible focA-pflB transcript.
Expression of the focA-pflB operon is remarkably complex. The operon encodes the central enzyme of fermentative metabolism, pyruvate formate-lyase (PFL), and a membrane protein, FocA, thought to transport formate (Suppmann & Sawers, 1994; Sawers & Clark, 2004). Expression of the operon is subject to redox-dependent transcriptional activation by FNR and ArcAP (Sawers & Suppmann, 1992; Sawers, 1993; Drapal & Sawers, 1995; Kaiser & Sawers, 1995). Transcription of the operon is also controlled from multiple promoters. Three of these promoters are located in the regulatory region 5' of the operon, and two of these (promoters 6 and 7) are anaerobically regulated by FNR and ArcAP, while the third is a weakly transcribed constitutive promoter termed 6a (Fig. 1; Sawers & Böck, 1989). The 5' ends of five further transcripts map within the operon. The 3' ends of all eight transcripts map to the end of the pflB gene (Sawers & Böck, 1989). Although the transcripts generated from promoters 6, 6a and 7 are the result of primary transcription events (Sawers, 1993; Kaiser & Sawers, 1997), recent studies have demonstrated that the majority of the five operon-internal transcripts result from specific processing of the longer primary transcripts (Sawers, 2005b). Surprisingly, these processing events appear to be RNA-polymerase-dependent and although it is currently uncertain what factor(s) is responsible for this processing, it is clear that it is not RNase E, RNase G or RNase III. Nevertheless, it is possible that one or more of these endonucleases is involved in the general turnover of these transcripts subsequent to their generation. Since nothing is known regarding the stage in the growth phase that focA-pflB operon transcription occurs and what controls the initiation of transcript turnover and degradation, this study aimed to shed light on these events. As well as identifying a role for RNase E in transcript turnover, this work also shows that transcription of the operon is restricted to the exponential phase of growth, that the anaerobically inducible promoters 6 and 7 are expressed at different stages of the growth phase and that their transcripts have different stabilities.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. Bacteria were grown routinely at 37 °C in the buffered rich medium TGYEP, pH 6.5, which included 20 mM glucose (Begg et al., 1977). Where indicated, pyruvate was added to 10 mM final concentration. Aerobic cultures were grown in flasks filled maximally to one-tenth of their volume, while anaerobic cultures were grown in stoppered bottles filled with medium to the top.
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Antibiotics were added to the following final concentrations (µg ml–1): ampicillin, 75; chloramphenicol, 15; tetracycline, 15. Media were solidified by the inclusion of 1.5 % (w/v) agar.
Analysis of RNA transcripts.
Two methods were used to isolate total RNA from cells. The standard method involved use of a Qiagen RNeasy kit, according to the manufacturer's instructions. In experiments where the half-life of the focA-pflB transcripts was analysed, cells were grown anaerobically to an OD600 of 0.5 and rifampicin was added to the cultures to a final concentration of 0.2 mg ml–1. To obtain the 0 min time-point, immediately prior to addition of rifampicin, a 5 ml aliquot of the culture was removed and rapidly added to 5 ml hot (65 °C) phenol (Aiba et al., 1981). At appropriate time-points after rifampicin addition, a 5 ml aliquot of the culture was removed and immediately added to 5 ml hot phenol for RNA preparation. In experiments where RNA was isolated from CH1826 (rne-1), cells were grown to an OD600 of approximately 0.5 at 30 °C and then cultures were shifted to 42 °C for 30 min prior to harvesting of cells for RNA preparation or addition of rifampicin to determine the chemical half-life of transcripts. The chemical half-life determinations were performed at least twice for each strain and representative gels are show for each experiment.
S1 nuclease mapping of focA-pflB transcripts was performed according to either Sawers & Böck (1989) or Sawers (2005b) using 50 µg total RNA. The BamHI DNA fragment used for hybridization was derived from plasmid p29 (Christiansen & Pedersen, 1981). Labelling of this fragment with [
-32P]ATP, as well as treatment of the fragment prior to S1 analysis, was carried out exactly as described previously (Sawers, 2005b).
Northern blotting of total RNA was performed according to Sawers & Böck (1989). A total of 10 µg total RNA from each sample was applied to a 1 % (w/v) agarose gel containing formaldehyde. The 2 kbp DNA probe used to detect the pflB transcripts is shown in Fig. 1
and was labelled by nick-translation (Sambrook et al., 1989) using [
-32P]dATP.
Other methods.
Protein concentration was determined by the Lowry method. SDS-PAGE was performed using 7.5 % (w/v) polyacrylamide gels according to the method of Laemmli (1970). Western blotting was carried out according to Towbin et al. (1979). Anti-PFL antiserum was diluted 1 : 10 000 and the antibody–antigen reaction was visualized using the ECL chemiluminescent method (Amersham Bioscience), exactly as recommended by the manufacturer. These experiments were repeated at least twice and representative gels are shown. Densitometric analyses were performed using SynGene Gene Tools analysis software.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Five of these transcripts result from processing of three longer transcripts, which originate in the upstream regulatory sequences (URS) (Fig. 1). These processing events are catalysed by a currently unknown factor(s), but they appear to be distinct from RNA turnover and degradation events (Sawers, 2005b). To determine precisely when during the growth phase the focA-pflB operon is expressed, total RNA from cells grown aerobically and anaerobically in the exponential and stationary phase was isolated and analysed by S1 nuclease protection (Fig. 2). Analysis of RNA from aerobic cells (Fig. 2, lane 1) grown to mid-exponential phase revealed low levels of transcripts 1–5, with barely detectable levels of transcript 6A (anaerobically induced, FNR-dependent transcript 6 is not present in aerobically grown cells: Sawers, 1993; Reyes-Ramírez & Sawers, 2006). Transcript 7 is never observed in aerobically grown cells (Sawers, 1993; Reyes-Ramírez & Sawers, 2006). Supplementation of the growth medium with pyruvate, which was shown previously to activate pflB-lacZ expression (Sawers & Böck, 1989), had only a marginal effect in slightly increasing the levels of transcript 6A (Fig. 2, lane 2). RNA isolated from cells grown aerobically with glucose into late stationary phase had a slightly altered transcript pattern where the level of transcript 6 or 6a (this gel system does not distinguish these transcripts) increased approximately threefold relative to the level in exponential-phase cells (Fig. 2, lane 3). This is probably due to increased transcription from promoter 6, as no such increase in transcript level was observed when glucose was omitted from the growth medium (see lane 4) and transcript 6a has been shown to be constitutively transcribed (Kaiser & Sawers, 1997).
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Anaerobic growth to exponential phase showed substantial induction of transcript levels (Fig. 2, lanes 5 and 6), which is consistent with previous observations (Sawers & Böck, 1989). While the levels of all transcripts increased co-ordinately relative to levels during aerobic growth, transcript 7 was only detectable under anaerobic conditions. Pyruvate had no discernible effect on the overall transcript pattern (Fig. 2, lane 6).
In cells grown anaerobically to late stationary phase, transcripts 4, 5, 6 and 7 were not detectable and transcripts 1, 2 and 3 were dramatically reduced in level compared with exponentially grown cells. This finding indicates that focA-pflB operon transcription is virtually non-existent in late stationary phase. This result contrasts with what was observed in aerobically grown cells and suggests that transcription initiation was reduced under these conditions in anaerobic cells and/or that the transcripts were degraded.
To determine more precisely in the growth phase when the focA-pflB transcript levels started to decrease during anaerobiosis, RNA was prepared from cells throughout growth, from early exponential to early stationary phase (Fig. 3). S1 nuclease protection analysis revealed that even in cells that were emerging from the lag phase (Fig. 3b, lane 1), anaerobic focA-pflB transcription was activated. Moreover, the pattern of transcripts 1–6 was similar until late-exponential phase (Fig. 3b, lane 5), indicating that there was no difference in the level of co-transcriptional processing. Interestingly, expression of transcript 7 peaked at early to mid-exponential phase (Fig. 3b, lanes 2 and 3). On the other hand, the intensity of transcript 6, as determined by densitometric analysis, peaked at late exponential phase (Fig. 3b, lane 5). As soon as cells entered stationary phase, transcript 7 was no longer detectable and the levels of transcripts 4, 5 and 6 were substantially reduced (Fig. 3b, lane 6). This suggested either that transcription of the operon was reduced or that turnover of the transcripts was significantly increased, or that both of these events contributed to reduced levels of the transcripts.
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). The level of the full-length pflB transcript was clearly reduced in stationary phase in accord with what was observed in the S1 nuclease protection experiment.
A Western blot using anti-PFL antiserum revealed that the level of PFL polypeptide was approximately fivefold lower in the lag phase (Fig. 3d, lane 1) compared with the maximal level observed in late exponential phase (Fig. 3d, lane 5). The level of PFL polypeptide began to decrease in early stationary phase, but was still present at significant levels, suggesting that the half-life of the protein is long. Notably, the Western blot revealed a protein doublet, the lower band of which, termed PFL', is characteristic of radical-bearing enzyme that has undergone oxygenolytic cleavage (Knappe & Sawers, 1990; Wagner et al., 1992) and indicates that the polypeptide in early stationary phase was biologically active.
Differential half-lives of focA-pflB transcript 5' ends
The half-lives of the 5' ends of seven of the eight focA-pflB transcripts were determined using rifampicin to inhibit DNA-dependent RNA polymerase transcription initiation (see Methods) and S1 nuclease protection to analyse the complete set of transcripts (Fig. 4). RNA was analysed from cultures grown to the mid-exponential phase, equivalent to an OD600 of 0.5 (see sample 2 in Fig. 3a). The transcripts exhibited different turnover profiles in response to rifampicin treatment. Whilst transcript 6 was rapidly degraded with a half-life of 1 min (see Fig. 4a, b), the half-life of transcript 7 was shown reproducibly to be between 5 and 6 min. This finding further emphasizes the different regulation of these transcripts. Transcript 5 also had a similar turnover profile to that of transcript 6 and thus had a similar half-life of approximately 1 min. The remaining transcripts exhibited a precursor–product relationship, in particular transcript 1, the level of which increased during the course of the experiment (Fig. 4a). These findings indicated that transcript 1 was a degradation-intermediate for at least some of the longer transcripts.
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).
RNase E is responsible for focA-pflB transcript turnover
While the RNases III, E and G have been shown not to be involved in the initial co-transcriptional processing of the focA-pflB transcripts (Sawers, 2005b), it was important to determine whether any of them had a role in turnover of the transcripts. The half-lives of the 5' ends of the transcripts were therefore analysed in various RNase mutant backgrounds (Fig. 5a). Transcripts 4, 5 and 6 were no longer detectable in the wild-type, and the rng and rnc mutants, after 10 min treatment with rifampicin. Indeed, the transcript profiles were essentially identical (Fig. 5a). In contrast, in a mutant with a temperature-sensitive RNase E enzyme, transcripts 4, 5 and 6 could readily be detected after 10 min treatment with rifampicin. Densitometric analysis of the transcripts indicated that the half-lives of transcripts 5 and 6 had increased from 1 min in the wild-type to approximately 5 min in the rne-1 mutant (data not shown). Moreover, transcripts 1 and 2 had neither diminished in level nor accumulated intermediates on the degradation pathway. This finding indicates that RNase E has an important role minimally in the turnover of the 5' ends of the focA-pflB transcripts.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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One of the unusual features of the focA-pflB operon is the long URS, which comprises minimally 416 bp with an untranslated region of 346 bases between the 5' end of transcript 7 and the GUG translation initiation codon of the focA gene (Sawers & Böck, 1989). Within this URS are three promoters, the weak, constitutive P6a promoter (Kaiser & Sawers, 1997), and the anaerobically inducible P6 and P7 promoters (Sawers, 1993; Drapal & Sawers, 1995). Both P6 and P7 are regulated by FNR and ArcAP, but while the upstream P7 promoter is absolutely dependent on both FNR and ArcAP, the strong P6 promoter is still functional in the absence of ArcAP. Until this study, this was the only regulatory distinction between these promoters. The transcription start site of the P7 promoter is far upstream of the focA gene and the function of this promoter in governing operon expression has been an enigma, mainly because of its location, but also because the activity of the promoter appears to be constrained (Drapal & Sawers, 1995). Here it is clearly shown that the level of the P7 transcript peaks in the early exponential phase of growth, with the level becoming reduced in the mid- to late exponential phase. In contrast, the level of P6-generated transcript slowly increased until the late exponential phase of growth, where it peaked, and then decreased rapidly upon entry into stationary phase. Based on this experiment alone, it is not possible to state unequivocally that the temporal difference in P6 and P7 transcript levels is a consequence of a difference in transcription activation, since the absolute transcript level observed is the sum of transcription plus turnover. The results of the transcription inhibition experiment, however, revealed that transcript 7 has a comparatively long half-life of approximately 5 min, while that of transcript 6 was only of the order of 1 min. Whilst it cannot be excluded that specific transcript stability may change throughout the growth phase, this finding suggests that the differences in temporal regulation observed for transcripts 6 and 7 are through transcriptional control, indicating that these promoters are responding in their activity to different signals. Since both are regulated positively by ArcAP and FNR, the difference in their regulation could be due to either promoter 7 functioning only when both ArcAP and FNR are maximally active, or a further factor to which this particular promoter responds.
It is intriguing that transcript 7, which has a 346 base untranslated region, has a significantly longer half-life than transcript 6, which lies downstream of it and is within 26 bases of the focA gene. This difference in stability might be conferred by the additional 320 bases of AU-rich sequence at the 5' end of the P7 transcript.
The rapid degradation of transcripts 4 and 5 is also slowed markedly by the temperature-sensitive rne-1 mutation, suggesting that they are also substrates for RNase E. At least in the case of transcripts 1 and 2 it appears clear that they are likely intermediates in the degradation of the longer focA-pflB transcripts (Fig. 6) and they act as a barrier to further rapid degradation of the full-length pflB transcript. The factor(s) determining the comparative longevity of these transcripts is also currently unclear. Northern and Western blotting experiments indicated that the pflB transcripts and the PFL protein are relatively long-lived species in the growing anaerobic cell. This correlates well with the key role that PFL plays in primary metabolism during both fermentative and anaerobic respiratory growth (Sawers & Clark, 2004). The formate channel FocA is required in lower amounts and presumably the amount synthesized from the weakly expressed but long-lived dicistronic focA-pflB transcript is sufficient to meet the requirements of the cell to export and reimport formate (Suppmann & Sawers, 1994). Further work will be required to determine the precise level of FocA in the cell.
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), since in an rne-1 mutant grown at the non-permissive temperature, the characteristic pattern of eight transcripts (including transcript 6a) is observed. Nevertheless, it appears that RNase E contributes to the production of the comparatively stable transcripts 1 and 2, since their abundance in the rne-1 mutant was significantly reduced compared with the wild-type. It will be of interest to determine whether it functions alone or as part of the ‘degradosome’ in its capacity to turnover the focA-pflB transcript.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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Received 28 February 2006;
revised 9 April 2006;
accepted 11 April 2006.
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X174 with HinfI. The sizes of the respective fragments are shown on the right of the figure.