Temperature-dependent processing of the cspA mRNA in Rhodobacter capsulatus

Stephanie Jäger1,{dagger}, Elena Evguenieva-Hackenberg1 and Gabriele Klug1

1 Institut für Mikrobiologie und Molekularbiologie, Justus-Liebig-Universität Giessen, Heinrich-Buff-Ring 26, D-35392 Giessen, Germany

Correspondence
Gabriele Klug
Gabriele.Klug{at}mikro.bio.uni-giessen.de


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
The expression of genes for cold-shock proteins is proposed to be regulated primarily at the post-transcriptional level by increase of mRNA stability after transition to low temperatures. Destabilization of the Escherichia coli cold-induced cspA transcript at 37 °C as well as stabilization upon cold shock is known to depend on the unusually long (159 nt) 5'-untranslated region. Determination of the cspA mRNA 5'-end from Rhodobacter capsulatus revealed a shorter distance between the start of transcription and the start codon for translation. The cspA mRNA of R. capsulatus was shown to be stabilized at low temperatures to a greater extent than other investigated transcripts. To address the mechanism of decay of the cspA transcript, it was incubated with purified degradosome of R. capsulatus. Endoribonucleolytic in vitro cleavage in the 5'-untranslated region as reported for the cspA transcript of E. coli in vivo was not observed. Instead, the data indicated that the cspA mRNA decay in R. capsulatus is mediated by endoribonucleolytic cleavages within the cspA coding region.


Abbreviations: PAA, polyacrylamide; UTR, untranslated region

{dagger}Present address: Institut für Pathologie, Klinikum der Philipps-Universität, Baldingerstraße, D-35043 Marburg, Germany.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Micro-organisms possess the ability to adapt to sudden changes in their habitat. A rapid downshift in temperature is known to induce the production of a specific set of proteins, the cold-shock or cold-stress proteins (CSPs) (for a recent review see Gualerzi et al., 2003). The major cold-shock protein in Escherichia coli, CspA, was identified in 1987 (Jones et al., 1987). CspA and homologous proteins are small (~7 kDa) mostly acidic proteins and are present in more than 60 psychrophilic, mesophilic and thermophilic bacterial species of the 96 bacterial genomes in the NCBI Entrez Genome Database (www.ncbi.nlm.nih.gov). Induced expression of CspA homologues in E. coli is not exclusively dependent on cold shock but also on growth phase or glucose supply (Yamanaka & Inouye, 1997). Only four of nine CspA-homologous proteins (CspA–I) from E. coli are cold-shock inducible, namely CspA, CspB, CspG and CspI (reviewed by Phadtare et al., 2000). In contrast to E. coli, the Gram-positive soil bacterium Bacillus subtilis only contains three CspA homologues, CspB, CspC and CspD (Graumann & Marahiel, 1999).

The CspB protein from B. subtilis and CspA from E. coli adopt the structure of a five-stranded {beta}-barrel fold containing the conserved RNA binding motifs RNP1 and RNP2 (Schindelin et al., 1993, 1994). The amino acid sequence of CspA and other cold-shock proteins shares more than 40 % identity with the cold-shock domain (CSD) of eukaryotic Y-box proteins, which are able to specifically bind RNA and single-stranded DNA (Lee et al., 1994). While heat-shock proteins as chaperones manage the correct folding of proteins at normal and elevated temperatures, cold-shock proteins are thought to enhance mRNA degradation and translation efficiency and are therefore called ‘RNA chaperones' (reviewed by Graumann & Marahiel, 1998). CspA and homologous proteins exhibit a nonspecific, cooperative binding to linear RNA that inhibits formation of RNA secondary structures after transcription and thereby stimulates RNase degradation and translation (Jiang et al., 1997; reviewed by Graumann & Marahiel, 1998). Translation initiation of cold-shock proteins at low temperatures is enhanced by the downstream box (DB), a 15 nt sequence which is complementary to 16S rRNA and has been found downstream of the start codon of cspA, cspB, cspG, cspI, csdA and rbfA (reviewed by Phadtare et al., 2000).

The regulation of cold-shock genes seems to be mediated mainly at post-transcriptional level. No cold-shock-specific sigma factor or repressor protein has been identified so far. An AT-rich upstream element was localized in front of the -35 region of the E. coli cspA promoter that enhances transcription at 15 °C as well as at 37 °C (Mitta et al., 1997). The cspA promoter seems to be active at 37 °C but efficient translation is reduced due to extreme instability of the transcript, with a half-life of less than 12 s at 37 °C in contrast to more than 20 min at 15 °C (Brandi et al., 1996; Goldenberg et al., 1996; Fang et al., 1997). Destabilization of the transcript at 37 °C as well as stabilization at low temperatures is known to depend on the unusually long 5'-untranslated region (UTR) (Mitta et al., 1997). An extremely long 5'-UTR is also characteristic for cspB, cspG, cspI and csdA and for the cold-shock genes of B. subtilis. In E. coli, the remarkably rapid degradation at 37 °C is at least partially due to action of endoribonuclease E consequently reducing translation of CspA (Fang et al., 1997). In addition, premature termination or pausing during transcription of the 5'-UTR at 37 °C might be responsible for the low production of CspA (Fang et al., 1999). This region contains the 11 bp cold box, which is bound by CspA and CspE (Bae et al., 1997, 1999). Binding of CspA to the cold box of its own mRNA destabilizes the elongation complex of RNA polymerase, which results in attenuation of transcription, whereas binding of CspE to the DNA leads to pausing of the RNA polymerase.

The cold-shock response has been studied extensively in E. coli and B. subtilis; however, only little is known about the regulation in other bacterial species. The facultatively photosynthetic {alpha}-proteobacterium Rhodobacter capsulatus is a free-living bacterium that is highly exposed to variations in temperature in its aqueous habitats. Our previous studies with this bacterial species revealed that the degradation of the puf operon encoding pigment-binding proteins of the photosynthetic apparatus depends on rate-limiting cleavage by RNase E (Klug et al., 1992; Fritsch et al., 1995). Recently we showed that R. capsulatus contains an mRNA-degrading machinery that is similar to but also distinct from that of E. coli (Jäger et al., 2001). To elucidate whether mRNA processing is also involved in the regulation of the cold-shock response in R. capsulatus, we have addressed the regulation of the cspA gene from this bacterium through post-transcriptional mechanisms. We have shown that the cspA mRNA is strongly stabilized at low temperatures in comparison to other investigated transcripts. Our data suggest that the cspA mRNA is degraded by RNase E-mediated endoribonucleolytic processing in the cspA coding region; rate-limiting cleavage in the long 5'-UTR was not observed.


   METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Bacterial strains and growth conditions.
Rhodobacter capsulatus was cultivated aerobically in minimal malate medium at 32 °C (Drews, 1983). As indicated, the cultures were shifted to cold shock at 10 °C. For cloning purposes, E. coli strains were routinely grown in Standard I medium (Merck) at 37 °C. If appropriate, antibiotics were added to the growth medium at the following concentrations: kanamycin 25 µg ml-1 and ampicillin 200 µg ml-1.

Plasmid construction.
Cloning procedures were carried out by standard procedures (Sambrook et al., 1989). Restriction enzymes and T4 DNA ligase were obtained from New England Biolabs. PCR primers were obtained from Carl Roth GmbH and PCR products were generated with Vent DNA polymerase (New England Biolabs). The constructed plasmids were cloned in E. coli strain JM109 (Yanisch-Perron et al., 1985). For in vitro degradation assays, the cspA 5'-UTR and the cspA gene together with 5'- and 3'-UTR corresponding to the complete cspA mRNA were cloned under T7 control. For this purpose the oligonucleotide PR-cspA290+/EcoRI 5'-GGAATTCTAATACGACTCACTATACGTGAACTTGGACTTTCC-3' (starting at the cspA mRNA 5'-end determined by primer extension analysis) and the oligonucleotides PR-cspA403-/HindIII 5'-CCCAAGCTTCATATCTCTCTCCTCGTATTG-3' (starting at the 3'-end of the cspA 5'-UTR) and PR-cspA661-/HindIII 5'-CCCAAGCTTAAAGAAAAAAGCCCGCGTGG-3' (starting at the cspA mRNA 3'-end determined by RNase protection analysis), respectively, were used during PCR. The restriction sites are underlined and the T7 promoter is indicated in bold. After EcoRI/HindIII restriction, the PCR fragments were cloned into the EcoRI/HindIII-cut vector pUC18 (Yanisch-Perron et al., 1985), resulting in plasmids pUC18csp5'UTR and pUC18cspA, respectively.

RNA isolation and quantification of mRNA by Northern blot analysis.
Bacteria from 20 ml of an aerobic culture of R. capsulatus B10 (ATCC 33303) were harvested and total RNA was isolated as described by Nieuwlandt et al. (1995). To determine mRNA half-lives, during exponential growth phase at an OD660 of 0·4–0·6 rifampicin was added to the cultures (300 µg ml-1) and cells were collected at the indicated time points. Northern hybridization was performed as described by Heck et al. (1996). Total RNA (10 µg per lane) was electrophoresed on 1 % (w/v) agarose, 2·2 M formaldehyde gels and transferred to Biodyne B membrane (Pall) using a vacuum blot apparatus (Appligene). cspA-, puf- and puc-specific DNA fragments of R. capsulatus were radiolabelled with [{alpha}-32P]dCTP using nick translation (nick translation kit, USB). The oligonucleotide used for hybridization with fragmented R. capsulatus 23S rRNA was 5'-CTTAGATGTTTCAGTTCCC-3', corresponding to the 23S rDNA positions 187–205 (E. coli numbering). Ten picomoles of oligonucleotide was labelled with [{gamma}-32P]ATP using T4 polynucleotide kinase (New England Biolabs). The DNA fragments and the oligonucleotide used for hybridization were purified using micro columns (Probe Quant G-50, Amersham Biosciences). Per hybridization reaction 0·5–2x106 c.p.m. were used. The radiolabelled bands were quantified using a phosphor imaging system (Personal Molecular Imager FX, Bio-Rad) and the appropriate software (Quantity One, Bio-Rad).

In vitro degradation assays.
The 115 nt cspA5'UTR RNA and the 373 nt cspA RNA were transcribed in vitro from the HindIII-linearized plasmids pUC18csp5'UTR and pUC18cspA in the presence of [{alpha}-32P]UTP using T7 RNA polymerase (Promega) (Rauhut et al., 1996). RNase E and the degradosome were purified from R. capsulatus strain 37b4 (DSMZ 938) grown semi-aerobically (100 µM dissolved oxygen) at 32 °C and in vitro degradation assays were performed as described by Jäger et al. (2001). Approximately 4000 c.p.m. of labelled RNA was incubated with 0·3 µl of degradosome fractions at 32, 17 and 37 °C. The reaction was carried out in buffer optimized for RNase E (Fritsch et al., 1995) supplemented with 5 mM ATP to enhance helicase and phosphorolytic exoribonuclease activity of the degradosome. After separation on 7 M urea, 6 % polyacrylamide (PAA) gels the generated radiolabelled RNA fragments were quantified as described above. As molecular mass marker, RNA century marker (Ambion) was transcribed in vitro in the presence of [{alpha}-32P]UTP according to the manufacturer's recommendations.

Primer extension and RNase protection analysis.
The 5'-ends of the cspA transcript were determined by primer extension analysis as described by Heck et al. (1996) using the oligonucleotide PR-cspA-PE 5'-CTCCTCGTATTGGCCGAAG-3' hybridizing to the cspA transcript approximately 7–25 nt upstream of the start codon. Mapping of 5'-ends corresponding to RNase E cleavage sites in the 373 nt cspA transcript by primer extension analysis was performed using the oligonucleotide PR-cspA661-/HindIII 5'-CCCAAGCTTAAAGAAAAAAGCCCGCGTGG-3' hybridizing to the transcript's 3'-end. The 373 nt cspA mRNA was transcribed in vitro in the absence of [{alpha}-32P]UTP and incubated with degradosome fraction in buffer optimized for RNase E (Fritsch et al., 1995) at 32 °C prior to the primer extension reaction. The primer extension products were analysed on a 7 M urea, 6 % PAA sequencing gel side by side with the products of the sequencing reactions using the same labelled primer. DNA sequences were obtained by dideoxy sequencing (Sanger et al., 1977) using [35S]dATP{alpha}S and the T7 Sequencing Kit (Pharmacia).

The 3'-ends of the cspA transcripts were detected by RNase protection analysis using the HybSpeed RPA-Kit (Ambion) according to the manufacturer's recommendations. For generation of the antisense-RNA, a region 49 nt upstream to 139 nt downstream of the cspA stop codon was amplified by PCR using the oligonucleotides PR-cspA556+/EcoRI 5'-GGAATTCGATCTCGGGGCGCGACGG-3' and PR-cspA678-/HindIII 5'-CCCAAGCTTCGTCGGCGATCTGCAGG-3' (restriction sites underlined). After EcoRI/HindIII restriction, the PCR fragment was cloned into EcoRI/HindIII-cut vector Bluescript SK+, resulting in plasmid pBlcspA3'. The 0·2 kb antisense RNA was generated by T7 in vitro transcription of the EcoRI-linearized plasmid pBlcspA3' in the presence of [{alpha}-32P]UTP. The protected products were analysed on a 7 M urea, 6 % PAA sequencing gel in parallel with the products of the sequencing reactions. DNA sequences were obtained with the oligonucleotide PR-cspA556+ 5'-CGATCTCGGGGCGCGACGG-3' hybridizing to the antisense strand approximately 100 nt upstream of the detected RNA 3'-ends.


   RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Determination of the 5' and 3' ends of the cspA transcript from R. capsulatus
We determined the 5'- and 3'-ends of the R. capsulatus cspA transcript by primer extension and RNase protection analysis, respectively, using total RNA from cells grown at 32 °C or after 20–30 min transition to cold shock at 10 °C. Only one 5'-end could be detected, located 111 nt upstream of the start codon AUG, which may represent the transcriptional start point (Fig. 1a). Therefore, the 5'-UTR of the R. capsulatus cspA transcript consists of 114 nt, whereas the unusually long E. coli 5'-UTR has a length of 159 nt. Comparison of the putative promoter region of the R. capsulatus cspA gene revealed homology to the E. coli sigma-70 as well as to the E. coli cspA promoter in the -35 region, whereas homology in the putative -10 region is not obvious (Fig. 1b). By using RNase protection analysis, six 3'-ends of the cspA mRNA could be mapped (Fig. 2a). They are located in a U-rich region approximately 50 nt downstream of the stop codon UGA. We propose that a GC-rich DNA region of dyad symmetry spanning from 24 to 44 nt downstream of the stop codon of cspA followed by the 10 nt T-rich region functions as a transcriptional terminator of the cspA transcript (Fig. 2b). The RNA structure representing a rho-independent or intrinsic terminator was predicted by computer analysis using the mfold program version 2.3 (http://www.bioinfo.rpi.edu/applications/mfold/old/rna) (Zuker, 1989, 2003; Jaeger et al., 1989). The stability of the RNA stem–loop is calculated to {Delta}G values of -14·9 kcal mol-1 (62·3 kJ mol-1) at 32 °C and -19·3 kcal mol-1 (80·7 kJ mol-1) at 10 °C by the mfold program. During transcription, formation of the RNA secondary structure followed by the U-rich region results in transcription termination (von Hippel, 1998).



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Fig. 1. (a) Determination of the cspA mRNA 5'-end at 32 °C and after 20 min cold shock at 10 °C. Primer extension analysis was performed using an oligonucleotide hybridizing to the cspA transcript approximately 7–25 nt upstream of the start codon. Sequencing reactions obtained with the same oligonucleotide were run on a sequencing gel side by side with the products of the primer extension reaction and are labelled A, C, G and T. The detected 5'-end is indicated by an asterisk. (b) Comparison of the putative cspA promoter region of R. capsulatus (in bold) with the consensus sequences of the E. coli vegetative sigma factor (sigma-70) promoter and the E. coli cspA promoter (Yura et al., 1993; reviewed by Phadtare et al., 2000). The start point is indicated as +1.

 


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Fig. 2. (a) Determination of the cspA mRNA 3'-ends at 32 °C and after 30 min cold shock at 10 °C. RNase protection analysis was performed using an antisense RNA generated via T7 in vitro transcription corresponding to the cspA sense RNA approximately 100 nt upstream and 40 nt downstream of the detected 3'-ends. Sequencing reactions were obtained with an oligonucleotide hybridizing to the cspA coding strand 100 nt upstream of the detected 3'-ends corresponding to the 3'-end of the antisense RNA. Reactions were run on a sequencing gel side by side with the products of the RNase protection reaction and are labelled A, C, G and T. The detected 3'-ends are indicated by asterisks and the palindromic stem region of the putative rho-independent terminator of transcription is marked by arrows. (b) RNA secondary structure of the putative rho-independent terminator of transcription of the R. capsulatus cspA gene determined by using the mfold program version 2.3. The stability of the stem–loop structure is calculated to {Delta}G values of -14·9 kcal mol-1 (62·3 kJ mol-1) at 32 °C and -19·3 kcal mol-1 (80·7 kJ mol-1) at 10 °C.

 
Effect of temperature on the cspA transcript level
In order to quantify the effect of cold shock on the cspA expression, we performed Northern blot analyses of total RNA from R. capsulatus wild-type B10. Using the cspA-specific 0·2 kb DNA probe hybridizing to the complete cspA coding region, we detected a transcript with a size of 0·4 kb (Fig. 3a), which is in agreement with the predicted length of the cspA transcript based on the experimentally determined 5'- and 3'-ends. During temperature decrease from 32 °C, the optimal growth temperature of R. capsulatus, to cold shock at 10 °C, we observed a weak increase of the cspA transcript levels that reached a maximum of 1·5–1·8 fold (Fig. 3b). In contrast, a 2–3-fold decrease in the amount of transcripts of the puf and puc operon encoding pigment-binding proteins of the photosynthetic apparatus in R. capsulatus was detectable (not shown). The moderate induction of the cspA transcript after shift to 10 °C indicates either a rather constant level of transcription or different transcriptional activities compensated by altered mRNA stability.



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Fig. 3. Transcript levels of the R. capsulatus 0·4 kb cspA mRNA following cold shock at 10 °C. (a) Northern blot analysis of total RNA from R. capsulatus wild-type B10. RNA was isolated from R. capsulatus B10 grown at 32 °C (0 min) and at the indicated times after a temperature shift to 10 °C, separated by agarose gel electrophoresis, blotted onto nylon membrane and probed with the cspA-specific 0·2 kb probe hybridizing to the complete cspA coding region. The autoradiograph below shows the same blot rehybridized with a probe specific for fragmented 23S rRNA (14S rRNA) of Rhodobacter. (b) The mRNA bands were quantified by phosphor imaging and normalized with respect to the rRNA amount. Values were plotted as a function of time.

 
Effect of temperature on the stability of the cspA transcript
To test the effect of cold shock on the stability of the cspA transcript, we added rifampicin to cultures grown at 32 °C or 20 min after the transition to 10 °C. While the mean half-life of the 0·4 kb cspA transcript was 3·7±0·4 min at 32 °C, it increased significantly, about 13 fold, to 47±4 min at 10 °C (Fig. 4a, Table 1). To test, whether this effect is restricted to the cspA transcript or rather represents a general effect of temperature on mRNA stability, we determined the stability of transcripts of the puf and puc operon. After shift from 32 °C to 10 °C, we observed a sixfold increase of stability (4±0·8 min to 25±3 min) for the 2·7 kb pufBALMX processing product (not shown, Table 1) and a twofold increase (30±1 min to 60±10 min) for the 0·5 kb pufBA processing product (Fig. 4b, Table 1). The 0·5 kb pucBA transcript of R. capsulatus was stabilized threefold upon temperature downshift (15±4 min to 44±6 min) (Fig. 4b, Table 1). The calculated half-lives at 32 °C are consistent with previous determinations of R. capsulatus puf and puc mRNA stability (Klug, 1991). These data indicate a general mRNA stabilization at lower temperatures as a result of increased stability of RNA secondary structures or a reduced activity of ribonucleases. Nevertheless, the remarkable stabilization of the cspA mRNA upon cold shock represents a specific effect of temperature on cspA half-life.



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Fig. 4. Half-life determination of different R. capsulatus mRNA species at 32 °C and 10 °C. (a) Northern blot analysis of the R. capsulatus 0·4 kb cspA RNA at 32 °C and 10 °C. R. capsulatus wild-type B10 cells were cultivated overnight at 32 °C before shifting one culture to cold shock at 10 °C. After 20 min at 10 °C and 32 °C, the antibiotic rifampicin was added to the cultures (0 min). Total RNA was isolated from samples taken at various time points and the cspA-specific transcript was monitored by Northern hybridization using the 0·2 kb cspA probe. The autoradiograph below shows the same blot rehybridized with a probe specific for fragmented 23S rRNA (14S rRNA) of R. capsulatus. The half-life was determined by quantification of the mRNA bands by phosphor imaging. The mRNA amount was normalized with respect to the rRNA amount. The values were plotted on a semi-logarithmic scale as a function of time; the 0 min time points were set to 100 %. The calculated half-lives are indicated. (b) Half-life determination of the 0·5 kb pufBA and the 0·5 kb pucBA transcript at 32 °C and 10 °C by Northern blot analysis using total RNA from R. capsulatus wild-type B10. Specific transcripts of R. capsulatus were monitored on the same Northern membrane using a 1·7 kb probe hybridizing to genes Q, B and A of the puf operon, a 0·6 kb probe hybridizing to the genes B and A of the puc operon and the probe specific for fragmented 23S rRNA (14S rRNA). Half-lives were determined as described and are indicated.

 

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Table 1. Stability of different transcripts after cold shock from 32 °C to 10 °C

Half-lives were determined by analysis of at least three different Northern blots; the results are means±the maximum deviation.

 
In vitro degradation of the cspA 5'-UTR and the cspA transcript
To investigate the degradation of the cspA mRNA in relation to temperature, the whole cspA transcript and the 5'-UTR were separately cloned under T7 control and transcribed in vitro. Both transcripts were tested in in vitro degradation assays at different temperatures using purified degradosome of R. capsulatus grown semi-aerobically at 32 °C. An mRNA-degrading, high molecular mass complex, termed the degradosome, was initially identified and characterized in E. coli (Carpousis et al., 1994). We purified an analogous complex from R. capsulatus, which comprises endoribonuclease E, the key enzyme for mRNA degradation, two ATP-dependent RNA helicases, the transcription-termination factor rho and the 3'->5' exoribonuclease PNPase as a minor component of the complex (Jäger et al., 2001). Incubation of the 115 nt cspA5'UTR RNA with degradosome fraction revealed only slight degradation of the transcript at 32 °C and 37 °C, resulting in two degradation products of ~105 and ~80 nt and minor fragments of smaller size (Fig. 5a). At the lower temperature of 17 °C we observed almost no degradation of the RNA. In contrast, incubation of the 373 nt cspA transcript with the degradosome fraction resulted in rapid RNA degradation (Fig. 5b). The half-life of the cspA RNA monitored in the in vitro degradation assay was decreased twofold at 37 °C and increased eightfold at 17 °C, respectively, in comparison to 32 °C. The results of the assays shown in Fig. 5 suggest that the degradation of the cspA transcript is due to RNase E-mediated endonucleolytic cleavages in the coding region. To localize the endonucleolytic cleavage sites, primer extension analysis was performed. Primer extension reactions with the 373 nt cspA transcript, which was previously incubated with degradosome fraction, were loaded side by side with the control reaction (transcript incubated with buffer only) (Fig. 6a). The detected cleavage sites are shown schematically in Fig. 6(b), and their position in the cspA mRNA sequence is marked in Fig. 6(c).



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Fig. 5. In vitro degradation of the 115 nt cspA5'UTR RNA (a) and the 373 nt cspA RNA (b). Approximately 4000 c.p.m. of [{alpha}-32P]UTP internally labelled RNA was incubated with RNase E-containing degradosome fractions for the time and at the temperature indicated. Fragments were separated on denaturing PAA gels. C, RNA in the absence of added protein for 32 min at 37 °C; M, RNA molecular mass marker. Approximate sizes of the major degradation products are indicated. The calculated in vitro half-life of the 373 nt cspA RNA at 17 °C, 32 °C and 37 °C is indicated.

 


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Fig. 6. (a) Mapping of 5'-ends corresponding to endonucleolytic cleavage sites in the 373 nt cspA transcript by primer extension analysis using an oligonucleotide hybridizing to the transcript's 3'-end. Sequencing reactions obtained with the same oligonucleotide were run on a sequencing gel side by side with the products of the primer extension reaction and are labelled G, A, T and C. The in vitro-transcribed 373 nt cspA transcript was incubated with RNase E-containing degradosome fraction for 1 min (lane 1) and 5 min (lane 2), at 32 °C prior to the primer extension reaction. Lane 3 represents the control reaction with the transcript incubated for 5 min in buffer. The detected 5'-ends are indicated by arrows; the arrow numbers correspond to the position of the detected cleavage site from the 5'-end of the cspA transcript. (b) Schematic representation of the positions of the detected endonucleolytic cleavage sites in the cspA transcript. (c) Precise positions of the endonucleolytic cleavage sites in the cspA mRNA sequence marked by arrows. The start and the stop codon are indicated in bold; the region of dyad symmetry of the putative rho-independent terminator of transcription is underlined.

 

   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
Gene expression of the cold-shock protein CspA and several homologues is thought to be regulated mainly at the post-transcriptional level by increased mRNA stability at low temperatures. In the present study, we investigated the level and the stability of the R. capsulatus cspA mRNA, whose translation product shows homology of 80 % to the major cold-shock protein of E. coli. After transfer of R. capsulatus cultures from 32 °C to cold shock at 10 °C, a rather moderate increase of the amount of the monocistronic 0·4 kb cspA transcript was detectable by Northern analysis. However, a decreasing amount of the oxygen-dependent transcripts of the puf and puc operon was detectable. Variations in the R. capsulatus cspA transcript level correlating with growth phase were not observed (not shown). In E. coli, massive presence of the CspA protein is detectable at 37 °C in early exponential growth phase, and destabilization of the cspA transcript does not start before mid- to late-exponential phase (Brandi et al., 1999). The 13-fold increase of the half-life of the R. capsulatus cspA mRNA at 10 °C indicates a post-transcriptional regulation at the level of mRNA stability but also implies that cspA transcription is reduced at lower temperatures. Increasing stability after cold shock by a factor of 2–6 was also observed for the oxygen-dependent puf and puc transcripts of R. capsulatus, indicating that this effect is not restricted to cspA. Temperature most likely has a general influence on the stability of RNA secondary structures and the activity of ribonucleases. Nevertheless, stabilization of the R. capsulatus cspA mRNA by a factor of 13 points to a specific stabilization upon cold shock.

A dramatic stabilization of the cspA mRNA was observed in E. coli after transfer to low temperature. The transcript exhibits a half-life of 12 s at 37 °C that increases 100-fold to more than 20 min at 15 °C (Brandi et al., 1996; Goldenberg et al., 1996; Fang et al., 1997). Similarly, the mRNA of the cold-regulated RNA-binding protein gene rbpA1 in the cyanobacterium Anabaena variabilis is stabilized upon cold shock (Sato & Nakamura, 1998). At 38 °C, a half-life of 5 min was detected, which increased fourfold to 18·5 min after transfer to 22 °C. However, changes in the half-life of other transcripts upon cold shock were not investigated in A. variabilis. Stability of the E. coli bulk mRNA and the specific transcript rrn, the leader sequence of the rrn rRNA, was determined at 37 °C and after cold shock (Brandi et al., 1996; Goldenberg et al., 1996). The results indicated that the extent of stabilization of the cspA mRNA after transfer to the low temperature exceeds that displayed by other E. coli transcripts. Extensive investigations of the E. coli csp genes resulted in identification of several conserved regulatory regions, e.g. the cold box or the upstream element (Bae et al., 1997; Mitta et al., 1997). An alignment of the cspA gene from R. capsulatus and E. coli revealed no region homologous to either the cold box or the AT-rich upstream element in the R. capsulatus cspA flanking regions (not shown).

Our data reveal that the 5'-end of the cspA mRNA is located 111 nt upstream of the start codon. The absence of further primer extension signals and homology of the region upstream of the detected 5'-end to the sigma-70 promoter consensus and the cspA promoter of E. coli implies that the detected 5'-end represents the transcription start point. While nt 31–36 upstream of the cspA 5'-end of R. capsulatus, whose DNA has a rather high G+C content, show significant homology to both -35 regions, nt 8–13 upstream of the cspA 5'-end resemble the E. coli -10 regions in only two of six nucleotides. Primer extension analysis of total RNA from R. capsulatus cells grown under aerobic (chemotrophic) conditions at 32 °C and 10 °C showed that positioning of the cspA mRNA 5'-end is not influenced by temperature. The 5'-UTR of the R. capsulatus cspA transcript consists of 114 nt, in contrast to 159 nt in E. coli (Fang et al., 1997). The cspA1 mRNA of Yersinia enterocolitica and the rbpA1 transcript of Anabaena variabilis also contain a 5'-UTR of approximately 150 nt (Neuhaus et al., 1999; Sato & Nakamura, 1998). In contrast, the 5'-UTR of the cold-inducible cspH gene from Salmonella enterica consists of only 23 nt (Kim et al., 2001). In comparison to the 5'-UTR of other investigated genes from R. capsulatus, e.g. the rpoD gene consisting of 77 nt (Pasternak et al., 1996), the groESL operon with a length of 60 nt (Hübner et al., 1996), or the lepB gene consisting of only 16 nt (Klug et al., 1997), the R. capsulatus cspA 5'-UTR can be termed extended.

Endoribonuclease E shows cleavage preference for single-stranded AU-rich regions (Ehretsmann et al., 1992; McDowall et al., 1994, 1995). In vitro degradation of the R. capsulatus cspA 5'-UTR revealed that no rate-limiting RNase E cleavage occurs in this region, although a few single-stranded AU-rich regions exist in the 115 nt cspA 5'-UTR. In E. coli and A. variabilis, it was supposed that altered stability and transcription, respectively, of the investigated cold-inducible transcripts cspA and rbpA1 is correlated with their 5'-UTR (Mitta et al., 1997; Sato & Nakamura, 1998). The A. variabilis rbpA1 5'-UTR contains a regulatory inverted repeat that serves as a target site for DNA-binding proteins involved in repression of transcription at high temperatures (Sato & Nakamura, 1998). The 5'-UTR of the E. coli cspA mRNA seems to regulate the degradation of the whole transcript and there is evidence for temperature-dependent RNase E cleavage in this region (Fang et al., 1997). Three base substitutions in a single-stranded AU-rich region around the Shine–Dalgarno sequence alter the secondary structure and markedly stabilize the cspA transcript at 37 °C. Furthermore, a substantially higher amount of cspA mRNA was detected in cells harbouring a temperature-sensitive RNase E mutation, but no direct evidence for in vitro cleavage of the transcript by RNase E exists. RNase E is known to attach to the RNA 5'-end prior to cleavage (Emory & Belasco, 1990) and binding is inhibited by both stem–loop structures and triphosphates at the 5'-end (Emory et al., 1992; Mackie, 1998). Recent investigations revealed that the cspA cold box region of E. coli forms a stem–loop structure at mRNA level, which is located 2 nt downstream of the 5'-end (Xia et al., 2002). Deletion of this secondary structure results in destabilization of the whole transcript. In contrast, the R. capsulatus cspA mRNA shows no homology to the cold box region, which might result in a shorter cspA 5'-UTR in this bacterial species.

Putative changes in the composition of the E. coli degradosome have been reported (Beran & Simons, 2001), but no variations in composition and degradative activity of the degradosome correlated with temperature were observed in R. capsulatus (S. Jäger & G. Klug, unpublished data). Particularly at 32 °C and 37 °C, in vitro degradation of the full-length cspA transcript using purified degradosome of R. capsulatus grown at 32 °C resulted in rapid generation of two major products of approximately 162 and 148 nt, as estimated from their migration in the gel. At the lower temperature of 17 °C, earlier generation of the 148 nt fragment could be monitored. The detected cspA RNA fragments most likely correspond to 5'-end products arising solely due to endonucleolytic cleavage at position 146, or at position 159 of the transcript (see Fig. 6b). The lack of other major cspA RNA fragments in Fig. 5(b) can be explained by very efficient endonucleolytic cleavage of the transcript at all sites represented in Fig. 6(b), and the generation of multiple small fragments. The endonucleolytic cleavages are probably performed by RNase E, although degradosome but not purified RNase E was used during in vitro degradation. The endonucleolytic cleavage sites at positions 309 and 312 are localized upstream of the putative terminator stem–loop structure (Fig. 6c). In vivo cleavage at these positions may serve to remove this secondary structure, which protects the transcript from exonucleolytic degradation. RNase E cleavage at each of the detected positions would lead to the immediate inactivation of the cspA transcript.

In conclusion, the data presented strongly suggest that the cold-shock-dependent stabilization of the R. capsulatus cspA mRNA is not regulated by rate-limiting cleavage in its 5'-UTR. Our in vitro experiments show that the full-length cspA transcript is cleaved endonucleolytically within the coding region, most probably by RNase E. This indicates that various mechanisms exist for the temperature-dependent processing of cold-shock transcripts in bacteria.


   ACKNOWLEDGEMENTS
 
This work was supported by the DFG graduate programme ‘Biochemie von Nukleoproteinkomplexen’ and the Fonds der Chemischen Industrie.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
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Received 23 July 2003; revised 11 December 2003; accepted 12 December 2003.



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