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
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ABSTRACT |
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Present address: Institut für Pathologie, Klinikum der Philipps-Universität, Baldingerstraße, D-35043 Marburg, Germany.
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INTRODUCTION |
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The CspB protein from B. subtilis and CspA from E. coli adopt the structure of a five-stranded -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 -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.
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METHODS |
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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 0·40·6 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 [
-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 187205 (E. coli numbering). Ten picomoles of oligonucleotide was labelled with [
-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 0·52x106 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 [-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 [
-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 725 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 [
-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
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 [-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.
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RESULTS |
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DISCUSSION |
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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 3136 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 813 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 ShineDalgarno 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 stemloop 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 stemloop 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 stemloop 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.
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ACKNOWLEDGEMENTS |
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Received 23 July 2003;
revised 11 December 2003;
accepted 12 December 2003.
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