Departamento de Genética y Biología Molecular, Centro de Investigación y de Estudios Avanzados del IPN, Apartado Postal 14-740, México DF 07000, Mexico1
Author for correspondence: Gabriel Guarneros. Tel: +52 55 5747 3338. Fax: +52 55 57477100. e-mail: guarnero{at}lambda.gene.cinvestav.mx
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ABSTRACT |
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Keywords: bicistronic mRNA, mRNA stability, pth, ychF, RNase E
Abbreviations: Pnp, polynucleotide phosphorylase; Pth, peptidyl-tRNA hydrolase
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INTRODUCTION |
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E. coli pth has been the paradigm for a group of homologous ORF sequences conserved in eubacteria and eukaryotes (De La Vega et al., 1996 ; Schmitt et al., 1997
). In the E. coli chromosome, pth is flanked by genes of unknown function: ychF (named gtp1 in Galindo et al., 1994
) located 3' to pth, and ychH on the 5' end (orf2 in Galindo et al., 1994
). A polypeptide encoded by ychH has not been shown, but ychF encodes a homologue to a conserved GTP-binding protein (Gtp1 in Galindo et al., 1994
). As the concentration of Pth in the cell is critical to prevent peptidyl-tRNA-mediated lethality, we aimed to understand how pth expression is controlled. Our evidence indicates that both 5' and 3' sequences flanking pth affect the concentration of pth transcripts and Pth expression. However, except for limiting endonuclease RNaseE activity, Pth concentration is highly stable to changes in the growth conditions of the wild-type and of various mutant strains.
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METHODS |
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Plasmid constructs.
pRJ5·5 was constructed by cloning the EcoRI (27·1 min)KpnI (27 min) chromosomal segment in clone (246) 12A3(-) of Kohara (Kohara et al., 1987 ; Rudd, 1992
) into the EcoRI and KpnI sites of vector pGEM-4 (Promega), a vector convenient to obtain RNA probes through in vitro transcription using T7 RNA polymerase. Construct pRJ1·6 was obtained by subcloning the EcoRIEcoRV 1·6 kb segment from plasmid pRJ5·5 into the EcoRI and SmaI sites of pGEM-4. pRJ1·0 was obtained through cloning, into the EcoRI and SmaI sites of pGEM-4, of a PCR fragment containing pth and short flanking segments amplified from the pRJ5·5 template. The primers used were 5'-TACGTTATCTGAATTCGATACGCAGTTT-3', a sequence located upstream from the putative pth promoter (Dutka et al., 1993
) modified to contain an EcoRI site (underlined), and 5'-GAATTTGTCGATATCGCCGGTCTGG-3', harbouring the EcoRV site (underlined) located within ychF (Fig. 1B
, coordinate 1·6). pRJ0·8 was created by subcloning the 0·8 kb EcoRINruI fragment (Fig. 1B
, coordinates 0 and 0·8, respectively) into the EcoRI and SmaI sites in pGEM4.
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RNA probes for Northern blotting were synthesized by in vitro transcription and labelled with [-32P]UTP (see below). For probe P, used in Figs 2
and 5B
(upper panel), the template was pRJ0·8 restricted with DdeI (Fig. 1B
), whereas for probe F (Fig. 2
) the template was pRJ1·6 restricted with DraI (Fig. 1C
). The in vitro transcription mixtures contained, in 20 µl: 40 mM Tris/HCl (pH 8·0); 10 mM MgCl2; 5 mM DTT; 50 mM KCl; 300 µM of three NTPs; 10 µCi [
-32P]UTP (3000 Ci mmol-1, 111 TBq mmol-1; Amersham Pharmacia Biotech); 50 mg BSA ml-1; 10 units RNasin (Promega); 2 µg template DNA; and 5 units T7 RNA polymerase (New England BioLabs). They were incubated for 30 min at 37 °C. The reaction was stopped with 10 units RNase-free DNase (Boehringer Mannheim, Germany) for 15 min at 37 °C. Finally, 2 µl of 0·5 M EDTA, 80 µl 3 M sodium acetate (pH 5·2) and 10 µg glycogen (Boehringer Mannheim) were added. The stopped reactions were phenol/chloroform (1:1; v/v) extracted, precipitated with 2·5 vols ethanol at -20 °C for at least 20 min and finally resuspended in 10 µl water. For hybridization, the RNA probe was denatured with 40% formamide at 65 °C for 10 min and used as indicated above. The specific activity of the probes used was about 3x106 c.p.m. ng-1.
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The DNA probe for ß-lactamase (bla) transcripts (Fig. 5, lower panel) was obtained by PCR amplification on a pGEM4 template. Thirty femtomoles of DNA template was added to the amplification mixture [in 50 µl; 40 mM Tris/HCl (pH 8·0), 5 mM MgCl2, 10 mM DTT, 50 mM NaCl, 150 µM 3 dNTPs, 50 µCi [
-32P]dCTP (6000 Ci mmol-1, 222 TBq mmol-1; Amersham Pharmacia Biotech), 1 unit Taq DNA polymerase (Applied Biosystems) and 10 pmol of primers]. The primers were 5'-GTATTCAACATTTCCGTG-3', beginning within the second codon of bla, and 5'-CAATGCTTAATCAGTGAG-3' complementary to the final bla ORF sequence. Amplification was carried out for 50 cycles (95 °C, 30 s; 55 °C, 30 s; 72 °C, 1 min) after an initial denaturation period of 1 min at 95 °C. The amplified product was denatured at 95 °C and added to the hybridization mixture described above.
S1 nuclease analysis.
For S1 endonuclease protection analysis, the DNA probes (Fig. 1C, a and b) and
X184 DNA (Fig. 3B
, markers) were labelled at the 5'-ends by the phosphate exchange reaction. Ten picomoles of DNA was dissolved in the exchange mixture [in 20 µl; 40 mM Tris/HCl (pH 8·0), 10 mM MgCl2, 5 mM DTT, 50 mM NaCl, 50 µCi [
-32P]ATP (3000 Ci mmol-1, 111 TBq mmol-1; Amersham Pharmacia Biotech) and 10 units T4 polynucleotide kinase (New England Biolabs)] and incubated at 37 °C for 30 min. The reaction was stopped at 65 °C for 10 min and extracted with phenol/CHCl3. The solution was precipitated with 2 µl 3 M sodium acetate (pH 5·2) and cold ethanol. Probe c (Fig. 1C
) was labelled by 3'-repair at the ApaLI site; 10 pmol of the DNA fragment was dissolved in the repair mixture [in 20 µl: 40 mM Tris/HCl (pH 8·0), 10 mM MgCl2, 5 mM DTT, 50 mM KCl, 50 mg ml-1 BSA, 600 µM of dNTPs except dCTP, 10 µCi [
-32P]dCTP (6000 Ci mmol-1, Amersham Pharmacia Biotech) and 10 units DNA polymerase Klenow fragment (New England Biolabs)]. The mixture was incubated at 37 °C for 30 min. Finally, the labelled probe c was extracted, precipitated and redissolved in water as described for probes a and b.
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Western blotting.
The immunodetection assays in Fig. 5(A) were carried out as described elsewhere (Cruz-Vera et al., 2000
). The samples were resolved through an SDS-polyacrylamide gel: 10% for polynucleotide phosphorylase (Pnp), or 15% for Pth. The antisera used were raised by immunization of rabbits with a purified preparation of Pth (Cruz-Vera et al., 2000
) or Pnp protein (García-Mena et al., 1999
).
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RESULTS |
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Transcriptional initiation of pth and ychH
The reported sequence between ychH and pth in the E. coli chromosome contains three 70-like promoter sequences (Fig. 3A
; Hawley & McClure, 1983
; Wosten, 1998
). One
70 promoter presumably controls initiation of pth transcripts and the other two promoters, oriented in the opposite direction, could initiate transcripts containing ychH sequences. To investigate the activity of these promoters, we looked for the predicted transcripts by nuclease S1-protection assays. Total RNA extracted from strain C600 transformed with pRJ5·5 and 5'-end 32P-labelled DNA probes a and b were used (Fig. 1C
; see Methods). Probe a, a 2·4 kb EcoRIApaLI fragment comprising ychH, pth and most of ychF, yielded four protected fragments, visualized as two strong and two weak bands (Fig. 3B
, lane 2). However, probe b, 0·5 kb longer than a (Fig. 1C
), yielded only one fragment, which migrated nearly as band IV revealed by probe a (Fig. 3B
, lane 5). Using the nuclease S1-protection assay, which is about 100-fold more sensitive than Northern blot assay, similar fragments were observed with RNA extracted from untransformed cells (not shown). Therefore, the protecting transcripts were not initiated at vector promoters. These results show that the ends of the RNAs protected by fragments I, II and 750 nt are located within the 0·5 kb segment between the ApaLI and ClaI sites (Fig. 1C
). Fragment IV should be protected by a transcript divergent to the transcripts that protect fragments I, II (Fig. 1C
) and should contain ychH sequences (Fig. 3A
). The calculated sizes of the protected fragments revealed by probe a indicate that: (1) the faint signal of fragment I, above 1353 bp, is protected by RNA1 (Fig. 1A
) the 5'-end of which is located between pth and ychH; (2) the strong band, of about 1000 bp, of fragment II is protected with RNA2 (Fig. 1A
), the 5'-end of which is located between pth and ychF; and (3) the fragment of 750 nt, also a weak signal, corresponds to a transcript spanning from a site somewhere within ychF to the end of probe a.
Start sites of pth transcripts
To define the start sites of the pth transcripts, we determined their 5'-ends by primer extension on an RNA preparation of strain C600 transformed with pRJ5·5 (see Methods and Fig. 3C). A unique signal was detected at the cytidine located seven bp downstream from the -10 box of the putative promoter for pth (Fig. 3A
, +1). It is very likely that this signal corresponds to a transcriptional initiation site because: (1) in S1 protection assays, only one protected DNA fragment was visualized, implying that the primer extension signal was not due to an unspecific AMV reverse transcriptase drop-off (data not shown); and (2) the 5'-end cytidine was confirmed by primer extension on in vitro transcripts generated by RNA polymerase
70 (result not shown). These data strongly suggest that the identified Ppth promoter (Fig. 3C
) indeed controls pth transcription.
Transcriptional start for ychF transcripts
Evidence for the existence of a transcriptional promoter, specific for ychF, was derived from a plasmid construct that expressed protein Gtp1 even in the absence of a pth promoter region (data not shown). To investigate the presence of a transcriptional promoter in front of ychF, we attempted to identify the 5'-end of the corresponding transcripts through primer extension experiments. The assay, again, was conducted on RNA prepared from cells transformed with pRJ5·5 (see Methods). Results showed that the strongest signal detected corresponded to the adenine located at 6 bp downstream from a consensus -10 box of a putative promoter (Fig. 4A, s1, and Fig. 4B
). Other minor signals were identified at one adenine (s2), at 16 bp, and two thymines, at 11 and 17 bp, downstream from the box (Fig. 4B
). To find out which of these 5'-ends corresponded to ychF transcription initiation sites, we performed in vitro experiments using RNA polymerase
70 in the presence of [
-32P]ATP or [
-32P]UTP. Results showed the incorporation only of adenine at the 5'-end of the transcripts. Primer extension assays of the in vitro transcripts revealed only the ends corresponding to the adenines in positions 6 and 16 (not shown). Taken together, these data show the presence of PychF specific for ychF.
3'-ends of transcripts containing ychF sequences
We investigated the termination sites of the transcripts originated at Ppth and PychF. Results in Fig. 3(B) had suggested that the 3'-ends of ychF transcripts were located within the ApaLIClaI DNA segment (Fig. 1B
). To locate the 3'-ends of those transcripts precisely, we performed S1-nuclease protection using the ApaLIClaI DNA fragment 32P-labelled at the ApaLI end (see Fig. 1C
, probe c, and Methods). The RNA extracted from strain C600 transformed with pRJ5·5 yielded only one protected DNA fragment (Fig. 4C
, lane 2). From this result and the fragment size, it seems that the 3'-end of the transcripts is unique and located about 50 nt past the EcoRV site at coordinate 2·7 (Fig. 1B
). The sequence of this region (Oshima et al., 1996
) does not show any recognizable rho-independent transcription terminators (Lesnik et al., 2001
). However, two pairs of inverted repeats were recognized beyond the ychF termination codon. In addition, we identified a short ORF of 21 codons that overlapped with the second inverted repeat (Fig. 4D
).
Effect of ychF sequences on Pth protein concentration
The pth transcript is found in two types of RNA: a long homogeneous bicistronic pthychF message and a smear of transcripts from 200 to 800 nt (Fig. 2). Since pth ORF is 650 nt long, the smear could result from 3'-exonucleolytic degradation of longer pth transcripts. To investigate the efficiency of Pth expression from both types of transcripts, we compared the levels of Pth protein from pRJ5·5, which contains the complete pthychF operon, and pRJ1·6, which carries a deletion of most of the ychF 3' region. Results showed that the concentration of Pth protein in extracts from cells transformed with pRJ5·5 was about fivefold higher than that in extracts from cells transformed with pRJ1·6 (Fig. 5A
; compare lanes 1 and 3, upper panel). This effect is specific to Pth, since the concentrations of Pnp, an unrelated protein, remained unchanged in both transformants (lower panel). Trans-expression of ychF from an overproducing construct did not affect Pth concentration (result not shown). This implies that ychF sequences affect pth expression in cis.
Effect of ychF sequences on pth transcripts
To investigate how the ychF region affected Pth levels, we tested the effect of ychF deletions on pth mRNA from pRJ1·6. We carried out Northern blot assays on RNA samples from C600 transformants using probe P. The results showed a 1200 nt RNA in pRJ1·6, instead of the 2000 nt RNA1 observed with pRJ5·5, as expected from the ychF deletion (Fig. 5B, upper panel, compare lanes 3 and 1). Also visible in pRJ1·6 is the smear of partially degraded pth message not present in the pRJ0·8 construct, carrying a pth deletion (lane 4). A quantitative analysis of radioactive probe hybridized in lanes 1 and 3 showed that the pth transcripts from pRJ1·6 are just 20% of those from pRJ5·5. This calculation has taken into account variations in the mean copy number of plasmid constructs per cell, as it has been normalized to the levels of ß-lactamase transcripts also encoded in the constructs (Fig. 5B
, lower panel). As the constructs used carry the same original pth promoter, it was likely that the variations in pth transcript concentrations in the transformants were due to the effect of distal ychF sequences on pth transcript stability. In fact, the half-life of the pthychF complete pRJ5·5 transcripts was 2 min compared with less than 0·5 min for the pRJ1·6 transcripts (data not shown).
Effect of RNase E on the expression of pth from the bicistronic transcript
To investigate whether a particular endoribonuclease affected RNA1 and/or RNA2 stability, we used strains carrying a specific mutation in endoribonuclease E (RNase E) or in RNase III. A strain rnc105, a mutant affected in RNase III activity (Bardwell et al., 1989 ), carrying pRJ5·5 did not show any differences in concentration or in the 5'-ends of the pth transcripts relative to those in the wild-type strain. The mutation ams1 determines a thermosensitive RNase E at 43 °C (McDowall et al., 1993
). The half-lives of RNA1 and RNA2, as revealed by probe F, increased dramatically at 43 °C in C600ams1, relative to those at 32 °C (Fig. 6A
, B
). pth sequences probed with P also showed an increased stability at 43 °C (not shown). Interestingly, the stability of the transcripts from pRJ1·6, carrying pth (RNA3) but deleted for most of ychF, was not affected (not shown). These results suggest that RNase E processes sequences of the ychF transcript, reducing pth mRNA stability. The low stability of pth mRNA is accompanied by a low Pth concentration at 32 °C when RNaseE is active (Fig. 6C
, upper panel). The concentration of Pnp was also higher at 43 °C than at 32 °C in the ams1 strain, as expected from the fact that the pnp messenger is processed by RNase E (Hajnsdorf et al., 1994
). The protein concentrations at 32 °C versus 43 °C remained constant for both Pth and Pnp in the wild-type strain (Fig. 6
, lower panel).
Effect of the PychH region on pth expression
We inquired whether the presence of the ychH region affected pth expression. The levels of Pth protein expressed from two constructs and accumulated in the cells were compared. One of the constructs, pRJ1·0, harbours the DdeIEcoRV insert comprising pth and the initial sequences of ychF. The other, pRJ1·6, carries a longer segment from site EcoRI before ychH to the same EcoRV site in pRJ1·0 (Fig. 1D). The additional 200 bp segment in pRJ1·0 upstream of Ppth includes the putative ychH promoters (Fig. 3A
). The results of a Western blot assay (Fig. 5A
upper panel, lanes 2 and 3) showed that the levels of Pth accumulated in transformants for pRJ1·0 are, at least, twice those for pRJ1·6. This difference in Pth protein concentration is paralleled by the pth transcript levels (Fig. 5B
upper panel, compare lanes 2 and 3). Since the stability of the pth transcripts from both pRJ1·0 and pRJ1·6 is the same, and the ychH expression from a plasmid did not affect Pth expression (data not shown), we conclude that ychHpth intercistronic sequences affect pth transcription capacity. These sequences may be the putative ych promoters (Fig. 3A
) or part of the RNA polymerase binding site for Ppth (Nickerson & Achberger, 1995
).
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DISCUSSION |
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The messages of pth and the 3'-proximal gene, ychF, are found as either bicistronic or monocistronic transcripts (Figs 1 and 2
). The pth-only transcripts are a smear of RNA-containing proximal pth sequences as if they were products of exonucleolytic decay (Figs 2
and 5B
). However, we do not know whether the decay substrates are terminated pth monocistronic transcripts or processed bicistronic messages. If the latter case is correct, processing is not performed by RNase E (see below). It is likely that all pth-containing transcripts originate from Ppth, the only promoter identified upstream from the pth initiation codon (Fig. 3C
; García-Villegas et al., 1991
). The ychF-only transcript is an abundant message initiated from a previously unidentified promoter, PychF, located between the pth and ychF ORFs.
The complete pthychF bicistronic messenger is an efficient source for Pth accumulation; however, in the absence of distal ychF sequences, the pth messenger generates scant Pth protein levels (Fig. 5A). This result indicates that the ychF message stabilizes pth mRNA (Fig. 5B
). The effect is in cis, as trans expression of ychF did not affect Pth protein accumulation (not shown). Apparently, there is no evolutionary advantage to keep the pthychF pair together in bacterial chromosomes. The gene sequence ychHpthychF observed in E. coli is typical of enterobacteria (McClelland et al., 2001
; Parkhill et al., 2001
). Other sequenced genomes such as Haemophilus (Fleischmann et al., 1995
), Pseudomonas (Stover et al., 2000
) and Mesorhizobium (GenBank: XL465) maintain the pair pthychF, whereas the rest of the sequenced bacterial genomes keep pth-, ychH- and ychF-homologous genes dispersed in different regions.
In a thermosensitive RNase E mutant, incubated at a non-permissive temperature, the mono- and bicistronic transcripts containing ychF (RNA1 and RNA2) are stabilized, but the heterogeneous pth transcripts are unaffected (RNA3, data not shown). We interpret this result to mean that at least one RNase E processing site is located within ychF. The site may be in the transcript beyond (3' to) the EcoRV site (coordinate 1·6, Fig. 1B) because the 5'-ends previous to the EcoRV site are the same in the presence or absence of RNase E activity (not shown). Endonucleolytic cleavage of mRNA is usually followed by 3'
5' exonucleolytic degradation (Higgins et al., 1993
). Thus, RNase E activity may co-ordinately regulate the expression of both pth and ychF from the bicistronic transcript. Negative regulation by RNase E processing of ribonuclease messengers has been described for pnp (Hajnsdorf et al., 1994
), rnb (Zilhao et al., 1995
) and rne (Jain & Belasco, 1995
).
The in vivo concentration of the ychF-only transcript is much higher than that of pthychF (Fig. 2). These concentrations probably represent the transcription initiation frequencies at PychF and Ppth because both transcripts are equally stable (Fig. 6A
). This notion was reinforced by in vitro
70 RNA polymerase transcription; PychF is a stronger promoter than Ppth (not presented). Also, the degree of identity to the -10 and -35 consensus promoter boxes and the base composition of the spacer between boxes suggest that PychF may be a better promoter than Ppth (Hawley & McClure, 1983
). Although the spacer in Ppth corresponds to the consensus 17 bp, it includes 10 G:C pairs that could reduce the transcriptional efficiency of the promoter (Auble & deHaseth, 1988
). In contrast, PychF features an atypical 24 bp spacer but contains 5 bp inverted repeats flanking a thymine tract (Fig. 4B
), a sequence described for the promoter Pm of phage Mu. During complex formation of Pm DNA and RNA polymerase, a distortion occurs at the spacer allowing DNA strand separation at the thymine tract. This event favours the transition from a closed to an open complex and the initiation of transcription (Artsimovitch et al., 1996
).
The ychH transcript probably originated from either of the two promoters identified by sequence in the ychHpth intergenic region and oriented divergently from Ppth (Fig. 3A, B
). Deletion of a DNA segment containing the ychH promoters in pRJ1·0 results in a stronger expression of pth mRNA and Pth protein (Figs 5A
, B
). Therefore, these promoters, or other element(s) in the intergenic sequence, may be inhibitory for pth expression.
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ACKNOWLEDGEMENTS |
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Received 31 May 2002;
revised 18 July 2002;
accepted 24 July 2002.