Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India
Correspondence
Umesh Varshney
varshney{at}mcbl.iisc.ernet.in
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ABSTRACT |
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INTRODUCTION |
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In E. coli, four genes encode initiator tRNAs. Three of these, metZ, metW and metV, are located in a single locus at 63·5 min and the fourth one, metY, is located at 71·5 min (Berlyn, 1998). In E. coli K-12, the genes located at 63·5 min code for tRNA1fMet and the one at 71·5 min encodes a variant, tRNA2fMet. The two species differ by a single nucleotide at position 46 in the variable loop by which the tRNA1fMet possesses 7mG and the tRNA2fMet an A (RajBhandary & Chow, 1995
). tRNA1fMet represents the major form (
7580 %) of the tRNAfMet in the cell. On the other hand, tRNA2fMet is a minor component (Mandal & RajBhandary, 1992
), and a disruption of its gene (metY) with a kanamycin resistance gene produces a mutant strain that shows the same growth rate as the wild-type strain (Kenri et al., 1992
). However, replacement of the tRNA1fMet genes (metZWV) with a chloramphenicol resistance gene results in a mutant strain with a slow growth phenotype, the extent of which varies with the growth temperature (Kenri et al., 1991
).
We generated a T35A36 (termed U35A36) mutation in a plasmid-borne copy of metY to introduce a 34CUA36 anticodon in the encoded tRNA2fMet. The tRNA2fMet (U35A36) thus produced initiates from a UAG initiation codon (Varshney & RajBhandary, 1990). Both in vitro and in vivo studies have shown that the initiator tRNA mutants containing U35A36 mutations are aminoacylated with Gln (Schulman & Pelka, 1985
; Seong et al., 1989
). Further, N-terminal sequence analysis of the translated products using such initiator tRNA mutants has confirmed that Gln is inserted in response to a UAG initiation codon, with no evidence of initiation with Met (O'Connor et al., 2001
). However, when MetRS is overproduced in E. coli, a limited aminoacylation of tRNA2fMet (U35A36) by Met does take place, especially when its recognition by GlnRS is compromised by mutations in the acceptor stem (Varshney & RajBhandary, 1992
).
Recently, Rothschild and co-workers demonstrated that N-terminal protein labelling efficiency could be drastically improved using such initiator suppressors' in vitro (Mamaev et al., 2004). Using the in vivo amber initiation assays, we have shown that the creation of a strong base pair at the 1 : 72 position (as in the G72 mutation, Fig. 1B
) results in a loss of initiation activity, primarily because of a severe defect in formylation of the tRNA2fMet (U35A36/G72) and the non-availability of its formylated form for initiation (Lee & RajBhandary, 1991
; Varshney et al., 1991a
,b
). However, overproduction of methionyl-tRNA synthetase, methionyl-tRNA (fMet) formyltransferase or IF2 (Varshney & RajBhandary, 1992
; Mangroo & RajBhandary, 1995
), or generation of an intragenic mutation (C to T), resulting in a U1 : G72 wobble base pair at the top of the acceptor stem (Thanedar et al., 2000
), rescued the initiation defect of tRNA2fMet (U35A36/G72).
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METHODS |
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Preparation of cell-free extracts.
The pCATam1 derivatives with or without pACQS were introduced into E. coli by transformation. The transformants were grown to exponential phase and the cells from 2 ml cultures were harvested. The cell pellet was thoroughly resuspended in 200 µl TME (25 mM Tris/HCl, pH 8·0, 2 mM -mercaptoethanol, 1 mM Na2EDTA), lysed by sonication and subjected to centrifugation at 10 000 r.p.m. at 4 °C for 30 min in a microfuge. The supernatant was transferred to a new tube, quantified for total proteins using Bradford's method, mixed with an equal volume of 2x storage buffer (20 mM Tris/HCl, pH 8·0, 10 mM
-mercaptoethanol, 200 mM NaCl, 80 % glycerol, v/v) and stored at 20 °C.
Immunoblot analysis.
Cell-free extracts (15 µg total protein) were resolved by 12 % SDS-PAGE and electroblotted onto a PVDF membrane (Amersham). The membrane was blocked with 1 % BSA in Tris/HCl buffered saline, TBS (20 mM Tris/HCl, pH 7·5, 0·9 % NaCl), overnight and then incubated for 4 h at room temperature with anti-chloramphenicol acetyl transferase and anti--lactamase rabbit antibodies (1 : 3000 dilutions). After three washings with TBS/Tween 20 (0·2 %, v/v), the blot was incubated with alkaline phosphatase-conjugated goat anti-rabbit IgG (1 : 3000 dilution) for 4 h at room temperature. After three washings with TBS/Tween 20 (0·2 %, v/v), the blot was developed with p-nitro blue tetrazolium chloride and 5-bromo-4-chloro-3-indolyl phosphate in 0·1 M Tris/HCl (pH 9·0) and 4 mM MgCl2 to visualize the bands (Sambrook et al., 1989
).
Assay for chloramphenicol acetyl transferase (CAT) activity.
The cell-free extracts (0·21 µg total protein) were assayed in 50 µl reaction volumes containing 150 µM Cm, 10 µM [14C]Cm (specific activity 12·5 mCi mmol1, 463 MBq mmol1), 500 mM Tris/HCl (pH 8·0) and 400 µM acetyl-CoA. The reactions were carried out at 37 °C for 20 min, extracted with 500 µl ethyl acetate and processed by TLC on silica gel plates (Merck) using CHCl3 and methanol (95 : 5, v/v) as the mobile phase (Shaw, 1983). The plates were dried, exposed to a phosphor-imaging screen and quantified using a BioImage analyser (BAS1800, Fuji Films). Enzyme activity was expressed as nanomoles of acetyl chloramphenicol (1 acetyl+3 acetyl) formed per minute per milligram of total protein.
P1 transductional cross.
P1 phage lysate preparations and transductions were performed as described by Miller (1972).
Northern blot analysis.
Total tRNA from various strains was isolated under acidic conditions, separated on 6·5 % polyacrylamide acid urea (8 M) gels at 4 °C, and electroblotted onto a Nytran membrane (Varshney et al., 1991a). A 5'-32P end-labelled oligodeoxyribonucleotide complementary to positions 2947 of tRNA2fMet (U35A36) was used as probe. The probe possessed two mismatches from the tRNA2fMet, and three from the tRNA1fMet sequence complements. To generate the tRNA2fMet marker, the tRNA preparation was treated with 100 mM Tris/HCl (pH 9·0) (Sarin & Zamecnik, 1964
).
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RESULTS |
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Isolation and characterization of CmR strains from E. coli CA274
In our earlier studies, we used a tRNA2fMet (U35A36/G72) mutant to characterize suppressors that resulted in initiation from CATam1 mRNA (Varshney & RajBhandary, 1992; Thanedar et al., 2000
). In the present study, we used overnight cultures of E. coli CA274 harbouring pCATam1metYCUA/G72G73 and screened for a CmR phenotype. Of the several colonies that grew on the antibiotic plate, two, named Su15 and Su31, were selected for further studies. Multiple rounds of growth in antibiotic-free medium resulted in curing of the resident plasmid from these strains, and the cured strains showed a CmS phenotype (e.g. Fig. 4A
, sectors 4 and 8).
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Mapping of mutation(s) in Su15 and Su31
The observation that the suppressors (Su15 and Su31) were able to grow on Cm in the presence of a CATam1 reporter, even in the absence of any plasmid-encoded tRNAfMet, led us to believe that one of the initiator tRNA genes (genomic copy) in these strains might have been mutated to effect initiation from the CATam1 reporter. In order to genetically map the mutation by a candidate gene approach, we carried out P1 transductional crosses using various CAG strains marked with Tn10 (TcR) at 69·3 min, 71·8 min or 72·5 min in the vicinity of metY (71·5 min) encoding tRNA2fMet (Fig. 3A). As shown in Table 2
, replacement of the 71 min region of the suppressors (harbouring the plasmid pCATam1) with that from a CAG12072(71·8 min) strain resulted in the loss of CmR by a co-transduction frequency [(TcR+CmS)/TcR] of about 70 %. The crosses with CAG12152and CAG12153strains (69·3 and 72·5 min, respectively) did not result in any transductants that were CmS. These observations suggested that the mutation in Su15 and Su31, which conferred CmR, must be in the vicinity of 71·8 min. As metY is located at 71·5 min, to further narrow down the mutation, we amplified by PCR and sequenced the metY genes from both the suppressors. Each of them revealed two mutations corresponding to positions 35 and 36 of the anticodon resulting in a 34CAU36 to 34CUA36 change (Fig. 3B
). Analysis of several other genes from this locus, including infB (which encodes IF2) at 71·4 min, revealed no mutations (data not shown). Further, back crosses onto the parent strain (CA274, harbouring pCATam1) using P1 phage raised from the Su15 and Su31 (TcR+CmR, marked at 71·8 min in the above experiment) resulted in an identical level of CmR in the presence of pCATam1; it was concluded that mutations in the metY gene alone resulted in the suppressor phenotype of Su15 and Su31 (data not shown).
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Efficiency of initiation
To determine the utility of the Su15 and Su31 strains for various applications, we assessed the efficiency of these strains in initiation from a CATam1 reporter by phenotypic and biochemical assays for CAT activity. As shown in Fig. 4A, the Su15 and Su31 strains themselves were sensitive to growth on Cm plates [sectors 8 and 4, respectively; compare panel I(i) with panels (ii)(v)]. However, in the presence of the pCATam1 plasmid, the single copy of the chromosomally located metYCUA in Su15 and Su31 initiated from the CATam1 reporter and sustained growth not only at a concentration of 50 µg Cm ml1 [panel (ii)] in the medium (which was used to isolate them) but also at 100 and 150 µg Cm ml1 [sectors 2 and 3, panels (iii) and (iv), respectively]. In fact, at longer incubation times of about 24 h, growth was detectable even at 200 µg Cm ml1 [panel (v)]. The observation that the single-copy metYCUA gene supported growth of the host up to 200 µg Cm ml1 clearly supports the view that it affords efficient initiation from an amber initiation codon. However, to analyse the effect of GlnRS overproduction on initiation activities of Su15/Su31, we carried out CAT activity assays. These assays also allowed a quantitative comparison between the initiation efficiencies of Su15 and Su31 with that of the multicopy plasmid-based system. As shown in Fig. 4B
, in the absence of overproduced GlnRS, Su15 and Su31 produced a CAT activity which converted
5060 nmol Cm min1 (mg total cell protein)1 to acetyl-Cm (lanes 7 and 8). This activity was increased to
90100 nmol Cm min1 (mg total cell protein)1 in the presence of overproduced GlnRS (lanes 3 and 4). Under the same assay conditions, the CAT activity from the multicopy plasmid-based amber initiation system was
1100 nmol Cm min1 (mg total cell protein)1 (lanes 2 and 6). Interestingly, the difference between the initiation efficiencies of Su15/Su31 and the multicopy plasmid system correlated well with the copy number of the ColE1 origin of replication (pBR322-derived pCATam1-based plasmids) (Sambrook et al., 1989
; Atlung et al., 1999
). More importantly, these observations now allow us to choose the desired level of initiation from a UAG initiation codon for regulated expression of genes, especially those that encode proteins toxic to the host.
To determine if the initiation activities correlated well with the availability of fGln-tRNA2fMet (U35A36) in the cell, we performed Northern blot analysis of the total tRNA prepared under acidic conditions. As shown in Fig. 4C, in the Su15 and Su31 strains, approximately 52 % of the tRNA2fMet (U35A36) accumulated as fGln-tRNA2fMet (U35A36) (lanes 3 and 5). However, overproduction of GlnRS resulted in a slight increase in the steady-state accumulation of fGln-tRNA2fMet (U35A36) to
61 % (lanes 4 and 6). Such an increase in the steady-state accumulation of the fGln-tRNA2fMet (U35A36) agrees well with the increase in their initiation efficiency (Varshney et al., 1991a
). The wild-type form of tRNA2fMet accumulated as fMet-tRNA2fMet quantitatively (lanes 1 and 2). It may be noted that the signals arising from the CA274 samples (lanes 1, 2 and 7) are less intense. This is merely because the hybridization probe [complementary to the tRNA2fMet (U35A36)] contains two mismatches from the wild-type sequence.
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DISCUSSION |
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Although the efficiency of termination at nonsense codons within the coding region can be minimized to some extent by placing them in an appropriate sequence context (Tate & Mannering, 1996), a drawback of the elongator tRNA-based suppressors is that suppression is not complete and results in the release of incomplete polypeptides due to competition from release factors. In another scenario, the suppressor tRNAs compete with the release factors for binding to the natural termination codons in the A site of the ribosome, resulting in translation of the cellular mRNAs beyond the termination codon. Both of these situations can be toxic to cells (Snyder & Champness, 1997
). Furthermore, although not studied systematically, some of the elongator tRNA-based suppressors may also interfere with the autoregulatory mechanisms involving termination codons within ORFs as well as the mechanism that leads to the insertion of selenocysteine in proteins (Craigen & Caskey, 1986
; Mansell et al., 2001
). Notably, these drawbacks are essentially a consequence of the requirement of the elongator tRNA-based suppressors for recognition of the nonsense codons in the ribosomal A site.
The initiation of protein synthesis from an mRNA occurs from an initiation codon located in a specialized region, the translation initiation region (TIR). While the presence of the ShineDalgarno sequence (SD sequence) within the TIR is one of the most important elements that determines the efficiency of initiation, several other features, such as the sequence context within which an initiation codon is located and its spacing from the SD sequence, contribute to efficient utilization of the prokaryotic mRNA in translation (reviewed by Gold, 1988). The observation that the base pairing between the initiating codon and the anticodon of the initiator tRNA, and not the AUG sequence per se, is responsible for initiation, allowed us to design a plasmid-based system for initiation from a UAG termination codon (Varshney & RajBhandary, 1990
), and by virtue of their (initiator tRNA) binding at the ribosomal P site, they remedy the limitations of the elongator tRNA-based suppression system. In fact, in the plasmid-based system that we described, the initiator tRNA (with CUA anticodon) was overproduced from a multicopy plasmid without any detectable toxic consequences to bacterial growth. In the initiator tRNA-based systems, the issue of production of the incomplete peptide does not arise. Further, as the natural termination codons are generally not located in a sequence context (TIR) required to foster initiation, the chances of inappropriate initiation are also minimized.
Although the plasmid-based system using such initiator suppressor tRNA was described some time ago (Varshney & RajBhandary, 1990), the fact that plasmid vectors are needed to introduce various test genes into the bacteria means that it was desirable to isolate/generate E. coli strains in which the initiator tRNA was altered in the chromosomal background. The characterization of the Su15 and Su31 strains in this report bridges this gap.
A question that the present study raises is whether or not such strains could also be generated by introducing mutations in any of the initiator tRNA genes located at 63·5 min. It is possible that the mechanism that led to the generation of Su15/Su31 or a directed approach (Datsenko & Wanner, 2000) could result in the isolation of a strain in which initiation from an amber codon would occur because of mutation in the metZ, metW or metV genes at 63·5 min. However, it may be noted that the tRNA sequences in this cluster have not diverged over the evolutionary time-scale. On the other hand, the tRNA sequence encoded by the metY locus (at 71·5 min), which is distantly located, has diverged, at least at position 46, from those located at 63·5 min. Recently, it was suggested that the multiple copies of rRNA gene sequences are prevented from diverging from each other by a gene-conversion process. In addition, genes located near to each other are corrected more efficiently than those located distantly (Hashimoto et al., 2003
). Therefore, it is possible that the initiator tRNA genes located at 63·5 min are under selective pressure to maintain the intactness of their sequences. Also, the distantly located metY may be the only locus that can be allowed to accumulate mutations. Thus, at a first approximation, it would seem that the metY-based system for initiation from a termination codon would be a stable one. Hence, the characterization in this study of the E. coli strains that we isolated by serendipity is of notable significance. Interestingly, the Su15 and Su31 strains were isolated more than six years ago in the laboratory, and continue to be stable even in the absence of any selective pressure and, consistent with the studies of Kenri et al. (1992)
, have no apparent growth defect. However, we have not yet studied the natural fitness of these strains vis-à-vis their parent strain (E. coli CA274). Such studies in future may lead to an understanding of the possible selective advantage of the presence of multiple copies of the initiator tRNA genes in E. coli.
The strains Su15 and Su31 that utilize tRNA2fMet (U35A36) initiate with formyl-glutamine, which is also the most efficient non-methionine amino acid to initiate protein synthesis (Mayer et al., 2001). Therefore, this strain should be the most useful for general purposes to obtain initiation from an amber initiation codon. Recently, it was shown that initiation from a UAG codon is highly comparable to initiation from AUG (Mayer et al., 2003
). Interestingly, as we have shown (Fig. 4
), the efficiency of initiation from an amber initiation codon in these strains can be enhanced by simultaneous expression from a plasmid-borne copy of the GlnRS gene. Also, given that plasmid-based systems are available for initiation with formylated forms of valine, isoleucine and phenylalanine, etc. (Chattapadhyay et al., 1990
; Pallanck & Schulman, 1991
), it should be feasible to generate more strains for initiation with other non-methionine amino acids by using the general approach described in this study. We believe that the generation of such a library of strains for the expression of transcriptionally controlled genes with non-AUG initiation codons would be a valuable addition to the field of nonsense suppression genetics and open up further opportunities for its application.
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ACKNOWLEDGEMENTS |
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Received 26 January 2005;
revised 23 February 2005;
accepted 23 February 2005.
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