Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, 560012, India1
Author for correspondence: Umesh Varshney. Tel: +91 80 394 2686. Fax: +91 80 360 2697 or 0683. e-mail: varshney{at}mcbl.iisc.ernet.in
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ABSTRACT |
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Keywords: tubercle bacilli, Thermus thermophilus, elongation factor G, polysome binding, termination complex
Abbreviations: CD, circular dichroism; EcoEFG, E. coli EFG; EcoRRF, full-length E. coli RRF; EFG, elongation factor G; frr, locus coding for RRF; frrts, locus coding for temperature-sensitive RRF; ESI-MS, electron spray ionization-mass spectroscopy; MtuEFG, M. tuberculosis EFG; MtuRRF, full-length M. tuberculosis RRF; RFF, ribosome recycling factor; TthRRF, full-length T. thermophilus RRF
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INTRODUCTION |
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Ribosome recycling factor (RRF) is an essential factor for protein synthesis in bacteria and in eukaryotic organelles (Janosi et al., 1996 ; Kaji et al., 1998
). RRF is required for the disassembly of the post-termination complex. Interestingly, in eukaryotes RRF is only needed in the organelles, making it a novel drug target (Janosi et al., 1996
; Kaji et al., 1998
). Also, in Staphylococcus aureus the levels of RRF increase upon infection of animal cells (Lowe et al., 1998
). Furthermore, in brucellosis (caused by Brucella melitensis) the sera from infected sheep show antibodies against RRF, suggesting this protein to be a virulence factor (Vizcaino et al., 1996
). Thus, RRF may even be of interest in developing subcellular vaccines. The RRFs from different bacteria are highly conserved in their primary structure (Fig. 1
), and the three-dimensional structure of the RRFs from four organisms (Thermotoga maritima, Thermus thermophilus, Escherichia coli and Aquifex aeolicus) are known (Selmer et al., 1999
; Toyada et al., 2000; Kim et al., 2000
; Yoshida et al., 2001
). These structures show that the overall architecture of the different RRFs, consisting of two domains, is also highly conserved and mimics tRNAs in its size and shape. Domain I is represented by three long
-helices and domain II is composed of a ß
ß sandwich. The two domains are connected to each other by two loops. Domains I and II represent the long and short arms, respectively, of the L-shaped tRNA.
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Recently, we have shown that, like TthRRF, M. tuberculosis RRF (MtuRRF) also failed to complement an frrts strain of E. coli LJ14. However, simultaneous expression of the elongation factor G (EFG) and the RRF from M. tuberculosis resulted in the rescue of the temperature-sensitive phenotype of the LJ14 strain, highlighting the importance of specific interactions between the two proteins (Rao & Varshney, 2001 ). To understand the mechanism of action of MtuRRF further, in this study we have generated a mutant of MtuRRF that lacks the last six amino acids of the C-terminal end (equivalent to
C5 of T. thermophilus), investigated its biochemical and biophysical properties and carried out functional analyses in E. coli.
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METHODS |
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Construction of C6MtuRRF.
The mutant lacking six amino acids from the C-terminal end of MtuRRF (C6MtuRRF) was generated by PCR from pTrcMtuRRF. PCR was carried out with Pfu DNA polymerase as described previously for MtuRRF (Rao & Varshney, 2001
) using a forward primer (5'-GCGCCCATGGTTGATGAGGCTCTCTTC-3') containing an NcoI site and a reverse primer (5'-AGCAAAGCTTATTCTTTGTGTTTAACC-3') which incorporated a stop codon at position 180 (G to amber) and a HindIII site. The PCR product was digested with NcoI and HindIII and cloned into the respective sites of the ColE1 origin-of-replication-based vectors pTrc99C and pET11d to generate pTrc
C6MtuRRF and pET
C6MtuRRF, respectively. The same fragment was also subcloned into pACDH containing a pACYC origin of replication to yield pACDH
C6MtuRRF. Incorporation of the fragment was confirmed by complete DNA sequencing.
Purification of MtuRRF and C6MtuRRF, N-terminal sequencing and electron spray ionization-mass spectroscopy (ESI-MS).
MtuRRF and C6MtuRRF were purified from E. coli BL21(DE3) harbouring either pETMtuRRF or pET
C6MtuRRF, respectively. The transformants were inoculated into 2xYT (Sambrook et al., 1989
) (2 l) and induced with 0·5 mM IPTG at the mid-exponential phase of growth (OD600 value of between 0·3 and 0·4). Cells were harvested, sonicated and used to make the S100 lysate. The S100 lysate was subjected to streptomycin sulfate (0·9%) precipitation, and the supernatant was subjected to ammonium sulfate precipitation (90% saturation). The precipitate was recovered by centrifugation, dissolved in 1 ml of 20 mM Tris/HCl (pH 7·4) and dialysed against the same buffer for 12 h. Dialysed sample was loaded onto a Superdex 75 column (Amersham Pharmacia Biotech). Proteins were eluted with 20 mM Tris/HCl (pH 7·4), 500 mM NaCl and 10% (v/v) glycerol, and the fractions enriched for RRF (as analysed by SDS-PAGE) were pooled, dialysed against 20 mM Tris/HCl (pH 7·4) and subjected to Mono Q column chromatography. The proteins were eluted with a linear gradient of 01 M NaCl in 20 mM Tris/HCl (pH 7·4) and 10% glycerol buffer. The fractions enriched for RRF were dialysed against 20 mM HEPES (pH 5·0) and loaded onto a hydroxyapatite column (Bio-Rad); the RRF was eluted using 20 mM HEPES (pH 5·0), 200 mM NaCl and 10% glycerol. The protein microsequence and ESI-MS analyses were carried out by the respective facilities at the Indian Institute of Science, Bangalore, India.
Purification of E. coli and M. tuberculosis EFG.
EFGs were purified from E. coli BL21(DE3) using the T7 RNA polymerase expression constructs pETMtuEFG and pETEcoEFG (Rao & Varshney, 2001 ).
Gel electrophoresis.
Proteins were electrophoresed on 15 and 12% polyacrylamide gels containing 0·1% SDS (SDS-PAGE) and visualized by Coomassie brillant blue R-250 staining (Laemmli, 1970 ). The non-denaturing PAGE (native PAGE) was performed in the same way but lacked SDS. Loading dye for native gels consisted of 50 mM Tris/HCl (pH 6·8), 10% (v/v) glycerol and 0·01% bromophenol blue.
Circular dichroism (CD) spectroscopy.
CD measurements were done on an automated JASCO-J715 spectropolarimeter using 0·2 cm and 1 cm path length quartz cuvettes for secondary and tertiary CD spectra, respectively. Samples were prepared in 20 mM potassium phosphate buffer (pH 7·0). Each spectrum was a mean of four scans with a slit width of 1 nm, response time of 4 s and a scan speed of 50 nm s-1.
Polysome preparation, in vitro ribosome recycling and polysome binding assays.
Polysomes were prepared from E. coli MRE600 and used in ribosome recycling assays (Tai & Davis, 1979 ; Girbes et al., 1979
; Rao & Varshney, 2001
). To perform binding assays, factor-free polysomes (OD260
2) were incubated in reaction volumes of 250 µl without or with RRFs in the RRF assay buffer (10 mM Tris/HCl, pH 7·4, 80 mM NH4Cl, 8·2 mM MgSO4, 1 mM DTT, 10 µM puromycin, 160 µM GTP) at 33 °C for 20 min and the reaction mixture was layered on a mini-column (1 ml) packed with Sepharose 4B matrix and centrifuged at 2500 r.p.m. for 3 min in a table-top centrifuge. The eluate was concentrated by vacuum drying, separated by SDS-PAGE (15%) and analysed by immunoblotting (Towbin et al., 1979
).
Immunoblotting.
The cell-free extracts (10 µg total proteins) or the ribosome samples (in the RRF binding studies) were separated by SDS-PAGE (12%) and electroblotted onto a PVDF membrane (Towbin et al., 1979 ) at 200 mA for 2 h. The blots were probed with anti-MtuRRF rabbit antibodies and detected using alkaline phosphatase-conjugated goat-anti-rabbit IgG with the substrates p-nitrotetrazolium blue chloride and 5-bromo-4-chloro-3-indolyl phosphate.
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RESULTS |
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CD spectroscopy
The X-ray structures of RRFs have shown that the two domains that mimic the arms of the L-shaped tRNA consist of well-defined secondary structural elements (Yoshida et al., 2001 ; Selmer et al., 1999
; Kim et al., 2000
; Toyada et al., 2000). Domain I consists of three long
-helices. This property of RRF makes it a suitable molecule for CD spectroscopic analysis to probe for any major structural changes that could result from the introduction of mutations. As seen in Fig. 3(a)
, the secondary CD profiles of the wild-type (MtuRRF) and the mutant (
C6MtuRRF) proteins are almost identical, suggesting that the deletion of six residues from the C-terminal end of MtuRRF does not result in its inappropriate folding. Furthermore, although the tertiary CD signals are weak (MtuRRF lacks tryptophans), the similar spectra for the two proteins (Fig. 3b
) suggest that even the overall architecture of the two proteins is similar.
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Complementation analysis of E. coli LJ14 with C6MtuRRF
As the deletion of the C-terminal five amino acids from TthRRF bestowed upon it the ability to complement an frrts strain of E. coli, it was of interest to us to investigate the phenotype of an equivalent mutant of MtuRRF. However, our analysis showed that C6MtuRRF failed to rescue the temperature-sensitive phenotype of E. coli LJ14 (frrts) even upon its induction with IPTG (Fig. 5a
, sector 2, compare growth at the permissive temperature to growth at the non-permissive temperature). Simultaneous overproduction of E. coli EFG (EcoEFG) also did not confer the ability to
C6MtuRRF to rescue the temperature-sensitive phenotype of E. coli LJ14 at the non-permissive temperature (Fig. 5a
, sector 3). In fact,
C6MtuRRF failed to complement E. coli LJ14 even when co-expressed with M. tuberculosis EFG (MtuEFG) (Fig. 5a
, sector 4). However, as reported earlier (Rao & Varshney, 2001
), the wild-type MtuRRF complemented E. coli LJ14 in the presence of MtuEFG (Fig. 5a
, sector 5) irrespective of induction with IPTG. To further verify these results, we monitored the growth of the transformants in liquid cultures (Fig. 5b
). Consistent with the plating experiment, the transformants harbouring the vectors or the
C6MtuRRF constructs alone or along with the plasmids harbouring EcoEFG or MtuEFG grew at the permissive temperature but not the non-permissive temperature. The immunoblot analysis of the cellular extracts of the transformants (grown at the permissive temperature) showed that
C6MtuRRF was produced in E. coli LJ14 (Fig. 5c
, lanes 16) and its expression increased upon induction with IPTG (Fig. 5c
, compare lanes 1, 3 and 5 with 2, 4 and 6). The levels of
C6MtuRRF produced upon induction with IPTG (Fig. 5c
, lanes 2, 4 and 6) were comparable to those of wild-type MtuRRF in uninduced cultures (Fig. 5c
, lane 7), suggesting that the failure of complementation of E. coli LJ14 by the mutant protein was not due to its inadequate production.
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DISCUSSION |
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It has been shown that RRF from P. aeruginosa complements E. coli LJ14 (frrts) (Ohnishi et al., 1999 ); however, TthRRF does not complement this mutant strain. Interestingly, a mutant lacking five amino acids from the C-terminal end of TthRRF did complement the frrts phenotype of E. coli (Fujiwara et al., 1999
; Toyoda et al., 2000). Furthermore, recent studies using EcoRRF have shown that deletion of up to seven amino acids from the C-terminal end of this protein still allows it to retain its activity (Fujiwara et al., 2001
). Therefore, these C-terminal residues are not absolutely critical for RRF function. Alignment of the available RRF sequences (a total of 49) showed that amino acid residues 178185 (E. coli numbering) belong to a highly conserved stretch of amino acids in RRFs. Does this high degree of conservation allude to the importance of these residues in RRF function?
In this study, based on the sequence comparison of MtuRRF and TthRRF (Fig. 1), we generated a mutant of MtuRRF that lacked the last six amino acids from the C-terminal end (
C6MtuRRF). However, unlike the C-terminally deleted TthRRF or EcoRRF mutants,
C6MtuRRF failed to function with EcoEFG in ribosome recycling in both in vivo and in vitro analyses. Surprisingly,
C6MtuRRF even failed to function with MtuEFG (Figs 5
and 6
). The biochemical and biophysical characterizations performed here suggest that the mutant protein is folded properly and retains a shape similar to that of the wild-type protein. Interestingly, the ribosome binding assays (Fig. 4
) show that while MtuRRF binds to E. coli ribosomes, the mutant protein is compromised for its binding to ribosomes. And, while such binding studies have not been carried out with the equivalent mutants of TthRRF and EcoRRF, we suggest that the loss of ribosome binding activity of the
C9 mutants of EcoRRF (Fujiwara et al., 2001
; Toyoda et al., 2000) is predominantly a consequence of the loss of these highly conserved residues at the C-terminal end of the RRF. Thus, the conserved residues at the C-terminal end of the RRFs may facilitate in their direct binding to ribosomes and/or in their prolonged residency on the ribosomes, possibly by modulating the on and/or off rates.
T. thermophilus and E. coli belong to the Gram-negative group of bacteria, whereas M. tuberculosis belongs to the Gram-positive group, indicating that TthRRF is relatively closer to EcoRRF and, therefore, may already possess many of the elements that are needed to establish specific contacts with EFG and the ribosome. Alignment of the RRF sequences (Fig. 1) shows that out of 185 residues a total of 74 are conserved between MtuRRF and EcoRRF (
40% sequence identity) with a similarity score of
59% (109/185). However, a total of 81 residues are conserved between TthRRF and EcoRRF (
44% sequence identity) with a similarity score of
63% (116/185). Thus, there is slightly higher sequence similarity between EcoRRF and TthRRF than between EcoRRF and MtuRRF. It has been suggested that a deletion of five amino acids from the C-terminal end of TthRRF results in improved flexibility of the hinge region connecting domains I and II of RRF, which in turn may facilitate its function in E. coli (Toyoda et al., 2000
). Possibly, what the removal of the residues from the C-terminal end achieves is that it brings these interacting partners into a better configuration for a productive association, such that even the shorter residency times of TthRRF are now adequate. However, in MtuRRF, which in comparison to TthRRF is slightly less intimately related to EcoRRF, an equivalent deletion may not result in similar favourable changes. It should also be noted that the earlier study (Fujiwara et al., 1999
) utilized a temperature-sensitive strain of E. coli containing a different allele of frr than the one used in this study. Thus, at this stage alternative interpretations that relate to the strain effects can not be ruled out. Nevertheless, our studies do highlight the significance of RRF binding to ribosomes and the establishment of specific interactions between RRF and EFG for the disassembly of post-termination complexes.
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ACKNOWLEDGEMENTS |
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Received 2 April 2002;
revised 1 July 2002;
accepted 29 August 2002.
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