Lilly Research Laboratories, Infectious Diseases Research, Eli Lilly and Company, Indianapolis, IN 46285-0438, USA1
Author for correspondence: Genshi Zhao. Tel: +1 317 276 2040. Fax: +1 317 276 1743. e-mail: Zhao_Genshi{at}Lilly.com
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ABSTRACT |
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Keywords: Era, GTPase, RNA-binding activity, GTPase activation, carboxylic acids
Abbreviations: GST, glutathione S-transferase
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INTRODUCTION |
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Several lines of evidence suggest that Era is involved in cell cycle regulation and ribosome assembly and that the GTP-binding and hydrolysis activities of Era are essential for its biological function (Britton et al., 1997 , 1998
; Gollop & March, 1991b
; Lerner et al., 1992
, 1995
; Nashimoto, 1993
; Nashimoto et al., 1985
; Nashimoto & Uchida, 1985
). Mutations affecting the cellular level or function of Era led to a severe alteration in ribosome assembly, a drastic change in cell morphology, a significant reduction in cell viability and an apparent suppression of temperature-sensitive mutations in a number of the genes that are involved in DNA replication and chromosome partitioning (Britton et al., 1997
, 1998
; Gollop & March, 1991b
; Nashimoto, 1993
; Nashimoto et al., 1985
; Nashimoto & Uchida, 1985
). Data also suggest that Era might be involved in the regulation of energy metabolism, since the decrease of the cellular amounts of Era altered the ability of E. coli to utilize carbohydrates in a minimal medium (Lerner & Inouye, 1991
; Shimamoto & Inouye, 1996
). Together, these results suggest that Era is a multifunctional protein that is involved in the regulation of the cell cycle, protein synthesis and possibly energy metabolism. The mechanisms by which Era regulates these cellular processes, however, remain to be determined.
Era consists of two distinct domains (Chen et al., 1999 ; Zuber et al., 1997
). The N-terminal domain of the protein contains the three GTP/GDP-binding motifs that are shared by many different families of GTPases (March, 1992
; Pillutla et al., 1995
; Zuber et al., 1997
). The C-terminal domain of Era, in contrast, is highly conserved only among Era proteins and thereby is unique to the Era family (March, 1992
; Pillutla et al., 1995
; Zuber et al., 1997
). Thus, the C-terminal domain of Era, although its function is unknown, most likely plays a direct role in the regulation of the cell cycle and protein synthesis. Consistent with this view, the C-terminal domain of the Streptococcus pneumoniae Era protein appears to be essential for its biological function in the cell (Zhao et al., 1999
). Recently, we have shown that the S. pneumoniae Era protein is primarily associated with the E. coli 16S rRNA when expressed in E. coli (Meier et al., 1999
). The binding of 16S rRNA to Era appears to stimulate its GTPase activity (Meier et al., 1999
). The part of Era which is responsible for this RNA-binding activity and the mechanisms by which the RNA stimulates the Era GTPase activity, however, were unknown.
In this study, we purified and characterized the E. coli Era protein. The results show that, like the S. pneumoniae Era protein, the E. coli Era protein is also associated primarily with 16S rRNA. The EraRNA complex exhibited a GTPase activity higher than that of the Era protein free of RNA. Era was also shown to bind RNA in vitro. In addition, this study shows that a C-terminally truncated Era protein could not bind 16S rRNA when expressed in vivo. The GTPase activity of the Era protein free of RNA, but not the EraRNA complex and the C-terminally truncated Era protein, was stimulated by acetate and 3-phosphoglycerate. Together, our studies demonstrate that the C terminus of the Era protein is essential for the RNA-binding activity and that the GTPase activity of Era appears to be modulated by the RNA and carbohydrates, possibly by a similar mechanism.
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METHODS |
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Bacterial strains and culture conditions.
The following E. coli strains were used in this study: K-12, a wild-type strain (Zhao & Winkler, 1995 ), BL21(DE3) pLysS (Cmr) (Stratagene), XL1-Blue (Stratagene), LY66 [XL1-Blue/pRBP-13 (GSTera+, Apr)] (this study), LY68 [BL21(DE3) pLysS/pRBP-11 (era+, Apr)] (this study), LY70 [BL21(DE3) pLysS/pRBP-12 (Hisera+, Apr)] (this study), LY52 [XL1-Blue/pLY52 (GST
era+, Apr)] (Zhao et al., 1999
), LY41 [XL1-Blue/pLY41 (GSTera+, Apr) (Zhao et al., 1999
) and LY160 [XL1-Blue/pGEX-2T (GST+, Apr)] (Pharmacia).
For expression of era, all E. coli strains were grown, induced and collected as described previously (Meier et al., 1999 ). LY12 (a wild-type E. coli K-12 strain; Zhao & Winkler, 1995
) was grown overnight in LB at 35 °C with vigorous shaking. The overnight culture (9 ml) was inoculated into 250 ml fresh LB medium. The culture was harvested at an OD600 of 0·81·00 by centrifugation as described above.
Cloning of the E. coli era gene.
The E. coli era gene was cloned directly by PCR amplification of chromosomal DNA (Zhao et al., 1999 ) by using two primers based on the published sequence (Ahnn et al., 1986
; Britton et al., 1997
, 1998
). The 5' PCR primer (5'-CCGGAATTCAGATCTCATATGAGCATCGATAAAAGTTAC-3') was designed to begin at the ATG start codon of era and contained added EcoRI, BglII and NdeI sites for cloning purposes. The 3' PCR primer (5'-CCGGAATTCAGATCTTTAAAGATCGTCAACGTAACCGAG-3') was designed to end at the stop codon of era and contained added EcoRI and BglII sites after the stop codon. These primers were then used to amplify the era gene from E. coli as described previously (Zhao et al., 1999
) under the following conditions: denaturation at 94 °C for 30 s, annealing at 60 °C for 30 s and polymerization at 72 °C for 30 s for 25 cycles. Five PCR reaction products were combined and a portion of the pooled PCR products was digested with EcoRI. The EcoRI-digested PCR fragment was cloned into pBCKS+ (Stratagene) previously digested with EcoRI and dephosphorylated with calf intestinal alkaline phosphatase (Gibco-BRL). era from several pBCKS+ clones was sequenced and a clone containing the consensus era gene sequence (pRBP-10) was used to construct the following expression plasmids. pRBP-10 was partially digested with NdeI and then BglII. The NdeIBglII DNA fragment containing era was subcloned into pET-11a and pET-15b at the NdeI and BamHI sites (Novagen). The resulting constructs were designated pRBP-11 and pRBP-12, respectively. pRBP-10 was also digested with BglII and the BglII fragment of era was subcloned into pGEX-2T at the BamHI site. The resulting construct was designated pRBP-13.
Purification of native Era, HisEra, GSTEra, GSTEra and GST proteins.
The glutathione S-transferase (GST)Era fusion protein (GSTEra) of E. coli was purified from cells of LY66 by glutathioneSepharose affinity column chromatography (Pharmacia) as described previously (Zhao et al., 1999 ). The purified GSTEra was dialysed in dialysis tubing with a molecular mass cut-off of 50 kDa (Sigma) against 4 l 20 mM Tris/HCl, pH 8·0, 5 mM MgCl2 (buffer A) at 4 °C overnight. After dialysis, glycerol was added to the Era preparation at a final concentration of 16% (v/v) and the resulting preparation was stored in aliquots at -70 °C. Protein concentration was determined by using a Bradford Protein Assay Kit (Bio-Rad) with BSA as standard (Bradford, 1976
).
The native Era (non-tagged) protein of E. coli was purified from cells of E. coli LY68 by using a 3-step column chromatographic method (Zhao et al., 1999 ), dialysed by using dialysis tubing with a molecular mass cut-off of 25 kDa (Sigma) against 4 l buffer A overnight at 4 °C and stored in small aliquots at -70 °C as described above.
GST, and the GSTEra and GSTEra proteins of S. pneumoniae were purified from LY160, LY52 and LY41, respectively, by using a glutathioneSepharose affinity column as described for the GSTEra protein.
Histidine-tagged Era (HisEra) was purified from LY70 by using an affinity nickel column as described previously (Zhao et al., 1999 ).
Determination of RNA association with the Era protein of E. coli.
To examine the association of RNA with Era, an affinity-purified GSTEra preparation was subjected to anion-exchange column chromatography (Mono-Q column, Pharmacia) as described by Meier et al. (1999) . Fractions (1 ml each) were collected and analysed by using SDS-PAGE (10% gel), agarose gel electrophoresis and spectrophotometric scanning (200600 nm) on a Bio-Spec 1601 spectrophotometer (Shimadzu) as described by Meier et al. (1999)
.
Affinity-purified GST protein preparations were also subjected to analyses similar to those for the purified GSTEra protein as described above.
To examine if Era isolated directly from LY12 was also associated with RNA, a crude extract of LY12 grown to an OD600 of 0·5 was prepared in buffer A, treated or not treated with RNaseA (1 mg ml-1) at room temperature for 3 h and subjected to gel filtration column chromatography as described by Meier et al. (1999) . Fractions (2 ml each) were collected and 15 µl of each of the fractions was subjected to SDS-PAGE (Laemmli, 1970
). The resolved Era protein was transferred to a PVDF membrane and detected by Western blotting analysis as described previously (Zhao et al., 1999
) using polyclonal antibodies against SDS-denatured native Era protein of E. coli, which were prepared (Robert Sargent, Inc.) as described previously (Zhao et al., 1999
; Zhao & Winkler, 1995
).
Analysis of RNA associated with Era.
Purified GSTEra, native Era and GST proteins (4 mg each) were extracted with equal volumes of phenol/chloroform/isoamyl alcohol and the resulting material was collected by ethanol precipitation (Sambrook et al., 1989 ). The pellets (RNA) collected were air-dried, resuspended in 200 µl diethyl-pyrocarbonate-treated water and 10 µl of each was analysed by agarose gel electrophoresis (1·5% agarose containing 0·5 µg ethidium bromide ml-1; Sambrook et al., 1989
). E. coli rRNAs were isolated from LY12 by the same method as described by Sambrook et al. (1989)
.
To directly detect RNA associated with the protein, purified Era proteins (10 µg each) were run on an agarose gel as described above. To determine whether RNA was associated with Era proteins, purified Era proteins and phenol/chloroform-extracted material were treated with 1 mg RNaseA ml-1 (Boehringer Mannheim) or 0·2 mg DNaseI ml-1 (Gibco-BRL) for 15 min at room temperature and then run on an agarose gel as described above.
Determination of Era GTPase activity.
The GTPase activity of Era was assayed by HPLC as described previously (Zhao et al., 1999 ). Reaction mixtures (300 µl each) containing 50 mM Tris/HCl, pH 7·5, 5 mM MgCl2 and 510 µM Era were incubated at 23 °C. Reactions were initiated by the addition of GTP at concentrations ranging from 0 to 2 mM. Reaction mixtures (50 µl each) were injected into an HPLC column and separated under the conditions described by Zhao et al. (1999)
. The GDP produced was quantified by comparing its peak areas with those of GDP standards.
Identification and quantitation of the RNA associated with the purified GSTEra of E. coli.
To determine the identity of the RNA associated with the purified GSTEra protein, an affinity-purified GSTEra preparation was subjected to gel filtration column chromatography as described above. A void-volume fraction from the column, which contained the GSTEra protein associated with RNA, was used as a source of RNA. Total RNA was extracted from 1 ml of the void fraction by using an RNeasy Midi Prep Kit (Qiagen) and resuspended in 150 µl H2O.
Standards for 16S and 23S rRNA, for use in RT-PCR, were prepared as follows. A 500 bp DNA fragment internal to the E. coli 16S or 23S rRNA gene was cloned from E. coli genomic DNA by PCR as described by Sambrook et al. (1989) and Zhao et al. (1999)
by using the following primers: 5'-CGCGGATCCTGACGTTACCCGCAGAAGAAG-3' and 5'-CCATCGATAAGGTTCTTCGCGTTGCATCG-3' (for 16S rRNA), and 5'-CGCGGATCCTGACCGATAGTGAACCAGTAC-3' and 5'-CCATCGATTCTCCCGTGATAACATTCTCC-3' (for 23S rRNA) based on the published sequences (Brosius et al., 1978
, 1980
). Both 5' primers contained a BamHI site and the 3' primers contained a ClaI site for cloning purposes. The amplified DNA fragments were digested with BamHI and ClaI and cloned into pBlueScriptII KS+ (Stratagene). The resulting clones, containing a DNA fragment corresponding to a segment of either the 16S or 23S rRNA gene, were digested to completion with XhoI and 1 µg of each digested clone was used for in vitro transcription by using a MEGscript T7 kit according to the instructions of the manufacturer (Ambion). After transcription, the resulting RNA was purified by using an RNeasy Midi Kit (Qiagen). Under these conditions, 123 and 137 µg RNA was obtained from the clones containing fragments of 16S and 23S rRNA, respectively.
For RT-PCR, all RNA samples were treated with DNaseI (10 µg RNA, 20 units DNaseI, final volume 200 µl) at 25 °C for 15 min. The reaction mixtures were then mixed with 20 µl 25 mM EDTA and heated to 65 °C for 10 min. The final concentration of each RNA preparation was adjusted to 45·5 ng µl-1. RT-PCR was performed by using a GeneAmp EZ rTth RNA PCR Kit (Perkin Elmer) with the following primers: 5'-TTAACGCGTTAGCTCCGGAAG-3' and 5'-GCACGCAGGCGGTTTGTTAAG-3' (for 16S rRNA), and 5'-ACGAGGCGCTACCTAAATAGC-3' and 5'-CATGCTTAGGCGTGTGACTGC-3' (for 23S rRNA) based on the published sequences (Brosius et al., 1978 , 1980
). As a control, amplification reactions were done without any reverse transcription process. For quantitation purposes, amplification reactions were performed under the conditions in which the formation of amplified products increased exponentially using sample RNA and standard RNA preparations. We found that the exponential amplification of products was achieved within the first eight cycles of reaction. Therefore, all amplification reactions were performed for seven cycles. Reaction mixtures (50 µl each) contained 91 ng RNA, 1x EZ rTth RNA PCR buffer (Perkin Elmer), 0·3 mM each nucleotide (dATP, dCTP, dGTP and dTTP), 2·5 units rTth DNA polymerase, 2·5 mM manganese acetate, 0·5 µl [
-33P]dCTP [Easy Tides dCTP, 20004000 Ci mmol-1, 10 mCi ml-1 (1 Ci=37 GBq); NEN Life Sciences Products] and 0·15 pM each primer. Reverse transcription reactions were carried out at 60 °C for 30 min. PCR amplification reactions were carried out in triplicates for seven cycles under the following conditions: denaturation at 94 °C for 15 s and annealing and extension at 60 °C for 30 s. After seven cycles, 10 µl of each of the reaction mixtures was subjected to PAGE (420% gradient gel) in TBE buffer (Sambrook et al., 1989
). The resulting gels were exposed to a phosphorimager plate (Molecular Dynamics) for 23 h and then scanned. The amounts of cDNA produced were quantified by measuring the intensity of each band using Imagequant (Molecular Dynamics).
In vitro binding of Era to RNA.
To test if purified native Era protein binds to RNA, a purified native Era preparation (16 µg) was mixed with or without 300 µg total RNA prepared from cells of E. coli LY12 by phenol/chloroform extraction (Sambrook et al., 1989 ) in 200 µl 25 mM Tris/HCl, pH 7·5, 150 mM NaCl and 2·5 mM MgCl2. The reaction mixtures were incubated at 4 °C for 30 min and then subjected to gel filtration column chromatography as described above. Fractions (2 ml each) were collected and the presence of Era in the fractions was detected by Western blotting analysis (Zhao et al., 1999
).
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RESULTS |
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To further establish that RNA is associated with Era, we subjected a crude extract of a wild-type strain of E. coli (without overexpression of Era) to gel filtration column chromatography. We reasoned that if RNA is associated with Era, then the EraRNA complex should be present as a higher molecular mass species. As shown in Fig. 3, the Era protein isolated directly from the E. coli cells exhibited two peaks; one located in the void-volume fractions from the column with an estimated molecular mass of 600 kDa and one eluted at a position with a molecular mass of about 32 kDa (monomer) (Fig. 3
, top panel). However, when the crude extract was treated with RNaseA, the Era protein exhibited only one peak with a molecular mass of around 32 kDa (Fig. 3
, bottom panel). In addition, when a crude extract of E. coli LY68 (a native Era overexpresser strain) was chromatographed under identical conditions, the native Era protein was also present in two peaks similar to those of the wild-type E. coli cells (data not shown). Together, these findings suggest that the Era protein is complexed with RNA in E. coli and that the RNA associated with the native Era protein purified from the Era overexpresser strain was removed during purification.
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Purified native Era protein can bind to RNA in vitro
To examine whether purified Era protein could bind to RNA in vitro, we mixed a purified native Era preparation with total cellular RNA isolated from E. coli cells, subjected the reaction mixtures to gel filtration column chromatography and examined column fractions for the presence of Era by Western blotting analysis. As shown in Fig. 5, two peaks were observed; one eluted in the void-volume fractions with an estimated molecular mass of 600 kDa and the other eluted in the fractions indicating a molecular mass of 32 kDa. The presence of a significant amount of Era in the void-volume fractions suggests that Era was bound to RNA. However, which species of RNA was bound to Era was not known and could not be determined, since a mixture of RNAs was used and each individual rRNA was not available. Nevertheless, these results have confirmed that Era is indeed an RNA-binding protein. We also tested the purified native Era protein preparation and the Era and RNA reaction mixtures for their GTPase activity. Apparently, in the presence of RNAs, the GTPase activity (19 mmol min-1 mg-1) of Era seemed to be higher than that (12 mmol min-1 mg-1) of Era free of RNA. This appears to suggest that the binding of RNA to Era stimulates its GTPase activity.
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DISCUSSION |
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There are several lines of evidence showing that 16S rRNA is associated with the E. coli Era protein. The native Era protein was found to be present as two species of 600 and 32 kDa in a wild-type E. coli strain. The Era16S rRNA complex is predicted to be around 600 kDa, since the E. coli 16S rRNA has an estimated molecular mass of 510 kDa (Brosius et al., 1978 ). RNase treatment of a crude extract of E. coli converted the 600 kDa species into a 32 kDa low-molecular-mass species, a monomeric form of Era. Similarly, the affinity-purified GSTEra protein was also present as high-molecular-mass species complexed with RNA. The results of agarose gel electrophoresis established that the RNA was physically associated with the Era protein and exhibited a mobility slower than that of free 16S rRNA. When associated with RNA, the Era protein of E. coli exhibited a GTPase activity significantly higher than that of the GSTEra protein that was free of RNA. A purified native Era protein preparation was also shown to bind to RNA in vitro. In the presence of total cellular RNAs, the GTPase activity of Era appeared to be higher than that of Era free of RNA. The RNA associated with Era was identified primarily as 16S rRNA. Finally, Johnstone et al. (1999)
have also shown that the E. coli Era protein was able to bind RNA in vitro. In light of these findings, we conclude that RNA is indeed associated with the Era protein of E. coli and might be required for its high GTPase activity. Therefore, the results of this study have confirmed our previous finding that the Era protein of S. pneumoniae is primarily associated with E. coli 16S rRNA (Meier et al., 1999
) and have further established that the binding of 16S rRNA to Era is an intrinsic property of Era proteins.
GTPases play a variety of regulatory roles in the cell, in such processes as translation, cell differentiation and cell cycle regulation (Barbacid, 1987 ; Bourne et al., 1990
, 1991
; March, 1992
). Small GTPases, in particular, generally function as regulatory switches that can be converted from an active state to an inactive state by hydrolysis of GTP to GDP to regulate their effector enzymes (Barbacid, 1987
; Bourne et al., 1990
, 1991
; March, 1992
). Era appears to play a role in cell cycle control and translation (Britton et al., 1997
, 1998
; Gollop & March, 1991b
). Presently, it is not known whether the RNA-bound or RNA-free form of Era is responsible for this regulation, nor is the mechanism of this regulation known. One possibility is that the RNA-bound form of Era, which has a higher GTPase activity, is in an active state that signals cell division and possibly translation (Meier et al., 1999
). The RNA-free form of Era may be in an inactive state that signals a termination of cell division and translation.
Sequence analysis revealed that the C-terminal domain of Era is highly conserved within, and unique to, the Era family. Era also appears to bind to the cytoplasmic membrane and to partition between the cytoplasm and the cytoplasmic membrane of the bacterial cell (Gollop & March, 1991a ; Lin et al., 1994
; Wu et al., 1995
; Zhao et al., 1999
). The C-terminal domain of Era seems to possess some characteristics of the KH domain of the pre-mRNA-binding K protein that is known to bind RNA (Chen et al., 1999
; Johnstone et al., 1999
; Siomi et al., 1993
). In this study, we demonstrated that a C-terminally truncated Era protein of S. pneumoniae was unable to bind to RNA in vivo when expressed in E. coli. We have shown previously that the intact era gene of S. pneumoniae complemented an E. coli mutant deficient in the production of Era, without overexpression (Zhao et al., 1999
). This truncated era gene, however, failed to complement the same E. coli mutant when a sufficient level of the protein was produced (Zhao et al., 1999
). Consistent with these results, mutations in the C-terminal domain of the E. coli Era protein abolished the RNA-binding activity of the protein and these mutant era genes failed to complement the E. coli mutant strain deficient in the production of Era (Johnstone et al., 1999
). Together, these results suggest that the C-terminal domain of Era is essential for the binding of 16S rRNA to the protein and that this RNA-binding activity of Era is required for the biological function of the Era protein. Previously, our studies suggested that the C-terminal domain of Era is required for the membrane-binding activity of the protein (Zhao et al., 1999
). In light of the new findings that RNA is bound to the C terminus of Era, it is quite possible that this membrane-binding activity is due to its binding to ribosomes. Clearly, this hypothesis warrants further study.
The mechanism by which RNA stimulates the GTPase activity of Era remains to be elucidated. This study has, however, provided several lines of evidence suggesting that the mechanism by which the small carbohydrates and RNA modulate the GTPase activity of Era might be similar. First, acetate and 3-phosphoglycerate stimulated the GTPase activity of the Era protein free of RNA, but failed to stimulate the GTPase activity of the Era protein bound with RNA. These results clearly validate the finding that the RNA associated with Era stimulates the GTPase activity of the enzyme. Second, the GTPase activity of the C-terminally truncated Era protein could not be stimulated by acetate and 3-phosphoglycerate, indicating that the carbohydrates must bind to the C-terminal domain of Era to function. Third, the RNA is bound to the C-terminal domain of Era. Together, these results also suggest that the small carbohydrates and RNA modulate the GTPase activity of Era probably through their direct binding to and interaction with the C-terminal domain of the protein.
Acetate, a small carboxylic acid (Fraenkel, 1996 ; Holms, 1996
), has been shown to stimulate the activities of some mutant enzymes that require a carboxylic acid for efficient catalysis (Drueckes & Schinzel, 1996
). 3-Phosphoglycerate is also a small carboxylic acid (Fraenkel, 1996
; Holms, 1996
). On the basis of their structural similarities and their ability to stimulate the GTPase activity of Era, it appears that Era also requires a small carboxylic-acid-like molecule for efficient catalysis. Presently, we do not know why pyruvate, glycerate, lactate, 2-phosphoglycerate and phosphoenolpyruvate, also small carboxylic acids, failed to exert significant effects on the GTPase activity of Era. However, it is possible that their side chains, located at the C-2 position, might interfere with their interaction with Era. If Era requires a carboxylic-acid-like molecule for efficient catalysis, it is clear that this molecule must bind to the C-terminal domain of Era to function, since these carbohydrates did not modulate the GTPase activity of the C-terminally truncated Era protein. Thus, it is likely that the binding of this carboxylic-acid-like molecule to Era causes a conformational change that allows efficient catalysis to occur. It is also possible that the RNA, like the carbohydrates tested, is a negatively charged molecule and causes a similar conformational change upon binding to the C terminus of Era which allows the catalysis of the enzyme to proceed more efficiently. It should be noted that we cannot rule out the possibility that other mechanisms might be involved in the modulation of the GTPase activity of Era in the cell.
The physiological role of carbohydrates in the modulation of the Era GTPase activity in vivo is unknown. There are two observations that appear to suggest that Era might be involved in the regulation of bacterial energy metabolism. First, the utilization of carbohydrate intermediates such as pyruvate was increased in the cells in which the level of Era was severely reduced (Lerner & Inouye, 1991 ). Second, when a dominant negative mutant of Era was overexpressed in cells of a wild-type E. coli strain, the growth of this strain was inhibited in a minimal medium supplemented with TCA cycle intermediates (Shimamoto & Inouye, 1996
). In this study, we showed that 3-phosphoglycerate and glyceraldehyde 3-phosphate, key intermediates in glycolysis (Fraenkel, 1996
; Holms, 1996
), modulated the GTPase activity of Era although pyruvate did not. The physiological concentrations of 3-phosphoglycerate and glyceraldehyde 3-phosphate in the cell are not known, but probably are similar (Fraenkel, 1996
; Holms, 1996
). The concentration of acetate in the cell varies significantly depending on growth conditions and carbon sources (Holms, 1996
). The finding that acetate, 3-phosphoglycerate and glyceraldehyde 3-phosphate appear to exert similar levels of effects on the GTPase activity of Era suggests that their overall effects on the GTPase activity of Era in the cell might be minimal. Therefore, the RNA might play an important role in the regulation of the Era GTPase activity in the cell.
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ACKNOWLEDGEMENTS |
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Received 11 August 1999;
revised 16 December 1999;
accepted 19 January 2000.