An HflX-type GTPase from Sulfolobus solfataricus binds to the 50S ribosomal subunit in all nucleotide-bound states.
ABSTRACT HflX GTPases are found in all three domains of life, the Bacteria, Archaea, and Eukarya. HflX from Escherichia coli has been shown to bind to the 50S ribosomal subunit in a nucleotide-dependent manner, and this interaction strongly stimulates its GTPase activity. We recently determined the structure of an HflX ortholog from the archaeon Sulfolobus solfataricus (SsoHflX). It revealed the presence of a novel HflX domain that might function in RNA binding and is linked to a canonical G domain. This domain arrangement is common to all archaeal, bacterial, and eukaryotic HflX GTPases. This paper shows that the archaeal SsoHflX, like its bacterial orthologs, binds to the 50S ribosomal subunit. This interaction does not depend on the presence of guanine nucleotides. The HflX domain is sufficient for ribosome interaction. Binding appears to be restricted to free 50S ribosomal subunits and does not occur with 70S ribosomes engaged in translation. The fingerprint (1)H-(15)N heteronuclear correlation nuclear magnetic resonance (NMR) spectrum of SsoHflX reveals a large number of well-resolved resonances that are broadened upon binding to the 50S ribosomal subunit. The GTPase activity of SsoHflX is stimulated by crude fractions of 50S ribosomal subunits, but this effect is lost with further high-salt purification of the 50S ribosomal subunits, suggesting that the stimulation depends on an extrinsic factor bound to the 50S ribosomal subunit. Our results reveal common properties but also marked differences between archaeal and bacterial HflX proteins.
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ABSTRACT: Initiation is the step of translation that has incurred the greatest evolutionary divergence. In silico and experimental studies have shown that archaeal translation initiation resembles neither the bacterial nor the eukaryotic paradigm, but shares features with both. The structure of mRNA in archaea is similar to the bacterial one, although the protein factors that assist translational initiation are more numerous than in bacteria and are homologous to eukaryotic proteins. This chapter describes a number of techniques that can be used for in vitro studies of archaeal translation and translational initiation, using as a model system the thermophilic crenarcheon Sulfolobus solfataricus, growing optimally at about 80 degrees in an acidic environment.Methods in Enzymology 02/2007; 430:79-109. · 2.00 Impact Factor
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ABSTRACT: To probe the cellular phenotype and biochemical function associated with the G domains of Escherichia coli EngA (YfgK, Der), mutations were created in the phosphate binding loop of each. Neither an S16A nor an S217A variant of G domain 1 or 2, respectively, was able to support growth of an engA conditional null. Polysome profiles of EngA-depleted cells were significantly altered, and His(6)-EngA was found to cofractionate with the 50S ribosomal subunit. The variants were unable to complement the abnormal polysome profile and were furthermore significantly impacted with respect to in vitro GTPase activity. Together, these observations suggest that the G domains have a cooperative function in ribosome stability and/or biogenesis.Journal of Bacteriology 12/2006; 188(22):7992-6. · 3.19 Impact Factor
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ABSTRACT: The assembly of ribosomal subunits from their individual components (rRNA and ribosomal proteins) requires the assistance of a multitude of factors in order to control and increase the efficiency of the assembly process. GTPases of the TRAFAC (translation-factor-related) class constitute a major type of ribosome-assembly factor in Eukaryota and Bacteria. They are thought to aid the stepwise assembly of ribosomal subunits through a 'molecular switch' mechanism that involves conformational changes in response to GTP hydrolysis. Most conserved TRAFAC GTPases are involved in ribosome assembly or other translation-associated processes. They typically interact with ribosomal subunits, but in many cases, the exact role that these GTPases play remains unclear. Previous studies almost exclusively focused on the systems of Bacteria and Eukaryota. Archaea possess several conserved TRAFAC GTPases as well, with some GTPase families being present only in the archaeo-eukaryotic lineage. In the present paper, we review the occurrence of TRAFAC GTPases with translation-associated functions in Archaea.Biochemical Society Transactions 01/2011; 39(1):45-50. · 2.59 Impact Factor
JOURNAL OF BACTERIOLOGY, June 2011, p. 2861–2867
Copyright © 2011, American Society for Microbiology. All Rights Reserved.
Vol. 193, No. 11
An HflX-Type GTPase from Sulfolobus solfataricus Binds to the 50S
Ribosomal Subunit in All Nucleotide-Bound States?
Fabian Blombach,1* Helene Launay,2Violeta Zorraquino,1Daan C. Swarts,1Lisa D. Cabrita,2
Dario Benelli,3John Christodoulou,2Paola Londei,3and John van der Oost1
Laboratory of Microbiology, Wageningen University, Wageningen, Netherlands1; Institute of Structural and Molecular Biology,
University College London (UCL), and Birkbeck College, University of London, London, United Kingdom2; and
Dipartimento Biotecnologie Cellulari ed Ematologia, Universita ` di Roma Sapienza, Rome, Italy3
Received 27 December 2010/Accepted 26 March 2011
HflX GTPases are found in all three domains of life, the Bacteria, Archaea, and Eukarya. HflX from
Escherichia coli has been shown to bind to the 50S ribosomal subunit in a nucleotide-dependent manner, and
this interaction strongly stimulates its GTPase activity. We recently determined the structure of an HflX
ortholog from the archaeon Sulfolobus solfataricus (SsoHflX). It revealed the presence of a novel HflX domain
that might function in RNA binding and is linked to a canonical G domain. This domain arrangement is
common to all archaeal, bacterial, and eukaryotic HflX GTPases. This paper shows that the archaeal SsoHflX,
like its bacterial orthologs, binds to the 50S ribosomal subunit. This interaction does not depend on the
presence of guanine nucleotides. The HflX domain is sufficient for ribosome interaction. Binding appears to be
restricted to free 50S ribosomal subunits and does not occur with 70S ribosomes engaged in translation. The
fingerprint1H-15N heteronuclear correlation nuclear magnetic resonance (NMR) spectrum of SsoHflX reveals
a large number of well-resolved resonances that are broadened upon binding to the 50S ribosomal subunit. The
GTPase activity of SsoHflX is stimulated by crude fractions of 50S ribosomal subunits, but this effect is lost
with further high-salt purification of the 50S ribosomal subunits, suggesting that the stimulation depends on
an extrinsic factor bound to the 50S ribosomal subunit. Our results reveal common properties but also marked
differences between archaeal and bacterial HflX proteins.
GTPases of the translation factor-related (TRAFAC) class
regulate a broad range of ribosome-associated processes, from
the assembly of ribosomal subunits to the control of the mature
ribosome during all phases of translation (5, 19, 23). The
TRAFAC class is named after the family of classical transla-
tion factor GTPases, which includes the two elongation factors
EF-Tu and EF-G. Homologs of these two GTPases are found
in every organism from the three domains of life (Archaea,
Bacteria, and Eukarya). Their molecular functions in transla-
tion are well known. Several other TRAFAC GTPase families
are present in organisms from all three domains of life, al-
though they are less ubiquitous than the classical translation
factor GTPases (4, 6, 23). The biological function of these
widespread GTPases is often not well understood. Among
these is the family of HflX GTPases.
Bacterial and eukaryotic HflX GTPases are three-domain
proteins. They are composed of a central G domain flanked by
two putative ligand-binding domains. Escherichia coli HflX was
originally presumed to be involved in phage lambda lysogeny,
but recent experiments have dismissed this hypothesis (10). E.
coli HflX was shown to interact with the 50S ribosomal subunit
(18), suggesting that HflX might have a ribosome-associated
function, similar to the observations made for members of the
related family of Obg GTPases (14, 24, 39, 42). Both the
N-terminal and the C-terminal domain are required for stable
ribosome binding in vitro (18). The ribosome binding of E. coli
HflX is nucleotide dependent (18). Surprisingly, E. coli HflX
can bind and hydrolyze not only GTP but also ATP (10, 18, 34).
This finding was unexpected because HflX GTPases encom-
pass a sequence motif for specific recognition of the guanine
base that is conserved in the vast majority of the various
GTPase families. The GTPase and ATPase activities of Esch-
erichia coli HflX are activated upon binding to the 50S ribo-
somal subunit. However, the precise biological function of
HflX and the role of GTP hydrolysis therein remain unknown.
In addition, hflX deletion strains of several bacterial species
did not exhibit any phenotype, indicating that HflX is nones-
sential under the conditions tested (10, 11, 28).
Archaeal HflX GTPases are two-domain proteins; they lack
the C-terminal domain that is present in bacterial and eukary-
otic HflX GTPases. The recently published structure of the
archaeal HflX ortholog from Sulfolobus solfataricus (SsoHflX)
revealed that the N-terminal domain (hereinafter termed
“HflX domain”) exposes a positively charged surface that
might be involved in nucleic acid binding (40). This is analo-
gous to the RNA binding domain present in other multido-
main TRAFAC GTPases (22, 36). The two flexible regions of
the G domain, termed switch 1 and switch 2, are positioned at
the interface of the G domain and the HflX domain. Switches
1 and 2 usually undergo conformational changes in response to
the nucleotide-bound state of the G domain. In SsoHflX, the
switch 1 and switch 2 regions might therefore alter the position
of the two domains relative to each other upon the exchange of
nucleotides (17, 40).
To gain insight in the function of the archaeal HflX
* Corresponding author. Mailing address: Laboratory of Microbiol-
ogy, Wageningen University, Dreijenplein 10, 6703 HB Wageningen,
Netherlands. Phone: 31 317 483740. Fax: 31 317 483829. E-mail: fabian
?Published ahead of print on 8 April 2011.
GTPases, we investigated whether the archaeal HflX homolog
SsoHflX interacts with the large ribosomal subunit and how
this interaction is influenced by the presence of guanine nu-
cleotides. Our data suggest that interaction with the large ri-
bosomal subunit is a conserved feature of HflX GTPases.
However, considerable differences between archaeal and bac-
terial HflX GTPases were found concerning the ribosome de-
pendence of the GTPase activity.
MATERIALS AND METHODS
Heterologous expression and purification of SsoHflX and SsoHflX-H. Plas-
mids for the heterologous expression of SsoHflX and the truncated mutant
protein encompassing the isolated HflX domain (SsoHflX-H) have been de-
scribed previously (40). Heterologous expression was carried out in E. coli Ro-
setta (DE3) cells (Novagen) in LB medium according to standard methods, and
cells were stored at ?80°C until further use. For the production of15N-labeled
SsoHflX, LB medium was replaced by M9 medium containing15NH4Cl (Cam-
bridge Isotope Laboratories) as the sole nitrogen source and 0.4% (wt/vol)
glucose as the carbon source. For the purification of SsoHflX, 1 g cell paste was
resuspended in 4 ml buffer G (20 mM Tris-HCl, pH 7.4, 300 mM KCl, 10 mM
imidazole, 1 mM dithiothreitol [DTT], 7.5% glycerol) and passed thrice through
a French pressure cell at 16,000 lb/in2. Cell debris was removed by centrifugation
(37,000 ? g for 30 min at 4°C), and the supernatant was incubated at 70°C for 20
min, allowing the removal of the heat-unstable host proteins by centrifugation.
The heat-stable cell extract was loaded on a Ni affinity chromatography column.
After washing with at least 10 column volumes of buffer G, SsoHflX was eluted
with buffer G containing 500 mM imidazole. The protein was further purified by
gel filtration using a Superdex 75 HR 16/60 column (GE Healthcare) in buffer G
containing no imidazole. SsoHflX-H was purified likewise, but the heat incuba-
tion was omitted and, after Ni chromatography, the protein was purified by
heparin-agarose chromatography with a gradient from 0.2 to 2 M KCl. Protein
concentrations were determined using a protein assay (Bio-Rad) based on the
method by Bradford.
Fractionation of cell lysates on sucrose density gradients and in vitro trans-
lation. S. solfataricus strain P2 cells were grown on modified Brock medium
supplemented with 0.1% (wt/vol) tryptone and 0.4% (wt/vol) sucrose (41). Cell
lysates were prepared as described previously (1). 70S ribosomes were obtained
by chemical cross-linking of cell lysates programmed for translation as described
previously (1), with the following modifications: 100 ?l in vitro translation assays
contained 480 ?g cell lysate (referring to the protein concentration measured by
the Bradford protein assay), 4 ?g orf104 mRNA, 0.24 A260units bulk Sulfolobus
tRNA, 1.8 mM ATP, 0.9 mM GTP, 4 ?l 1 mM amino acid mixture without
methionine (Promega) in 20 mM triethanolamine (TEA)-KOH, pH 7.4, 20 mM
magnesium acetate (MgOAc), 10 mM KCl. Samples were incubated at 73°C for
30 min and placed on ice. 70S ribosomes were stabilized by the addition of 1%
(vol/vol) formaldehyde and further incubation for 30 min on ice. The samples
were then loaded onto 10.5-ml linear 10%-to-30% sucrose gradients in 20 mM
Tris-HCl, pH 7.4, 40 mM NH4Cl, 10 mM MgCl2, 1 mM DTT and centrifuged for
4 h at 36,000 rpm in a TST41.14 rotor (Kontron instruments). Gradients were
fractionated, and proteins were concentrated by trichloroacetic acid-deoxy-
cholate (TCA-DOC) precipitation. Pellets were dissolved in 25 ?l 2? SDS-
PAGE loading buffer.
Isolation of ribosomal subunits. Ribosomal subunits from S. solfataricus were
isolated as described previously (1). After separation on 10%-to-30% sucrose
density gradients, the isolated subunits were concentrated and purified from
sucrose by ultrafiltration (30,000 molecular-weight cutoff; Vivaspin). The con-
centration was determined based on the absorption at 260 nm using conversion
factors of 60 pmol 50S ribosomal subunit per A260unit.
Ribosome binding assays. Amounts of 80 ?l contained 80 pmol SsoHflX, 80
pmol of purified large ribosomal subunit, and 100 ?M respective nucleotide in 20
mM Tris–HCl, pH 7.4, 40 mM NH4Cl, 10 mM MgOAc, 1 mM DTT, 5% glycerol.
Samples were incubated at 50°C for 15 min and then loaded onto 10%-to-30%
sucrose gradients. Further processing of the samples was as described above.
Generation of anti-SsoHflX antiserum and labeling of antibodies. Rabbit
antiserum against SsoHflX was produced at Eurogentec (Belgium). Protein
A-agarose-purified antibodies were labeled with digoxigenin (DIG)-3-O-methyl-
carbonyl-ε-aminocaproic acid-N-hydroxysuccinimide ester (Roche) in a 1:25 mo-
lar ratio according to the manufacturer’s protocol.
Immunodetection of SsoHflX. After SDS-PAGE, proteins were blotted on
0.2-?m-pore-size nitrocellulose (Schleicher & Schuell) in 10 mM 3-(cyclohexyl-
amino)-1-propanesulfonic acid (CAPS) (pH 11.0), 10% (vol/vol) methanol by
wet transfer at 10 V overnight. Filters were blocked with 0.2% i-block (Applied
Biosystems) in TBST buffer (20 mM Tris-HCl, pH 7.9, 150 mM NaCl, 0.1%
[vol/vol] Tween 20). The DIG-labeled anti-SsoHflX antibodies were used in
500? dilution in combination with anti-DIG-alkaline phosphatase (AP) Fab
fragments (Roche) as secondary antibody according to the manufacturer’s pro-
tocol. Chemiluminescence was detected using CDP-Star reagent (New England
BioLabs) and Biomax light films (Kodak).
NMR of SsoHflX. Uniformly15N isotopically labeled SsoHflX was used to
assess binding to 50S ribosomal subunits using nuclear magnetic resonance
(NMR) spectroscopy. Both the15N-labeled SsoHflX and purified 50S ribosomal
subunits were buffer exchanged to 10 mM HEPES-KOH, pH 7.5, 40 mM NH4Cl,
10 mM MgCl2, 1 mM DTT, with the addition of 10% D2O as an NMR lock
solvent, and SsoHflX was concentrated to 26 ?M. All NMR experiments were
performed at 50°C.15N-1H heteronuclear correlation spectra of SsoHflX were
recorded using the SOFAST-heteronuclear multiple quantum coherence
(HMQC) sequence (31), with 100 increments in the15N dimension, a spectral
width of 32 ppm, a recycling delay of 300 ms, and 256 scans. A 120° PC9 (2.454
ms at 700 MHz) shaped pulse was used for the excitation, and a REBURP
shaped pulse (1.782 ms at 700 MHz) was used for the inversion. For heteronu-
clear stimulated echo (XSTE)–pulsed-field gradient (PFG) NMR (12), a 1-ms
square gradient varying from 5% to 95% of the maximum gradient strength
together with a 200-ms diffusion delay were employed.1H spectra were recorded
using an 18 ?M sample of 50S ribosomal subunit with various concentrations of
SsoHflX. Data processing was performed using Topspin 2.4 (Bruker) and
NMRPipe (9). Proton transverse relaxation was measured using the spin echo T2
Nucleotidase activity assays. Volumes of 20 ?l contained the amounts of GTP
or ATP (1.6 ?M to 250 ?M) indicated below, supplemented with a trace amount
(8.25 nM) of [?-32P]GTP or [?-32P]ATP in 20 mM Tris–HCl, pH 7.4, 50 mM
NH4Cl, 10 mM MgOAc, 8% glycerol, 1 mM DTT. The amount of SsoHflX (0.24
?M to 7.52 ?M) was adjusted in a manner such that approximately 10% of the
nucleotides were hydrolyzed in the individual assays to ensure reliable quantifi-
cation and minimize inhibition by GDP. Samples were incubated at 50°C for 20
min, after which 4 ?l was withdrawn and mixed with 10 ?l of ice-cold stop
solution (20 mM EDTA). One microliter of this mixture was subsequently
spotted onto polyethyleneimine (PEI)-cellulose thin-layer chromatography
plates, resolved, and detected as described previously (40). Signals were detected
on phosphor storage screens (Kodak), and the Quantity One software package
(Bio-Rad) was used to quantify the spots. The amount of nucleotide hydrolysis
was calculated for each individual lane, and average values were determined
from at least two parallel experiments. All values were corrected for the amount
of nucleotide hydrolyzed in samples of identical composition with buffer replac-
Localization of endogenous SsoHflX. To establish initial in-
sights into the physiological function of SsoHflX, we tested for
comigration of endogenous SsoHflX with 50S ribosomal sub-
units during cell lysate fractionation on a sucrose density gra-
dient because such an association had been observed previ-
ously for the E. coli HflX ortholog (18). Free 30S and 50S
ribosomal subunits can readily be obtained from the S. solfa-
taricus cell lysate using sucrose density gradient ultracentrifu-
gation, whereas 70S ribosomes are not stable and dissociate
into 30S and 50S ribosomal subunits during ultracentrifugation
(1). S. solfataricus 70S ribosomes can be obtained by program-
ming the cell lysate for translation using in vitro translation
assays and subsequently cross-linking with formaldehyde.
Without programming of the cell lysate, SsoHflX comigrated
mainly with the free 50S ribosomal subunit. An additional peak
of SsoHflX was observed further up in the gradient (Fig. 1A).
This is approximately the position where the thermosome was
detected on the Ponceau S-stained blot (not shown). The ther-
mosome is the most abundant protein in S. solfataricus and
sediments at 20S (30). This suggests that SsoHflX might be
2862BLOMBACH ET AL.J. BACTERIOL.
part of another high-molecular-weight complex besides the
putative complex formed with the 50S ribosomal subunit.
After the cell lysate was programmed for translation, most
30S and 50S ribosomal subunits were incorporated in 70S ri-
bosomes (Fig. 1B). SsoHflX comigrated with the remaining
free 50S ribosomal subunit but not with the 70S ribosomes.
Furthermore, SsoHflX could not be detected any longer at the
Reconstitution of SsoHflX-50S ribosomal subunit com-
plexes. The comigration of endogenous SsoHflX with the 50S
ribosomal subunit in sucrose density gradients suggested that
SsoHflX might bind directly to 50S ribosomal subunits. In
order to test this, binding assays with recombinant SsoHflX
and purified 50S ribosomal subunit were undertaken.
Complexes were reconstituted at 50°C, and free SsoHflX
was separated from SsoHflX-50S ribosomal subunit complexes
by sucrose density gradient centrifugation. SsoHflX was able to
bind stably to the purified 50S ribosomal subunit in the pres-
ence of either GDP or the nonhydrolyzable GTP analog
GppNHp (guanylylimidodiphosphate) (Fig. 2A and B). Apo-
SsoHflX was able to bind to the 50S ribosomal subunit as well
(Fig. 2C), but in a less stable manner than its binding in the
presence of guanine nucleotides. Although it is possible that a
fraction of the recombinant SsoHflX might still be in a nucle-
otide-bound state after purification, the fact that the apo-
SsoHflX crystal structure was obtained from a protein prepa-
ration purified under comparable conditions indicates that
such a fraction should be rather small. Similar results were
obtained for endogenous SsoHflX when ribosomes were iso-
lated from cell extracts incubated with the respective nucleo-
tides (data not shown).
A truncated mutant protein encompassing only the isolated
HflX domain (SsoHflX-H) was similarly able to form stable
complexes with the 50S ribosomal subunit, indicating that the
HflX domain contributes to the 50S ribosomal subunit binding
interface of SsoHflX (Fig. 2D). The results for control samples
lacking 50S ribosomal subunit particles demonstrated that the
observed migration of SsoHflX and SsoHflX-H into the su-
crose density gradient was entirely dependent on interaction
with the 50S ribosomal subunit (Fig. 2E and F).
SsoHflX binds to the surface of the 50S ribosomal subunit.
In order to investigate the dynamics of the 50S ribosomal
subunit binding by SsoHflX in more detail, NMR spectroscopy
experiments were carried out. Using pulsed-field gradient dif-
fusion NMR experiments designed to determine the diffusion
coefficient of the species that give rise to resonances, we found
that the diffusion coefficient of15N-labeled SsoHflX at 25°C
was 1.14 ? 10?10? 0.05 ? 10?10m2s?1(mean ? standard
deviation) (Fig. 3B). Using the Stoke-Einstein equation (13),
this corresponds to a particle with a hydrodynamic radius of 2
nm. The SsoHflX protomer has a molecular weight of 41.6
kDa, which if we assume a spherical particle would correspond
to a radius of 2.3 nm. We therefore concluded that SsoHflX
appears to be a monomer in solution, in line with previous
results from native mass spectrometry experiments (40).
Two-dimensional (2-D)1H-15N heteronuclear single quan-
tum coherence (HSQC) spectra of SsoHflX were recorded at
25°C and 50°C. The spectrum at 25°C shows extensive broad-
ening of resonances with an unresolved set of highly broadened
FIG. 1. Localization of endogenous SsoHflX in S. solfataricus cell
lysates after sucrose density gradient centrifugation. The upper panels
show the absorption profiles at 260 nm. The positions of ribosomal
subunits are indicated. The lower panels show the immunodetection of
SsoHflX. (A) Cell lysate after incubation at 70°C. (B) Cell lysate
programmed for translation. 70S ribosomes were stabilized by form-
FIG. 2. Binding assays for SsoHflX with purified 50S ribosomal
subunits in the presence of different guanine nucleotides. (A to C) A
1 ?M concentration of recombinant SsoHflX was mixed with 1 ?M 50S
ribosomal subunit in the presence of 100 ?M GppNHp (A) or 100 ?M
GDP (B) and in the absence of nucleotides (C). (D) Binding assay
carried out as described above with the C-terminal G domain deletion
mutant protein SsoHflX-H in the absence of nucleotides. (E and F)
Migration of SsoHflX (E) and SsoHflX-H (F) in the absence of 50S
ribosomal subunits as detected by silver staining.
VOL. 193, 2011 S. SOLFATARICUS HflX BINDING TO 50S RIBOSOMAL SUBUNIT2863
peaks in the central region of the spectrum, but temperature
elevation resulted in marked sharpening of the cross-peaks,
and at 50°C, a large number (approximately 191 of 355) re-
solvable cross-peaks were observed (Fig. 3A), including a sig-
nificant number in dispersed regions of the spectrum. Together
with fast relaxation observed for the1H signals (T2 ? 30 ms),
the temperature effect and the heterogeneity in cross-peak
linewidths are indicative of conformational exchange that the
protein is undergoing on the ms-100 ?s timescale. After the
addition of 10 mM GTP, the GTP proton H8 was observed to
undergo a progressive change over 12 h at 50°C, as expected
from the slow GTP hydrolysis rate of SsoHflX (40). The 2-D
1H-15N HSQC spectra showed no changes in chemical shift or
linewidth of the SsoHflX cross-peaks in the presence of GTP
and GDP (data not shown).
The investigation of the possible interactions of SsoHflX
with the 50S ribosomal subunits was performed at 50°C. The
1H spectrum of the S. solfataricus 50S ribosomal subunits is
shown in Fig. 4A. Despite the high molecular weight of this
particle, a significant number of resonances were observed,
and by analogy with studies on the E. coli ribosome (8), these
may arise from the L12 proteins in the GTPase-associated
region of the 50S ribosomal subunit. In order to monitor the
attachment of the observed protein to the 50S particle, a PFG-
diffusion NMR experiment at 25°C was used to determine the
diffusion coefficient of the observed signal, which was 4.3 ? 0.2
0.10?11m2s?1. This diffusion coefficient corresponds to a
particle radius of 5.6 ? 0.3 nm, which corresponds well to a
particle of the size of the 50S ribosomal subunit (radius of ca.
6 nm), indicating that the proteins that give rise to those
resonances are attached to the 50S ribosomal subunits.
Upon the addition of SsoHflX to the 50S ribosomal subunit,
the resonances in the 2-D spectrum of SsoHflX became broad-
ened completely beyond detection, indicating an interaction of
the protein with the 50S ribosomal subunit (Fig. 4A). The
proton signals from flexible parts of the 50S ribosomal subunit
were not affected by the binding of SsoHflX (Fig. 4A).
The addition of a 10-fold molar excess of SsoHflX over the
50S ribosomal subunit (in the absence of GTP or nonhydro-
lyzable GTP analogs) did not result in the detection of signal
from SsoHflX (Fig. 4B and data not shown). The complete
broadening indicates that all SsoHflX molecules in solution
appeared to be interacting with the ribosome during the 50-ms
acquisition time, i.e., there are no SsoHflX molecules that
remain free in solution for the entire acquisition time. There-
FIG. 3. Examination of SsoHflX by NMR spectroscopy. (A) 2-D1H-15N SOFAST-HMQC of SsoHflX at 50°C; (B) pulsed-field gradient diffusion
NMR characterization of SsoHfIX (diamonds) compared to the 50S ribosomal subunit (open circles) at 25°C. The logarithm (y axis) of normalized
resonance intensity (I/I0) is plotted against G2, the square of the gradient strength (x axis). I0is the intensity at low gradient strength. The diffusion profile
of SsoHflX is homogeneous, as indicated by the linear regression of ln(I/I0) as a function of G2. The diffusion coefficient extracted from the slope
of ln(I/I0) as a function of G2is 1.1 ? 10?10m2s?1. The diffusion profile of the 50S resonances also appears to be homogeneous and corresponds
to a diffusion coefficient of 4.2 ? 10?11m2s?1. Error bars indicate the uncertainty of the signal intensity based on the spectral noise.
FIG. 4. The interaction of SsoHflX with the 50S ribosomal subunit
(rib. s.u.) results in the broadening of the SsoHflX resonances.
(A) Overlays of the1H one-dimensional spectra of SsoHflX in the
presence of GTP (upper panel, black line), the S. solfataricus 50S
ribosomal subunit (lower panel, black line), and SsoHflX plus the 50S
ribosomal subunit in the presence of GTP (both panels, red line).
(B)1H projection of the1H-15N SOFAST-HMQC of SsoHflX plus 50S
ribosomal subunit in the presence of GTP at 50°C. The entire spectrum
of SsoHflX is broadened beyond detection.
2864 BLOMBACH ET AL. J. BACTERIOL.
fore, the complete broadening of the 10-fold molar excess
indicates a dissociation rate (koff) larger than 10 times 1/50 ms:
200 s?1. Cross-peaks from the GTPase were also broadened
beyond detection in the presence of 10 mM nonhydrolyzable
GTP analog GppNHp under conditions of a 2:1 molar ratio of
SsoHflX over the 50S ribosomal subunit.
Stimulation of GTPase activity by the large ribosomal sub-
unit. Previously, we assessed the GTPase activity of SsoHflX
by measuring Pirelease by a malachite green assay (40). Here,
we measured the GTPase activity of SsoHflX in a ribosome-
compatible buffer system and detected GTP hydrolysis at 50°C
by thin-layer chromatography. This method appeared to be
more robust concerning elevated levels of background GTP
hydrolysis occurring in the presence of ribosomal subunits of
The kcatvalue for SsoHflX in the absence of ribosomes was
9.2 ? 10?4? 0.03 ? 10?4s?1, and the Kmvalue was deter-
mined to be 5.3 ? 0.6 ?M (Fig. 5A). These values were similar
to those measured previously using the malachite green assay
(40), with a slightly increased substrate affinity that might be
explained by the greater accuracy of the thin-layer chromatog-
raphy system at low GTP concentrations. GTP hydrolysis by
SsoHflX was linear during the 20-min incubation, and no sign
of inhibition by the accumulating GDP was observed (Fig. 5B).
E. coli HflX binds and hydrolyzes GTP, as well as ATP (10, 18,
34). In order to clarify whether SsoHflX also possesses ATPase
activity, we tested SsoHflX for such an activity in a time course
experiment. However, ATP hydrolysis was insignificant com-
pared to the levels of GTP hydrolysis (Fig. 5B).
The GTPase activity of E. coli HflX increases strongly in the
presence of 50S ribosomal subunits, as well as empty and
poly(U)-programmed 70S ribosomes (18, 34). In order to fur-
ther investigate the functional conservation between bacterial
and archaeal HflX orthologs, we studied the effects of different
preparations of S. solfataricus ribosomes on the GTPase activ-
ity of SsoHflX. Sucrose cushion centrifugation at 500 mM
NH4Cl is not sufficient to remove all extrinsic factors from the
ribosomal subunits, but higher concentrations of NH4Cl lead
to partial disintegration of the subunits and loss of poly(U)-
directed translation activity (1, 25).
Unfractionated ribosomal subunits purified by sucrose cush-
ion centrifugation at 500 mM NH4Cl did stimulate the GTPase
activity of SsoHflX more than 10-fold at 1 ?M concentration
(Fig. 6A). Notably, the effect was less pronounced when the
concentration of NH4Cl during ribosome purification was re-
duced to 100 mM. To determine whether the effect is caused by
specific ribosomal subunits only, the ribosomal subunits were
separated on sucrose density gradients. Although these ribo-
somal subunits were purified by sucrose cushion centrifugation
under high-salt conditions for 6 h, some residual translation
factors still seemed to be present in the preparations, as a
relatively high background GTPase activity was observed (data
not shown). In agreement with the interaction of SsoHflX with
the 50S ribosomal subunit, stimulation of the GTPase activity
of SsoHflX was observed for the 50S ribosomal subunit but not
for the 30S ribosomal subunit (Fig. 6B). In the presence of
both subunits, the stimulation of GTPase activity of SsoHflX
was somewhat reduced compared to the activation by the 50S
ribosomal subunit alone, suggesting that the 30S ribosomal
subunit competes with the SsoHflX-50S ribosomal subunit
complex formation. When the sucrose cushion purification of
50S ribosomal subunits was prolonged to 13 h, the background
GTPase activity of the ribosome preparations was reduced
significantly. Surprisingly, this coincided with a significant de-
crease of the stimulatory effect on the GTPase activity of
SsoHflX (Fig. 6C). This result suggests that the stimulation of
the GTPase activity of SsoHflX might depend on an extrinsic
factor that binds tightly to the 50S ribosomal subunit and is
only partially removed during the high-salt washing steps. Al-
though rather unlikely, an alternative explanation would be
that SsoHflX itself stimulated the activity of an extrinsic
GTPase copurified with the 50S ribosomal subunit.
GTPases of the TRAFAC class fulfill multiple functions
associated with the process of translation. Recent studies of
several bacterial TRAFAC GTPases revealed their function in
ribosome biogenesis (3, 5, 16, 20, 21, 26–28, 33, 37, 38). Ar-
chaea possess several TRAFAC GTPases, but apart from the
classical translation factors, SsoHflX from the HflX family is
FIG. 5. Basal nucleotidase activity of SsoHflX. Samples were incu-
bated at 50°C for 20 min with the indicated concentrations of GTP. For
all samples, GTP hydrolysis was corrected for background occurring in
the absence of SsoHflX under otherwise identical conditions. (A) Sub-
strate concentration-dependent GTPase activity of SsoHflX. (B) Time
course experiment to test for ATPase activity of SsoHflX. Samples
contained 7.5 ?M SsoHflX and 100 ?M ATP (filled circles) or GTP
(open circles). NTP, nucleoside triphosphate. Error bars show 1 stan-
FIG. 6. Stimulation of the GTPase activity of SsoHflX by ribosomal
subunits in the presence of 250 ?M GTP. (A) Effect of partially
purified ribosomes on SsoHflX GTPase activity. GTPase activity of
SsoHflX was measured in the absence of ribosomes (1) or with 1 ?M
ribosomes purified by 6-h sucrose cushion centrifugation at 100 mM
NH4Cl (2) or 500 mM NH4Cl (3). (B) Stimulation of GTPase activity
of SsoHflX by isolated ribosomal subunit GTPase activity was mea-
sured in the absence of ribosomal subunits (1) or the presence of 1 ?M
50S (2), 0.6 ?M 30S (3), or a mixture of 50S and 30S (4) ribosomal
subunits. (C) Stimulation of GTPase activity of SsoHflX by increasing
amounts of highly purified 50S ribosomal subunits after 13 h of sucrose
cushion centrifugation at 500 mM NH4Cl. Error bars show 1 standard
VOL. 193, 2011 S. SOLFATARICUS HflX BINDING TO 50S RIBOSOMAL SUBUNIT2865
the first archaeal representative for which evidence for a trans-
lation-associated function has been provided. Our results re-
veal not only similarities but also marked differences between
archaeal and bacterial HflX GTPases concerning their molec-
The binding of E. coli HflX to the 50S ribosomal subunit
requires the presence of a nucleotide, but it occurs both in the
“active” (GTP-bound) and “inactive” (GDP-bound) state of
the GTPase (18). Here, we show that for both SsoHflX and E.
coli HflX, the binding of guanine nucleotides strengthens the
interaction with the 50S ribosomal subunit but that the regu-
lation of binding is less strict in the case of SsoHflX. No
conformational changes were observed between the structures
of nucleotide-free and GDP-bound SsoHflX (17, 40). A pos-
sible explanation for the stabilizing effect of guanine nucleo-
tides on the ribosome binding by SsoHflX would be that the
guanine nucleotide is part of the interface of the 50S ribosomal
subunit and SsoHflX.
We could not detect binding of endogenous SsoHflX to 70S
ribosomes during in vitro translation. Similarly, no binding to
70S ribosomes or polysomes has been found for E. coli HflX
(18). Therefore, the molecular role of HflX GTPases might be
limited to free 50S ribosomal subunits, but it cannot be ruled
out that the observed association of archaeal and bacterial
HflX orthologs with free 50S ribosomal subunits only, as ob-
served in cell lysates, does not reflect the in vivo situation.
Shields et al. (34) recently reported stimulation of E. coli HflX
nucleotidase activity by highly purified 70S ribosomes, as well
as by poly(U)-programmed ribosomes, suggesting that E. coli
HflX might also interact with 70S ribosomes during translation
E. coli HflX requires both the N-terminal HflX domain and
the relatively poorly conserved C-terminal domain for stable
binding to the 50S ribosomal subunit (18). Similarly, the HflX
domain of bacterial Chlamydophila pneumoniae HflX is re-
quired for stable 50S ribosomal subunit binding but not suffi-
cient on its own (29). Because archaeal HflX GTPases only
contain the HflX domain as a single putative ribosome-binding
domain, it most likely provides the major surface for interac-
tion with the archaeal 50S ribosomal subunit. The acquisition
of additional domains was certainly a major driving force dur-
ing evolution toward new biological functions of TRAFAC
GTPases, as is obvious from the great variety of domain archi-
tectures occurring in this class of proteins (23). In the case of
bacterial (and eukaryotic) HflX, the additional C-terminal do-
main might have taken over part of the ribosome binding and,
hence, may also have contributed to its tighter regulation as
observed for E. coli HflX. An alternative explanation would be
that this effect is due to differences in the binding site between
bacterial and archaeal 50S ribosomal subunits.
The binding site of SsoHflX on the 50S ribosomal subunit
does not affect the resonances from the ribosome, which pos-
sibly arise from the L12 stalk region by analogy with the E. coli
50S (8). This is in contrast to the ribosome binding by the
bacterial GTPase elongation factor EF-G. Upon the addition
of EF-G to ribosomes, the L12 stalk resonances are broadened
beyond detection, which is in line with the function of this
flexible complex as a landing platform for translation elonga-
tion factors (8).
HflX GTPases possess a canonical G4 motif in the amino
acid sequence of the G domain. The G4 motif normally pro-
vides binding specificity for guanine nucleotides. C. pneu-
moniae HflX possesses GTPase activity that is not inhibited by
ATP, indicating specificity for GTP (29). In contrast, E. coli
HflX has been shown to hydrolyze both GTP and ATP (10, 18,
34). SsoHflX possesses specificity for GTP, indicating that the
ATPase activity of E. coli HflX is not a general feature of HflX
GTPases. The rate of GTP hydrolysis by SsoHflX in the ab-
sence of ribosomes is similar to that reported for E. coli HflX
(kcat? 8.4 ? 10?4s?1), but the extent of the stimulation of
GTPase activity by ribosomes may differ, as the stimulation
observed for E. coli HflX was about 1,000-fold (34). Although
the data presented here might underestimate the extent of
GTPase activity stimulation for SsoHflX, they strongly suggest
that the GTPase activity of SsoHflX can be stimulated only to
a much lesser extent.
Two alternative mechanisms might explain the observed
stimulation of GTP hydrolysis by SsoHflX after binding of the
50S ribosomal subunit. The crystal structures of SsoHflX re-
vealed extensive interactions between the HflX and the G
domain, including several key elements of the G domain in-
volved in guanine nucleotide binding and hydrolysis. Deletion
of the HflX domain led to a significantly increased rate of GTP
hydrolysis (24-fold) (40). It therefore seems possible that the
binding of SsoHflX to the 50S ribosomal subunit loaded with
an unidentified extrinsic factor causes alterations in the inter-
domain interactions, providing the G domain with greater
structural flexibility required for more efficient GTP hydrolysis.
Unfortunately, the expression of the isolated G domain and its
purification using the buffer conditions described here led to a
very unstable protein that could not be detected after sucrose
density gradient centrifugation and appeared to have only a
3-fold-increased GTPase activity in comparison to that of
SsoHflX (data not shown).
Alternatively to an interference with the interdomain inter-
actions of SsoHflX, the 50S ribosomal subunit loaded with
some unknown extrinsic factor might play a direct role in GTP
hydrolysis by SsoHflX, analogous to the role GTP-activating
proteins play in GTP hydrolysis by p21 Ras-related GTPases.
The fact that this unidentified extrinsic factor appears to bind
strongly to the 50S ribosomal subunit and can be removed
completely only by several consecutive high-salt washes sug-
gests that SsoHflX might be required for the release of this
factor. Such a function has been described, for example, for the
GTPase Lsg1 from yeast, which is needed for the release of the
ribosome export factor Nmd3 (15). In archaea, a possible can-
didate is the translation factor aIF6, absent in bacteria but
present in eukaryotes. This protein is a ribosome anti-associ-
ation factor that binds strongly to the 50S subunits and may
control their entry into the translation cycle or help in ribo-
some recycling after termination (2). In eukaryotes, IF6 re-
lease from the 60S subunits requires either protein modifica-
tion (in mammals) or the intervention of a GTPase (in yeast).
It is therefore conceivable that archaeal HflX may control the
dissociation of aIF6 from the 50S subunits.
Since the binding of SsoHflX to the 50S ribosomal subunit
appears to be only weakly regulated by guanine nucleotides,
the highly transient binding that we observed at 50°C would
still allow for efficient scanning of the 50S ribosomal subunit
and factor release.
2866 BLOMBACH ET AL.J. BACTERIOL.
In bacteria, TRAFAC GTPases are thought to play an es-
sential role in coordinating ribosome assembly with the energy
state of the cell by sensing the concentrations of guanine nu-
cleotides, including the “alarmone” ppGpp (guanosine 3?,5?-
bispyrophosphate) (5). This coupling allows bacterial cells to
quickly redirect the cellular metabolism during the stringent
response. The stringent response in archaea apparently differs
from that in bacteria, as in Sulfolobus it is not linked to de-
creasing GTP concentrations (7), and the bacterial alarmone
ppGpp is probably generally absent in archaea (7, 32). An
important question is how archaea link ribosome biogenesis
with the energy state of the cell. It is likely that TRAFAC
GTPases play a key role in the regulation of ribosome assembly
in archaea as well (4). Here, we provide the first experimental
evidence for this in the case of the archaeal HflX homolog, one
of the few TRAFAC GTPases that archaea share with bacteria.
Besides conventional sucrose density gradient centrifugation,
we employed NMR to test for ribosome binding by SsoHflX,
thereby providing insight into the fast dynamics of the Sso-
HflX-50S ribosomal subunit interaction at close-to-physiolog-
ical temperatures. We believe that this approach is highly valu-
able for studies on the interaction of proteins with archaeal
ribosomes, as many archaeal species are thermophiles or hy-
perthermophiles and their ribosomes exhibit considerable ri-
gidity at lower temperatures.
We thank Jasper Akerboom (Howard Hughes Medical Institute
Janelia Farm Research Campus) for critical reading of the manuscript.
We thank John Kirkpatrick (UCL) for assistance with NMR experi-
This work was supported by NWO (ALW-Vici project 865.05.001 to
J.V.D.O.) J.C., L.D.C., and H.L. acknowledge support from the
BBSRC (9015651/JC), and J.C. acknowledges an HFSP Young Inves-
tigator Award (RGY67/2007).
1. Benelli, D., and P. Londei. 2007. In vitro studies of archaeal translational
initiation. Methods Enzymol. 430:79–109.
2. Benelli, D., et al. 2009. Function and ribosomal localization of aIF6, a
translational regulator shared by archaea and eukarya. Nucleic Acids Res.
3. Bharat, A., M. Jiang, S. M. Sullivan, J. R. Maddock, and E. D. Brown. 2006.
Cooperative and critical roles for both G domains in the GTPase activity and
cellular function of ribosome-associated Escherichia coli EngA. J. Bacteriol.
4. Blombach, F., S. J. Brouns, and J. van der Oost. 2011. Assembling the
archaeal ribosome: roles for translation factor-related GTPases. Biochem.
Soc. Trans. 39:45–50.
5. Britton, R. A. 2009. Role of GTPases in bacterial ribosome assembly. Annu.
Rev. Microbiol. 63:155–176.
6. Caldon, C. E., and P. E. March. 2003. Function of the universally conserved
bacterial GTPases. Curr. Opin. Microbiol. 6:135–139.
7. Cellini, A., et al. 2004. Stringent control in the archaeal genus Sulfolobus.
Res. Microbiol. 155:98–104.
8. Christodoulou, J., et al. 2004. Heteronuclear NMR investigations of dynamic
regions of intact Escherichia coli ribosomes. Proc. Natl. Acad. Sci. U. S. A.
9. Delaglio, F., et al. 1995. NMRPipe: a multidimensional spectral processing
system based on UNIX pipes. J. Biomol. NMR 6:277–293.
10. Dutta, D., K. Bandyopadhyay, A. B. Datta, A. A. Sardesai, and P. Parrack.
2009. Properties of HflX, an enigmatic protein from Escherichia coli. J.
11. Engels, S., et al. 2005. The transcriptional activator ClgR controls transcrip-
tion of genes involved in proteolysis and DNA repair in Corynebacterium
glutamicum. Mol. Microbiol. 57:576–591.
12. Ferrage, F., M. Zoonens, D. E. Warschawski, J. L. Popot, and G. Boden-
hausen. 2003. Slow diffusion of macromolecular assemblies by a new pulsed
field gradient NMR method. J. Am. Chem. Soc. 125:2541–2545.
13. Garcia de la Torre, J., M. L. Huertas, and B. Carrasco. 2000. HYDRONMR:
prediction of NMR relaxation of globular proteins from atomic-level structures
and hydrodynamic calculations. J. Magn. Reson. 147:138–146.
14. Gradia, D. F., et al. 2009. Characterization of a novel Obg-like ATPase in the
protozoan Trypanosoma cruzi. Int. J. Parasitol. 39:49–58.
15. Hedges, J., M. West, and A. W. Johnson. 2005. Release of the export adapter,
Nmd3p, from the 60S ribosomal subunit requires Rpl10p and the cytoplas-
mic GTPase Lsg1p. EMBO J. 24:567–579.
16. Himeno, H., et al. 2004. A novel GTPase activated by the small subunit of
ribosome. Nucleic Acids Res. 32:5303–5309.
17. Huang, B., et al. 2010. Functional study on GTP hydrolysis by the GTP-
binding protein from Sulfolobus solfataricus, a member of the HflX family.
J. Biochem. 148:103–113.
18. Jain, N., et al. 2009. E. coli HflX interacts with 50S ribosomal subunits in
presence of nucleotides. Biochem. Biophys. Res. Commun. 379:201–205.
19. Karbstein, K. 2007. Role of GTPases in ribosome assembly. Biopolymers
20. Kimura, T., et al. 2008. Ribosome-small-subunit-dependent GTPase inter-
acts with tRNA-binding sites on the ribosome. J. Mol. Biol. 381:467–477.
21. Kimura, T., et al. 2007. Interaction between RsgA and the ribosome. Nucleic
Acids Symp. Ser. (Oxf.) 2007(51):375–376.
22. Kukimoto-Niino, M., et al. 2004. Crystal structure of the GTP-binding pro-
tein Obg from Thermus thermophilus HB8. J. Mol. Biol. 337:761–770.
23. Leipe, D. D., Y. I. Wolf, E. V. Koonin, and L. Aravind. 2002. Classification
and evolution of P-loop GTPases and related ATPases. J. Mol. Biol. 317:
24. Lin, B., D. A. Thayer, and J. R. Maddock. 2004. The Caulobacter crescentus
CgtAC protein cosediments with the free 50S ribosomal subunit. J. Bacteriol.
25. Londei, P., A. Teichner, P. Cammarano, M. De Rosa, and A. Gambacorta.
1983. Particle weights and protein composition of the ribosomal subunits of
the extremely thermoacidophilic archaebacterium Caldariella acidophila.
Biochem. J. 209:461–470.
26. Matsuo, Y., et al. 2006. The GTP-binding protein YlqF participates in the
late step of 50 S ribosomal subunit assembly in Bacillus subtilis. J. Biol.
27. Matsuo, Y., T. Oshima, P. C. Loh, T. Morimoto, and N. Ogasawara. 2007.
Isolation and characterization of a dominant negative mutant of Bacillus
subtilis GTP-binding protein, YlqF, essential for biogenesis and mainte-
nance of the 50 S ribosomal subunit. J. Biol. Chem. 282:25270–25277.
28. Morimoto, T., et al. 2002. Six GTP-binding proteins of the Era/Obg family
are essential for cell growth in Bacillus subtilis. Microbiology 148:3539–3552.
29. Polkinghorne, A., et al. 2008. Chlamydophila pneumoniae HflX belongs to
an uncharacterized family of conserved GTPases and associates with the
Escherichia coli 50S large ribosomal subunit. Microbiology 154:3537–3546.
30. Ruggero, D., A. Ciammaruconi, and P. Londei. 1998. The chaperonin of the
archaeon Sulfolobus solfataricus is an RNA-binding protein that participates
in ribosomal RNA processing. EMBO J. 17:3471–3477.
31. Schanda, P., E. Kupce, and B. Brutscher. 2005. SOFAST-HMQC experi-
ments for recording two-dimensional heteronuclear correlation spectra of
proteins within a few seconds. J. Biomol. NMR 33:199–211.
32. Scoarughi, G. L., C. Cimmino, and P. Donini. 1995. Lack of production of
(p)ppGpp in Halobacterium volcanii under conditions that are effective in
the eubacteria. J. Bacteriol. 177:82–85.
33. Sharma, M. R., et al. 2005. Interaction of Era with the 30S ribosomal subunit
implications for 30S subunit assembly. Mol. Cell 18:319–329.
34. Shields, M. J., J. J. Fischer, and H. J. Wieden. 2009. Toward understanding
the function of the universally conserved GTPase HflX from Escherichia
coli: a kinetic approach. Biochemistry 48:10793–10802.
35. Sklenar, V., and A. Bax. 1987. Spin-echo water suppression for the generation of
pure-phase two-dimensional NMR-spectra. J. Magn. Reson. 74:469–479.
36. Teplyakov, A., et al. 2003. Crystal structure of the YchF protein reveals
binding sites for GTP and nucleic acid. J. Bacteriol. 185:4031–4037.
37. Tu, C., et al. 2009. Structure of ERA in complex with the 3? end of 16S
rRNA: implications for ribosome biogenesis. Proc. Natl. Acad. Sci. U. S. A.
38. Uicker, W. C., L. Schaefer, and R. A. Britton. 2006. The essential GTPase
RbgA (YlqF) is required for 50S ribosome assembly in Bacillus subtilis. Mol.
39. Wout, P., et al. 2004. The Escherichia coli GTPase CgtAE cofractionates
with the 50S ribosomal subunit and interacts with SpoT, a ppGpp synthetase/
hydrolase. J. Bacteriol. 186:5249–5257.
40. Wu, H., et al. 2010. Structure of the ribosome associating GTPase HflX.
41. Zaparty, M., et al. 2010. “Hot standards” for the thermoacidophilic archaeon
Sulfolobus solfataricus. Extremophiles 14:119–142.
42. Zhang, S., and W. G. Haldenwang. 2004. Guanine nucleotides stabilize the
binding of Bacillus subtilis Obg to ribosomes. Biochem. Biophys. Res. Com-
VOL. 193, 2011S. SOLFATARICUS HflX BINDING TO 50S RIBOSOMAL SUBUNIT 2867