JOURNAL OF BACTERIOLOGY, Oct. 2006, p. 6757–6770
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Vol. 188, No. 19
The Escherichia coli GTPase CgtAEIs Involved in Late Steps
of Large Ribosome Assembly†
Mengxi Jiang,1Kaustuv Datta,1Angela Walker,2John Strahler,2Pia Bagamasbad,1
Philip C. Andrews,2and Janine R. Maddock1*
Department of Molecular, Cellular and Developmental Biology1and Department of
Biological Chemistry,2University of Michigan, Ann Arbor, Michigan 48109
Received 30 March 2006/Accepted 31 May 2006
The bacterial ribosome is an extremely complicated macromolecular complex the in vivo biogenesis of which
is poorly understood. Although several bona fide assembly factors have been identified, their precise functions
and temporal relationships are not clearly defined. Here we describe the involvement of an Escherichia coli
GTPase, CgtAE, in late steps of large ribosomal subunit biogenesis. CgtAEbelongs to the Obg/CgtA GTPase
subfamily, whose highly conserved members are predominantly involved in ribosome function. Mutations in
CgtAEcause both polysome and rRNA processing defects; small- and large-subunit precursor rRNAs accu-
mulate in a cgtAEmutant. In this study we apply a new semiquantitative proteomic approach to show that
CgtAEis required for optimal incorporation of certain late-assembly ribosomal proteins into the large ribo-
somal subunit. Moreover, we demonstrate the interaction with the 50S ribosomal subunits of specific nonri-
bosomal proteins (including heretofore uncharacterized proteins) and define possible temporal relationships
between these proteins and CgtAE. We also show that purified CgtAEassociates with purified ribosomal
particles in the GTP-bound form. Finally, CgtAEcofractionates with the mature 50S but not with intermediate
particles accumulated in other large ribosome assembly mutants.
Although assembly of prokaryotic ribosomes from purified
ribosomal proteins (r-proteins) and rRNAs can occur indepen-
dently in vitro (51, 52, 75), accumulating evidence suggests
that, as in eukaryotes, in vivo prokaryotic ribosome biogenesis
depends on the aid of nonribosomal factors. The higher tem-
perature, increased Mg2?concentration, and longer incuba-
tion times necessary for in vitro relative to in vivo conditions
(51) imply that the likely role of accessory factors is to expedite
ribosome maturation by reducing the activation energies for
the rate-limiting reactions. Although not complicated by the
involvement of different cellular compartments, the prokary-
otic ribosome assembly process is far from simple, requiring
coordinated synthesis of 3 rRNAs (5S, 16S, and 23S) and 55
r-proteins, processing and modification of these components,
and their appropriate sequential unification to produce mature
ribosomes. The details of how this process is controlled tem-
porally, even spatially, in the small bacterial cell are incom-
More than 170 nonribosomal proteins that transiently asso-
ciate with different preribosomal particles have been identified
in Saccharomyces cerevisiae (19, 22, 38, 62), largely due to
progress in combining biochemical affinity purification meth-
ods with newly developed proteomic techniques (24, 25, 29, 54,
58, 61). By contrast, only a few such assembly factors have been
found in bacteria, and most were identified via conventional
genetic methods. These proteins consist of RNA-modifying
enzymes such as methyltransferases and pseudouridine syn-
thases, RNA-remodeling proteins such as RNA helicases,
chaperones, GTPases, and proteins with unknown functions (1,
5, 7, 10, 11, 18, 26, 32, 33, 48–50, 60, 72, 76). Understanding of
the molecular mechanisms by which these factors monitor and
influence the ribosomal assembly process and a comprehensive
picture of the interactions among these different constituents,
however, are still lacking.
Based on phylogenetic analysis, it is hypothesized that all
GTPases are derived from an ancestral GTPase with a role in
translation (39). The Obg subfamily is a class of highly con-
served small monomeric GTPases that appear to be involved
primarily in assembly of the large ribosomal subunit. In Sac-
charomyces cerevisiae, the mitochondrial Obg ortholog Mtg2p
is involved in the biogenesis of the large (54S) mitochondrial
ribosomal subunit (14), and the nucleolar Obg protein Nog1p
is important for pre-60S particle assembly (34, 62). In Bacillus
subtilis, Obg associates with the ribosome, and this association
can be stabilized by the addition of GTP (82). More specifi-
cally, Obg has been shown to bind ribosomal protein L13 by an
affinity blot assay (66). The Caulobacter crescentus Obg protein
CgtACcofractionates exclusively with the 50S ribosomal par-
ticle (42), and strains expressing a temperature-sensitive allele
of cgtAChad a reduced level of 50S subunits compared to the
wild type, even at the permissive temperature (15). Likewise,
Escherichia coli CgtAEassociates with the large ribosome sub-
unit (60, 80), interacts with rRNAs and several r-proteins, and
copurifies with the known 50S ribosome assembly factor CsdA
(60, 80). In a cgtAEmutant, the ribosome profile is perturbed
and a defect in 16S rRNA processing is observed (60). Fur-
thermore, CgtAEhas been genetically implicated in the assem-
bly of the 50S subunit based on its ability to suppress an rrmJ
mutant. RrmJ is an RNA methyltransferase that is involved in
late 50S ribosome assembly. The deletion of rrmJ causes slow
* Corresponding author. Mailing address: Department of Molecu-
lar, Cellular and Developmental Biology, University of Michigan, 830
North University, Ann Arbor, MI 48109-1048. Phone: (734) 936-8068.
Fax: (734) 647-0884. E-mail: firstname.lastname@example.org.
† Supplemental material for this article may be found at http://jb
growth and a polysome defect, both of which can be sup-
pressed by overexpression of CgtAE(72). All these data are
consistent with the role of Obg/CgtA proteins in ribosome
assembly and/or 70S coupling.
In this study we further characterize the association between
the ribosome and the E. coli CgtAEprotein and show that they
interact, with the GTP-bound form of CgtAEhaving a higher
affinity for the ribosome. Further, we demonstrate that CgtAE
is crucial for the late steps of 50S ribosome subunit assembly.
A mutant form of cgtAEdisplays an altered polysome profile
similar to those seen in late ribosome assembly mutants. A new
semiquantitative proteomic technique, iTRAQ (isotope tag for
relative and absolute quantitation), allows us to describe the
proteome of the accumulated 50S particles in the cgtAEmu-
tant. This analysis provides new insights into the precise func-
tion of CgtAEin ribosome assembly.
MATERIALS AND METHODS
Bacterial strains, culture conditions, and growth measurements. E. coli strains
used are listed in Table 1. E. coli cells were grown in Luria-Bertani (LB) broth
(10 g tryptone, 5 g yeast extract, 10 g NaCl per liter) or on LB agar plates (LB
plus 1.5% agar), containing antibiotics as required, at the indicated tempera-
tures. Culture growth was monitored by measuring the absorbance at 600 nm.
Antibiotics were used at the following concentrations: 100 ?g/ml ampicillin, 30
?g/ml kanamycin, 20 ?g/ml chloramphenicol, or 12 ?g/ml tetracycline.
Strains and plasmid construction. Full-length cgtAE?or the cgtAE(G80E
D85N) allele was amplified from MG1655 genomic DNA or pGK15 (35),
respectively, by PCR using primers YhbZ3-new (5?-GAGAATCATATGAAGT
TTGTTGATGAA-3?) and YhbZ-end-HindIII (5?-GATTAAGCTTTTATCAT
CAGTGATTAA-3?). The 1.17-kb PCR products were cloned into the pCR2.1-
TOPO TA cloning vector (Invitrogen Life Technologies), generating pMJ25 and
pJM3738, respectively. The cgtAEpromoter region was amplified using primers
YhbZ-prom-up (5?-GTCTGCAGGGTCGTGACCACACTCTG-3?) and YhbZ-
prom-down (5?-GCCATATGGTATTCCCTGCAAAGCGCATT-3?) and cloned
into the pCR2.1-TOPO vector. The resulting plasmid was designated pMJ27. To
place cgtAEalleles under the control of the cgtAEpromoter, the cgtAEalleles
were excised from pMJ25 or pJM3738 using NdeI and BamHI and were ligated
to pMJ27 digested with the same enzymes. The resulting plasmids, pMJ33 and
pMJ29, were digested with PstI and BamHI and were ligated to the low-copy-
number plasmid pGD103-bla, a derivative of pGD103 (16), digested with the
same enzymes. The subsequent constructs, pMJ31 and pMJ32, were verified by
sequencing and transformed into GN5002 and GN5003, respectively. The orig-
inal plasmids in these two strains, pGK14 and pGK15, were lost by growing the
transformants in LB supplemented with ampicillin and kanamycin and screening
for chloramphenicol sensitivity, thus generating JM3903 and JM3907 [strains
harboring cgtAE?and cgtAE(G80E D85N), respectively].
Preparation of cell lysates for ribosome profiles. E. coli strains were grown at
the following temperatures: 37°C for MG1655; 30°C for JM3903, JM3907,
JM4711, and JM4714; and 20°C for JM3733 and JM3734. Chloramphenicol
(FisherBiotech) was added to a final concentration of 200 ?g/ml 30 s before
harvest. Cells were harvested at an optical density at 600 nm (OD600) of 0.4 to
0.8 by centrifugation at 10,000 ? g and 4°C for 10 min in an SLA-1500 rotor
(Sorvall). The cell pellet was resuspended in 1 ml lysis buffer (10 mM Tris-Cl [pH
7.5], 10 mM MgCl2, 30 mM NH4Cl, 100 ?g/ml chloramphenicol) per 100 ml of
culture. The cell lysate was mixed with an equal volume of glass beads (300 ?m;
Sigma) and vortexed for 5 min at 4°C. The lysate was clarified by a 10-min
centrifugation at 32,000 ? g and 4°C in an SA-600 rotor (Sorvall). The super-
natant was carefully collected and quantified by UV absorbance at 260 nm.
Protein and ribosome purification. His-CgtAEwas purified from JM1138 as
previously described (80), with the following modification. After the nickel-
nitrilotriacetic acid column, the CgtAE-containing fractions were pooled, dia-
lyzed against core buffer (50 mM Tris [pH 8.0], 10% glycerol, 1 mM dithiothre-
itol), and loaded onto a 50-ml Toyopearl DEAE-650 M column (TosoHaas), and
CgtAEwas eluted with a 100-ml linear gradient of core buffer containing 0 to 400
mM NaCl. The relevant fractions were then purified over a 100-ml Sephadex
G-75 column as previously described (40). Finally, similarly dialyzed CgtAE
fractions were purified over a 1.3-ml UNO Q1 column (Bio-Rad) and eluted with
a 30-ml linear gradient of core buffer containing 0 to 1 M NaCl. The concen-
tration of purified CgtAEwas determined by a Bradford assay (Bio-Rad). Puri-
fied ribosomes were obtained as previously described (13).
Polyribosome fractionation. Sucrose gradients were formed using a gradient
maker (SG15 or SG50; Hoefer) under the indicated buffer conditions. Approx-
imately 13 OD260units of the cell lysates was loaded gently onto the top of the
10-ml 7 to 47% sucrose gradients, and the gradients were centrifuged in a
TABLE 1. Escherichia coli strains and plasmids used in this study
Strain or plasmidRelevant genotype Source or reference
?(lab)U169 ?80 ?(lacZ)M15 hsdR17 endA1 gyrA96 recA1 supE44 thi-1
WJW45 rna-30 ?csdA
MG1655 ?cgtAE::kan ? pGK14
MG1655 ?cgtAE::kan ? pGK15
MG1655 ?cgtAE::kan ? pMJ31
MG1655 ?cgtAE::kan ? pMJ32
JM3903 ? pJW3146
JM3907 ? pJW3146
ori-PUC kan bla
ori-pSC101 cat PBADcgtAE
ori-pSC101 cat PBADcgtAE(G80E D85N)
ori-PUC kan bla cgtAE
ori-PUC kan bla cgtAE(G80E D85N)
ori-PUC kan bla PCgtAE
ori-pUC kan bla PCgtAEcgtAE
ori-pUC kan bla PCgtAEcgtAE(G80E D85N)
ori-pSC101 bla PCgtAEcgtAE
ori-pSC101 bla PCgtAEcgtAE(G80E D85N)
6758JIANG ET AL.J. BACTERIOL.
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