Nonbridging phosphate oxygens in 16S rRNA important for 30S subunit assembly and association with the 50S ribosomal subunit.

Abstract

Ribosomes are composed of RNA and protein molecules that associate together to form a supramolecular machine responsible for protein biosynthesis. Detailed information about the structure of the ribosome has come from the recent X-ray crystal structures of the ribosome and the ribosomal subunits. However, the molecular interactions between the rRNAs and the r-proteins that occur during the intermediate steps of ribosome assembly are poorly understood. Here we describe a modification-interference approach to identify nonbridging phosphate oxygens within 16S rRNA that are important for the in vitro assembly of the Escherichia coli 30S small ribosomal subunit and for its association with the 50S large ribosomal subunit. The 30S small subunit was reconstituted from phosphorothioate-substituted 16S rRNA and small subunit proteins. Active 30S subunits were selected by their ability to bind to the 50S large subunit and form 70S ribosomes. Analysis of the selected population shows that phosphate oxygens at specific positions in the 16S rRNA are important for either subunit assembly or for binding to the 50S subunit. The X-ray crystallographic structures of the 30S subunit suggest that some of these phosphate oxygens participate in r-protein binding, coordination of metal ions, or for the formation of intersubunit bridges in the mature 30S subunit. Interestingly, however, several of the phosphate oxygens identified in this study do not participate in any interaction in the mature 30S subunit, suggesting that they play a role in the early steps of the 30S subunit assembly.

Full text

Page 1

Nonbridging phosphate oxygens in 16S rRNA important
for 30S subunit assembly and association with the 50S
ribosomal subunit
SRIKANTA GHOSH and SIMPSON JOSEPH
Department of Chemistry and Biochemistry, University of California, San Diego, La Jolla, California 92093-0314, USA
ABSTRACT
Ribosomes are composed of RNA and protein molecules that associate together to form a supramolecular machine responsible
for protein biosynthesis. Detailed information about the structure of the ribosome has come from the recent X-ray crystal
structures of the ribosome and the ribosomal subunits. However, the molecular interactions between the rRNAs and the
r-proteins that occur during the intermediate steps of ribosome assembly are poorly understood. Here we describe a modifi-
cation-interference approach to identify nonbridging phosphate oxygens within 16S rRNA that are important for the in vitro
assembly of the Escherichia coli 30S small ribosomal subunit and for its association with the 50S large ribosomal subunit. The
30S small subunit was reconstituted from phosphorothioate-substituted 16S rRNA and small subunit proteins. Active 30S
subunits were selected by their ability to bind to the 50S large subunit and form 70S ribosomes. Analysis of the selected
population shows that phosphate oxygens at specific positions in the 16S rRNA are important for either subunit assembly or for
binding to the 50S subunit. The X-ray crystallographic structures of the 30S subunit suggest that some of these phosphate
oxygens participate in r-protein binding, coordination of metal ions, or for the formation of intersubunit bridges in the mature
30S subunit. Interestingly, however, several of the phosphate oxygens identified in this study do not participate in any inter-
action in the mature 30S subunit, suggesting that they play a role in the early steps of the 30S subunit assembly.
Keywords: 16S rRNA; 30S subunit; assembly; phosphorothioate; ribosome
INTRODUCTION
The ribosome is a macromolecular complex made up of two
unequally sized subunits. In Escherichia coli, the 50S large
subunit consists of a 23S rRNA molecule (2904 nt), a 5S
rRNA molecule (120 nt), and more than 30 proteins. The
30S small subunit consists of a 16S rRNA (1542 nt) and 21
proteins. The X-ray crystal structures of the ribosomal sub-
units show that the rRNAs, which form the core of the
subunits, are stabilized by RNA–RNA interactions in addi-
tion to RNA–protein interactions (Ban et al. 2000; Schlu-
enzen et al. 2000; Wimberly et al. 2000). Specific bases,
ribose 2?-hydroxyl groups, and nonbridging phosphate oxy-
gens within the rRNAs form an array of intra- and inter-
molecular interactions that stabilize the structure of the
fully assembled ribosome. However, the molecular mecha-
nism of how the rRNAs associate with the ribosomal pro-
teins to form a supramolecular assembly is poorly under-
stood. In vivo, ribosome assembly occurs cotranscription-
ally and is orchestrated by assembly factors (Maki et al.
2002, 2003; Alix and Nierhaus 2003; Charollais et al. 2003).
Remarkably, both the 30S (Traub and Nomura 1968) and
the 50S subunits (Nierhaus and Dohme 1974) assemble in
vitro from their constituent rRNAs and ribosomal proteins
in the absence of assembly factors. These in vitro reconsti-
tuted 30S and 50S subunits are active in translation.
Ribosome assembly is a dynamic process involving the
interaction of more than 50 proteins with three large RNA
molecules that must ultimately fold into a compact particle
(for review, see Williamson 2003). Early assembly mapping
experiments of the 30S subunit from E. coli showed that this
complex process involves an ordered pathway with the co-
operative interaction of small subunit proteins with the 16S
rRNA (Held et al. 1974). Based on assembling mapping
Reprint requests to: Simpson Joseph, 4102 Urey Hall, Department of
Chemistry and Biochemistry, University of California, San Diego, 9500
Gilman Drive, La Jolla, CA 92093-0314, USA; e-mail: sjoseph@chem.
ucsd.edu; fax: (858) 534-7042.
Article published online ahead of print. Article and publication date are
at http://www.rnajournal.org/cgi/doi/10.1261/rna.7224305.
RNA (2005), 11:657–667. Published by Cold Spring Harbor Laboratory Press. Copyright © 2005 RNA Society.
657

Page 2

experiments, the ribosomal proteins are classified as pri-
mary, secondary, and tertiary binding proteins. Primary
binding proteins bind directly to 16S rRNA in vitro, al-
though binding may be cooperative in vivo. Secondary
binding proteins interact with 16S rRNA only after one or
more primary binding proteins have associated with the 16S
rRNA to form a ribonucleoprotein particle (RNP). Tertiary
binding proteins require prior binding of at least one sec-
ondary binding protein before they can associate with the
16S rRNA. Interestingly, the three major domains in 16S
rRNA can assemble independently to form ribonucleopro-
tein particles (Weitzmann et al. 1993; Samaha et al. 1994;
Agalarov et al. 1998). Kinetic analysis of some of these RNPs
has revealed the dynamics of 30S assembly (Recht and
Williamson 2001). More recently, the assembly of the 30S
subunit was examined using molecular mechanics (Stagg et
al. 2003). These studies revealed that the primary binding
proteins bind to more ordered sites in the 16S rRNA and
help to organize the binding sites for proteins that bind later
in the assembly pathway. However, very little is known
about the contribution of various functional groups in 16S
rRNA in the 30S assembly pathway. Chemical modification
of the bases in 16S rRNA inhibits in vitro assembly of the
30S subunit (Nomura et al. 1968; Asai et al. 1999) and
subunit association (Herr et al. 1979), indicating that they
play a role in 30S assembly and subunit association. Simi-
larly, the accessibility of the 16S rRNA backbone to ethyl-
nitrosourea (ENU) in free 16S rRNA, in the 30S subunit,
and in the 70S ribosome showed a distinct protection pat-
tern consistent with the idea that functional groups in the
RNA backbone are also important for assembly (Baudin et
al. 1989). Importantly, heterologous 16S rRNA can be re-
constituted in vivo with ribosomal proteins from different
species (Nomura et al. 1968; Asai et al. 1999). This indicates
that, in addition to the universally conserved bases in 16S
rRNA, backbone interactions may contribute to the binding
of small subunit proteins and for subunit association.
Previously, the accessibility of Rp-phosphate oxygens in
domain I of 23S rRNA within the 50S subunit was mapped
using phosphorothioate-substituted Thermus aquaticus 23S
rRNA transcripts (Maivali et al. 2002). Several phosphate
oxygens were protected within the 50S subunit, indicating
shielding by the ribosomal large subunit proteins. However,
they were unable to identify any phosphate oxygens in do-
main I of 23S rRNA that are important for 50S assembly. In
this study, we have used a similar modification-interference
approach to identify nonbridging phosphate oxygens within
the E. coli 16S rRNA backbone that are important for the in
vitro assembly of the 30S subunit and for association with
the 50S large subunit. First, we show that phosphorothio-
ate-substituted 16S rRNA reconstitutes in vitro to form 30S
subunits. Second, these 30S subunits associate with native
50S subunits to form 70S ribosomes. Analysis of the 70S
ribosome reveals that several nonbridging phosphate oxy-
gens within the 16S rRNA participate either in the assembly
of the 30S subunit or for subunit association or may be
involved in both events. Finally, X-ray crystal structures of
the 30S subunit and the 70S ribosome were used to deduce
possible roles for these nonbridging phosphate oxygens in
16S rRNA for 30S assembly and for subunit association.
RESULTS
Reconstitution of 30S subunit with
phosphorothioate-modified 16S rRNA
The reconstitution procedure of Ofengand and coworkers
was used to assemble the 30S ribosomal subunit from pu-
rified total proteins of the 30S subunit (TP30) and in vitro
transcribed 16S rRNA (Krzyzosiak et al. 1987). Incorpora-
tion of Rp-phosphorothioate substitutions in 16S rRNA
transcripts was accomplished during in vitro T7 RNA poly-
merase transcription by adding a low level of a single (?-S)
phosphorothioate nucleotide triphosphate. In order to re-
duce the possibility of thiophilic metal ion contamination
that may interfere with our final analysis, diethyl dithiocar-
bamate (DTC) was included in the reconstitution buffers.
DTC chelates and precipitates thiophilic metal ions but with
very little affinity for Mg2+(Derrick et al. 2000). Control
experiments showed that the presence of DTC in the re-
constitution buffers have no adverse effects on 30S assem-
bly. The reconstituted 30S subunits were purified on su-
crose density gradients (Fig. 1). Fractionation of the gradi-
ents and absorbance measurement at 254 nm show peaks
corresponding to the free 16S rRNA and the reconstituted
30S subunit (Fig. 1B). Since the free 16S rRNA migrates
close to the 30S subunit, it can potentially contaminate the
30S fraction. Therefore, after the 30S reconstitution proce-
dure, a cDNA primer complementary to 906–923 of 16S
rRNA was added to the reaction mixture and treated with
RNase H to cleave the free 16S rRNA into two fragments of
∼620 nt (3?-fragment) and 906 nt (5?-fragment). The
cleaved 16S rRNA fragments sedimented as two distinct
peaks away from the 30S subunits, thereby minimizing the
possibility of free 16S rRNAs contaminating the 30S subunit
(Fig. 1C). Control experiments showed that the 16S rRNA
in the 30S subunit is not cleaved by RNase H. In order to
further improve the separation between the free 16S rRNA
and the 30S subunits, the concentration of MgCl2in the
sucrose gradient was lowered from 15 mM to 7.5 mM (Fig.
1D). These modifications resulted in better separation be-
tween the free 16S rRNA and the 30S subunit.
Subunit association experiments were performed to
verify that the 30S peak represents authentic reconstituted
30S subunits. The 30S subunits reconstituted with phos-
phorothioate-modified 16S rRNA associate with the native
50S large subunits to form 70S ribosomes (Fig. 1I). Fur-
thermore, these 70S ribosomes are active in tRNA binding
(Table 1). The protein composition of the reconstituted
30S subunit was analyzed by two-dimensional gel electro-
Ghosh and Joseph
658
RNA, Vol. 11, No. 5

Page 3

phoresis. Reconstituted 30S subunit contains the same
complement of small subunit ribosomal proteins as the na-
tive 30S subunit (Fig. 2), indicating that ribosomal proteins
associate with phosphorothioate-substituted 16S rRNA to
form the 30S subunit. Next, the 16S rRNA extracted from
the reconstituted 30S subunits were analyzed for the pres-
ence of phosphorothioate by iodine cleavage followed by
primer extension. Primer extension analysis showed reverse
transcriptase stops corresponding to the cleavage pattern
expected from the incorporation of a specific phosphoro-
thioate-substituted nucleotide in 16S rRNA (data not
shown). These results demonstrate that 30S subunits can be
reconstituted with phosphorothioated 16S rRNA.
Nonbridging phosphate oxygens in 16S rRNA that are
important for 30S reconstitution
In order to identify nonbridging phosphate oxygens in 16S
rRNA that are important for 30S reconstitution, the level of
phosphorothioate substitution in the 16S rRNA transcripts
before reconstitution (total population) was compared to
the level in 16S rRNA extracted from reconstituted 30S
subunits (selected population). Sites of phosphorothioate
substitution in 16S rRNA can be determined at nucleotide
resolution by iodine cleavage and primer extension analysis.
Phosphorothioate substitution at positions in 16S rRNA
that are important for 30S reconstitution will be underrep-
resented in the selected 30S subunit and result in lower-
intensity reverse transcriptase stop bands relative to the to-
tal population. Surprisingly, primer extension analysis
showed no significant difference in band intensity between
the selected and the total population. This suggests that
even though attempts were made to remove free 16S rRNA
from the 30S fraction, the problem persists and trace
amounts of partially assembled 30S subunits are present as
contaminants in the 30S fraction, making it difficult to di-
rectly identify sites that are important for 30S reconstitu-
tion. To confirm this hypothesis, we treated 16S rRNA tran-
scripts, native 30S subunits, and reconstituted 30S subunits
that were purified by sucrose gradient centrifugation with
base-specific chemical probes and compared the protection
pattern by primer extension analysis (Fig. 3). Chemical
probing reveals the higher-order structure of the 16S rRNA
in the 30S subunit (Moazed et al. 1986). Chemical probing
showed that the protection pattern of 16S rRNA in the
reconstituted 30S subunit is similar to that in the native 30S
subunit; however, the level of protection is incomplete. Es-
pecially, regions in the 16S rRNA that interact with the
tertiary binding proteins show incomplete protection com-
pared to regions that bind the primary and secondary bind-
ing proteins (Powers et al. 1988; Stern et al. 1988a,c; Svens-
son et al. 1988). This further indicates that the reconstituted
30S subunits purified by a single sucrose gradient centrifu-
gation are a mixture of fully and partially assembled 30S
particles.
Several approaches were tried to further purify fully re-
constituted 30S subunits. First, we collected reconstituted
30S subunits from only the late part of the sucrose gradients
FIGURE 1. Sucrose gradient profile of in vitro reconstituted 30S sub-
units. (A) Separation of phosphorothioate-substituted 16S rRNA tran-
scripts and native 30S subunits as control markers on sucrose gradient.
(B) Separation of 30S subunits reconstituted using 16S rRNA tran-
scripts substituted randomly with adenosine phosphorothioate at
2.5% incorporation level. (C) Same as B but treated with RNase H to
cleave free 16S rRNAs. (D) Same as C but separated on sucrose gra-
dient containing 7.5 mM MgCl2. (E–H) Separation of native 30S sub-
units, 50S subunits, 70S ribosomes, and 30S subunits reconstituted in
vitro with phosphorothioated 16S rRNA, respectively, as a control
marker on sucrose gradients. (I) Association of in vitro reconstituted
30S subunits with native 50S subunits to form 70S ribosomes. The 30S
subunits were reconstituted using phosphorothioated 16S rRNA.
(16S) 16S rRNA; (30S) 30S small ribosomal subunit; (5?F) 16S rRNA
fragment corresponding from 1–906 (906 nucleotides); (3?F) 16S
rRNA fragment corresponding from 923–1542 (620 nucleotides);
(50S) 50S large ribosomal subunit; (70S) 70S ribosome. The shaded
areas indicate the positions of 30S, 50S, and 70S on the sucrose gra-
dients. The Y-axis is absorbance at 254 nm and the X-axis is fractions
from the top to the bottom of the sucrose gradient. A–D are 10%–30%
(w/v) linear sucrose gradients, and E–I are 10%–40% (w/v) linear
sucrose gradients. All gradients contain 15 mM MgCl2, except D as
indicated.
TABLE 1. tRNA-binding activity
Particle
Percent
activity
Native 70S
Reconstituted 70S (16S rRNA transcript)
Reconstituted 70S (phosphorothioated 16S
rRNA transcript)
100
23
24
100% activity of native 70S corresponds to 0.8 pmol of tRNA
bound per pmol of 70S.
Phosphate oxygens important for ribosome assembly
www.rnajournal.org
659

Page 4

(away from the 16S rRNA fractions) and repurified the 30S
subunits on a second sucrose gradient. A second approach
that we tried was to use tRNA binding to select the fully
reconstituted 30S subunits. Reconstituted 30S subunits
were purified on sucrose gradients, programmed with the
T4 gene32 mRNA fragment, and allowed to bind 3?-bio-
tinylated tRNAVal1in the P-site. The complex was irradiated
with UV light to form a site-specific cross-link between the
tRNAVal1and 16S rRNA in the 30S subunit (Ofengand et al.
1979; Prince et al. 1982). The 16S rRNA cross-linked to
3?-biotin-tRNAVal1was extracted and purified by binding to
magnetic streptavidin beads (von Ahsen and Noller 1995).
The third approach that we tried was to purify reconstituted
30S particles on sucrose gradients followed by purification
on a 3.5% native polyacrylamide gel (Dahlberg et al. 1969).
The band corresponding to the 30S subunit was excised and
the 16S rRNA was extracted by passive elution. The 16S
rRNA was cleaved with iodine and analyzed by primer ex-
tension analysis. However, no difference in band intensity
was observed between the selected and total population,
indicating that it is difficult to directly select fully assembled
30S subunits using these approaches.
To overcome this problem, we indirectly selected fully
assembled 30S subunits by their ability to bind 50S subunits
and form 70S ribosomes. More importantly, the 70S ribo-
somes are well separated from the free 16S rRNA and the
heterogeneous 30S peak in a sucrose gradient (Fig. 1I). The
30S subunits reconstituted with phosphorothioate-modi-
fied 16S rRNA were mixed with native 50S subunits to form
70S ribosomes. The 70S ribosomes were purified on sucrose
gradients, and the level of phosphorothioate substitution at
each position in 16S rRNA (selected population) was com-
pared to the level in the 16S rRNA transcript before recon-
stitution (total population). A decreased level of phospho-
rothioate modification was observed at several positions
within 16S rRNA that was isolated from the 70S ribosome
(Fig. 4). At positions U37, A1171, G1270, and G1272 the
effects were subtle but consistently observed in independent
experiments (data not shown).
The 5?-domain
The 5?-domain of 16S rRNA constitutes the body of the 30S
subunit (helix 1–18) (Fig. 5). Small subunit proteins S4, S5,
S12, S16, S17, and S20 bind to this region of the 16S rRNA
(Mizushima and Nomura 1970; Held et al. 1973; Brodersen
et al. 2002). In the 5?-domain of 16S rRNA, protection was
observed at Rp nonbridging phosphate oxygen 5? to U37,
G76, C95, C110, G198, G213, G220, U224, G230, G232,
A270, and C308. Position U37 is located at the junction of
five helices (3, 4, 16–18). Positions G76 and C95 in helix 6
form a feature called the “spur” that is solvent exposed in
the mature 30S subunit.
The central domain
The central domain of 16S rRNA constitutes the platform of
the 30S subunit (helix 19–27). Protection was observed at
positions G606, U619, A622, G626, G627, C739, C754,
C770, and G838. Small subunit proteins S6, S8, S11, S15,
S18, and S21 bind to the central domain of 16S rRNA
(Mizushima and Nomura 1970; Held et al. 1973; Brodersen
et al. 2002). In addition, the central domain forms several
intersubunit bridges with the 50S large ribosomal subunit
(Yusupov et al. 2001; Gao et al. 2003). The phosphate oxy-
gen at C770 in helix 24 of 16S rRNA is a part of the inter-
subunit bridge B2c and may play a direct role in subunit
association (Yusupov et al. 2001; Gao et al. 2003).
FIGURE 2. Analysis of 30S subunit proteins by two-dimensional gel
electrophoresis. (A) Protein composition of native 30S subunits. (B)
Protein composition of 30S subunits reconstituted in vitro using un-
modified 16S rRNA transcripts. (C) Protein composition of 30S sub-
units reconstituted in vitro using phosphorothioate-modified 16S
rRNA transcripts. Labels S1–S20 correspond to the 30S small subunit
proteins.
Ghosh and Joseph
660
RNA, Vol. 11, No. 5

Page 5

The 3?-domain
In the 3? major domain of 16S rRNA (helix 28–45), Rp
phosphate oxygens at positions A1152, G1164, A1171,
G1175, G1270, A1271, and G1272 are important for 30S
assembly and subunit association (Fig. 5). Proteins S2, S3,
S7, S9, S10, S13, S14, and S19 interact with the 3? major
domain of 16S rRNA to form the “head” of the 30S subunit
(Mizushima and Nomura 1970; Held et al. 1973; Brodersen
et al. 2002). Surprisingly, we do not observe any protections
in helices 44 and 45, which make up the 3? minor domain
of 16S rRNA. Helices 44 and 45 are lo-
cated at the subunit interface and form
four intersubunit bridges (B2a, B3, B5,
and B6) with the 50S large ribosomal
subunit (Yusupov et al. 2001; Gao et al.
2003). These intersubunit bridges, how-
ever, may not involve contacts with the
phosphate oxygens in helix 44 of 16S
rRNA. Ribosomal protein S20 contacts
the bottom tip of helix 44, but these
interactions may not be essential for 30S
assembly and subunit
(Brodersen et al. 2002).
association
DISCUSSION
The 30S ribosomal subunit assembles in
vitro using 16S rRNA from one species
and the ribosomal proteins from a dif-
ferent species (Nomura et al. 1968).
Even more remarkable is the fact that
heterologous 16S rRNA can be reconsti-
tuted in vivo with ribosomal proteins
from a species that diverged more than
350 million years ago (Niebel et al. 1987;
Asai et al. 1999). Thus, sequence-inde-
pendent contacts with the 16S rRNA
backbone are likely to dominate recon-
stitution and subunit association. Con-
sistent with these results, the 16S rRNA
backbone is shielded from chemical
probes in 30S subunits and 70S ribo-
somes (Baudin et al. 1989; Merryman et
al. 1999). X-Ray crystal structures of the
30S subunit show that the backbone of
16S rRNA forms numerous intramo-
lecular and intermolecular interactions
with the small subunit ribosomal pro-
teins (Brodersen et al. 2002). It is, how-
ever, difficult to determine the relative
importance of specific backbone con-
tacts for 30S subunit assembly and sub-
unit association from the crystal struc-
tures. Furthermore, it is possible that
either additional or even different contacts are required
during the intermediate states of 30S assembly that are no
longer present in the fully assembled 30S subunit. Indeed,
the 16S rRNA undergoes significant conformational
changes during assembly into the 30S subunit (Hochkeppel
and Craven 1977; Moazed et al. 1986; Powers et al. 1993;
Holmes and Culver 2004).
Previously, Nierhaus and coworkers identified nonbridg-
ing phosphate oxygens within 5S rRNA that are important
for incorporation into the 50S large subunit (Shpanchenko
et al. 1998). More recently, the bases in 23S rRNA that are
FIGURE 3. Chemical probing of reconstituted 30S subunit. Higher-order structure of 16S
rRNA transcripts (16), native 30S subunits (30), and reconstituted 30S subunits containing
phosphorothioate-substituted 16S rRNA (30*) were probed using kethoxal. (Lanes A,G)
Dideoxy sequencing lanes; (−) indicates no treatment with kethoxal; and (+) indicates treat-
ment with kethoxal. The filled arrowheads indicate positions in 16S rRNA protected by the
ribosomal proteins in native and reconstituted 30S subunits relative to 16S rRNA transcript.
Shown are protection by primary binding proteins (S4, S8, S17, and S20), secondary binding
proteins (S6, S11, S12, S16, and S18), and tertiary binding proteins (S2 and S21) (Powers et al.
1988; Stern et al. 1988a,c; Svensson et al. 1988). Sites of enhanced reactivity observed in native
and reconstituted 30S subunits relative to 16S rRNA transcripts are indicated by the unfilled
arrowheads.
Phosphate oxygens important for ribosome assembly
www.rnajournal.org
661