Interdependencies govern multidomain architecture
in ribosomal small subunit assembly
DEEPIKA CALIDAS and GLORIA M. CULVER
Department of Biology, University of Rochester, Rochester, New York 14627, USA
The 30S subunit is composed of four structural domains, the body, platform, head, and penultimate/ultimate stems. The
functional integrity of the 30S subunit is dependent upon appropriate assembly and precise orientation of all four domains. We
examined 16S rRNA conformational changes during in vitro assembly using directed hydroxyl radical probing mediated by
Fe(II)-derivatized ribosomal protein (r-protein) S8. R-protein S8 binds the central domain of 16S rRNA directly and
independently and its iron derivatized substituents have been shown to mediate cleavage in three domains of 16S rRNA,
thus making it an ideal probe to monitor multidomain orientation during assembly. Cleavages in minimal ribonucleoprotein
(RNP) particles formed with Fe(II)-S8 and 16S rRNA alone were compared with that in the context of the fully assembled
subunit. The minimal binding site of S8 at helix 21 exists in a structure similar to that observed in the mature subunit, in the
absence of other r-proteins. However, the binding site of S8 at the junction of helices 25–26a, which is transcribed after helix
21, is cleaved with differing intensities in the presence and absence of other r-proteins. Also, assembly of the body helps
establish an architecture approximating, but perhaps not identical, to the 30S subunit at helix 12 and the 59 terminus. Moreover,
the assembly or orientation of the neck is dependent upon assembly of both the head and the body. Thus, a complex
interrelationship is observed between assembly events of independent domains and the incorporation of primary binding
proteins during 30S subunit formation.
Keywords: small subunit; ribosome assembly; directed hydroxyl radical probing; S8; RNA folding; RNA structure; r-protein
The small subunit of the ribosome decodes the mRNA
during translation with extremely high fidelity, hence the
formation of appropriate structure in the mature subunit is
crucial for cellular viability and fitness (Powers and Noller
1990; Kurland et al. 1996). The tertiary structure of the
small subunit is subdivided into four structural domains:
body, platform, head, and penultimate/ultimate stem, at
the intersection of which lies the decoding center of the
30S subunit (Moazed and Noller 1990; Lee et al. 1997;
Lodmell and Dahlberg 1997; Clemons et al. 1999; Merryman
et al. 1999; Yusupov et al. 2001; Yusupova et al. 2001). The
domains within the tertiary structure of the 30S subunit are
formed by the association of ribosomal proteins (r-proteins)
with corresponding domains of 16S rRNA within the sec-
ondary structure (59, central, 39 major, and 39 minor do-
mains) and this process can be carried out in vitro (Held
et al. 1973, 1974; Cunningham et al. 1991; Culver and Noller
2000b). The aim of this study is to examine domain assembly
and orientation during in vitro 30S subunit assembly using
directed hydroxyl radical probing from Fe(II)-derivatized
The hierarchical and cooperative principles underlying
in vitro assembly of the 30S subunit have been revealed
through a set of ground-breaking experiments whose results
have been graphically expressed in the form of an assembly
map (Fig. 1A; Mizushima and Nomura 1970; Held et al.
1974; Grondek and Culver 2004). Concisely, r-protein as-
sociation leads to conformational change or altered stability
of 16S rRNA tertiary structures, which in turn enhance the
association of other r-proteins and subsequent folding events
(Sykes and Williamson 2009). For example, r-protein S15 is
an early or primary binding protein that associates directly
and independently with the central domain, leading to the
incorporation of mid-binding or secondary r-proteins S6,
S18 and, finally, late binding or tertiary proteins S11 and
S21, which together form the platform (see Fig. 1A). Each
Reprint requests to: Gloria M. Culver, Department of Biology,
University of Rochester, Rochester, NY 14627, USA; e-mail: gculver@
mail.rochester.edu; fax: (585) 275-2070.
Article published online ahead of print. Article and publication date are
RNA (2011), 17:263–277. Published by Cold Spring Harbor Laboratory Press. Copyright ? 2011 RNA Society.
FIGURE 1. Structure of r-protein S8 and its interactions with the 30S subunit. (A) Modified in vitro assembly map of the 30S subunit,
illustrating domain organization (Mizushima and Nomura 1970; Held et al. 1974; Grondek and Culver 2004). Each domain within the secondary
structure of 16S rRNA is assigned a specific color, and this scheme is extended to the r-proteins that bind 16S rRNA to form corresponding
domains in the tertiary structure, delineated by boxes with the names of the domains written below. The arrows symbolize the direction of
dependence of r-protein binding with the width symbolizing the strength of the dependence. R-proteins can be subdivided into early, middle, and
late binding proteins contained in a dark gray, light gray, and white box, respectively. (B) Localization of S8 within the central domain of 16S
rRNA. Hydroxyl radical footprinting data for S8 (Powers and Noller 1995) are represented by black circles, while S8 crystal structure contacts are
represented by magenta circles. Concentric rings of color indicate sites of overlap. (C) Localization of S8 in the three-dimensional structure of the
E. coli 30S subunit. S8 is depicted in black and other r-proteins were omitted for clarity. Each domain of 16S rRNA is color coded as in A. (D,E)
Tethered probing sites on S8. The positions of cysteine substitutes (Lancaster et al. 2000) are indicated by number and distinctly colored. D is
oriented as in C, E depicting the molecule rotated by 180° along the y-axis. All figures containing three-dimensional structures were prepared
using the PyMOL and the Protein Data Bank file 2AW7 (Schuwirth et al. 2005).
RNA, Vol. 17, No. 2
domain exhibits similar patterns of dependencies (see
Fig. 1A) and can be assembled independently in vitro
(Weitzmann et al. 1993; Samaha et al. 1994; Agalarov
et al. 1998, 1999).
Although S8 does not aid in incorporation of r-proteins
that bind the central domain, the association of S8 has
profound effects on central domain structure (Jagannathan
and Culver 2003). Initially the minimal binding site of S8
was localized to the junction of helices 20, 21, and 22, by
a number of studies, including RNase protection assays
(Schaup et al. 1973; Ungewickell et al. 1975; Zimmermann
et al. 1975; Muller et al. 1979), chemical cross-linking
(Wower and Brimacombe 1983), and determination of
binding affinity (Mougel et al. 1993; Wu et al. 1994).
However, data from X-ray crystallography (see Fig. 1B;
Lancaster et al. 2000; Schuwirth et al. 2005) as well as
solution (see Fig. 1B; Powers and Noller 1995) and directed
hydroxyl radical probing (Lancaster et al. 2000) expanded
our understanding of S8 interaction with 16S rRNA to
include a second site at the junction of helices 25, 26, and
26a (see Fig. 1B). The existence of two separate sites led to
speculation that S8 plays a role in compacting the central
domain during 59–39 assembly in vivo (Lancaster et al.
2000). Moreover, within the tertiary structure of the small
subunit, S8 is proximal to the junction of body and
platform (see Fig. 1C). This, together with its role in the
incorporation of r-proteins S5 and S12 that bind near the
decoding center of the 30S subunit, has led to conjectures
that S8 may function in the appropriate orientation of
domains during assembly via direct or indirect interactions
with 16S rRNA and other r-proteins (Jagannathan and
Culver 2003). In light of the data presented above, S8 ap-
peared to be an excellent candidate r-protein for a directed
hydroxyl radical probing study of interactions during 30S
The technique of directed hydroxyl radical probing has
many applications, including the localization of binding
sites, the determination of orientation of a protein with
respect to an RNA or DNA molecule (Culver and Noller
2000a; Lancaster et al. 2000; Chen and Hahn 2003; Xu et al.
2008), the characterization of the RNA or DNA environ-
ment of the Fe(II)-derivatized protein, and monitoring
conformational changes or dynamics associated with the
incorporation of r-proteins with rRNA (Jagannathan and
Culver 2003, 2004; Dutca and Culver 2008). In fact,
a previous study had been carried out using Fe(II)-S8 to
localize its binding site in the 70S ribosome (Lancaster et al.
2000). These probing experiments revealed that Fe(II)-S8
could cleave elements within the head, body, and platform
of the 30S subunit, thereby suggesting that probing me-
diated by Fe(II)-S8 would allow assembly of these domains
to be monitored.
In order to examine the architecture of these domains
during assembly, iron derivatized S8 proteins were assem-
bled into ribonucleoprotein particles (RNPs) with 16S
rRNA in the presence and absence of various r-proteins
and subjected to directed hydroxyl radical probing. Cleav-
age patterns in various complexes were compared with
a view to determining a minimal RNP that recapitulated
cleavage patterns observed in the 30S subunit and 70S
ribosome. The association of the 30S subunit with the 50S
subunit in the ribosome did not change cleavage patterns
significantly as compared to free small subunits (data not
shown). However, the architecture(s) of binary complexes
of Fe(II)-S8 and 16S rRNA alone (Fe(II)-S8/16S) are
distinct from that of the Fe(II)-S8 containing 30S subunits,
as evidenced by differences in cleavage patterns. While the
minimal binding site of S8 at the junction of helices 20–22
was organized in the binary complex, the cleavage pattern
at the junction of helices 25-26a differed between the two
sets of complexes. Thus, there is a variation in r-protein–
rRNA interaction across the two S8 binding sites. Also,
in both the 59 and 39 major domains Fe(II)-S8 mediated
cleavages in 30S subunits were absent in binary complexes.
Restoration of cleavage patterns required the assembly of
entire domains, highlighting the significance of long-range
interactions in assembly, even between independent events
(Weitzmann et al. 1993; Samaha et al. 1994; Agalarov et al.
Comparison of Fe(II)-S8 directed cleavage data
from 30S subunits and 70S ribosomes
Previous work utilized S8 proteins substituted at nine dif-
ferent positions by cysteine residues (Fig. 1D,E) and de-
rivatized with iron (Fe(II)-C19-S8, Fe(II)-C28-S8, Fe(II)-
Fe(II)-C73-S8, Fe(II)-C86-S8, Fe(II)-C107-S8). These pro-
teins and a cysteine-less control were used to determine the
location of S8 within the 70S ribosome by directed hy-
droxyl radical probing (Lancaster et al. 2000). The present
study examined 30S subunit assembly utilizing proteins
derivatized at eight positions. The derivatized protein omit-
ted in the present study was Fe(II)-C46-S8 as it demon-
strated only a weak cleavage pattern at the junction of helices
25–26a in the ribosome that overlapped with that of Fe(II)-
C19-S8, Fe(II)-C61-S8, and Fe(II)-C67-S8 (Lancaster et al.
2000). Cleavage patterns in both the 30S subunits and 70S
ribosomes were examined to identify any possible changes
due to subunit association or changes in protocol from
previous work. The cleavage patterns of several derivatized
proteins in 70S ribosomes were consistent with previous
data (data not shown). Additionally, patterns of cleavage in
30S subunits and 70S ribosomes were extremely similar
throughout 16S rRNA (data not shown), corroborating pre-
vious unpublished data (L Lancaster, unpubl.). This sim-
ilarity in cleavage patterns also correlated with the estima-
tion of probe-target distances elucidated in the study by
Interdependencies govern 30S subunit architecture
Lancaster et al. (2000). This estimation in turn was de-
pendent upon earlier studies establishing these correlations
using a tRNA molecule as a helical ruler (Joseph et al.
1997). The data obtained in this study are therefore con-
sistent with X-ray crystal structures of the 30S subunit
(Schuwirth et al. 2005).
Examining 30S subunit assembly using S8 as a probe
Comparison of cleavage patterns between a binary complex
of Fe(II)-S8 bound to 16S rRNA alone (Fe(II)-C19-S8/16S
rRNA, Fe(II)-C28-S8/16S rRNA, Fe(II)-C54-S8/16S rRNA,
Fe(II)-C73-S8/16S rRNA, Fe(II)-C86-S8/16S rRNA, Fe(II)-
C107-S8/16S rRNA, Cysteine-less/16S rRNA) and Fe(II)-S8
in the mature subunit (Fe(II)-C19-S8/30S, Fe(II)-C28-S8/
30S, Fe(II)-C54-S8/30S, Fe(II)-C73-S8/30S, Fe(II)-C86-S8/
30S, Fe(II)-C107-S8/30S, Cysteine-less/30S) yielded infor-
mation on the architecture of three major domains, the 59,
central, and 39 major domains in the presence of S8 alone.
Only in helix 21 within the central domain were similar
patterns of cleavage between binary complexes and 30S
subunits observed (Fig. 2A–C). For the other S8 binding
site within the central domain as well as the 59 and 39 major
domain, differences in cleavage patterns between the two
complexes were revealed (Figs. 2, 3, 4, 5).
Previous studies have demonstrated that differences in
cleavage at specific sites between two Fe(II)-derivatized
complexes assembled and probed simultaneously can be in-
terpreted as indicating alternative conformations or dy-
namics of 16S rRNA in those complexes (Jagannathan and
Culver 2004; Dutca and Culver 2008). Therefore, Fe(II)-S8
containing RNPs of different complexities were probed to
dissect assembly events that occur subsequent to S8 asso-
ciation with 16S rRNA and to reprise patterns of Fe(II)-S8
cleavage observed in 30S subunits.
Assembly of the central domain
An interesting dichotomy becomes apparent upon com-
parison of cleavage patterns in binary complexes and 30S
subunits across the central domain of 16S rRNA. All the
iron derivatized S8 proteins cleave elements of the central
domain of 16S rRNA in 30S subunits. Helix 21 is cleaved
along its length on both faces with a similar extent and
intensity in both binary complexes and 30S subunits, thus
indicating that a mature structure is present in the binary
complex (Figs. 2A–C, 6, 7). To elaborate, nucleotides 590–
593 and 651–653 are cleaved by Fe(II)-C28-S8 (Fig. 2B,
lanes 3,4; Fig. 2C, lanes 3,4), Fe(II)-C54-S8 (Fig. 2A, lanes
5,6; Fig. 2C, lanes 5,6) and Fe(II)-C61-S8 (Fig. 2A, lanes
7,8), while nucleotides 601–605 and nucleotides 630–634
are cleaved by Fe(II)-C107-S8 (Fig. 2B, lanes 9,10; Fig. 2C,
lanes 11,12) and Fe(II)-C73-S8 (Fig. 2B, lanes 5,6; Fig. 2C,
lanes 7,8), respectively, in binary RNPs as in 30S subunits
and 70S ribosomes (Lancaster et al. 2000). Fe(II)-C107-S8
also cleaves between nucleotides 590–593 and 651–653 in
both binary complexes and 30S subunits. This is consistent
with previous enzymatic and mutational analyses that had
been carried out on the binding site of S8 at helix 21 (Stern
et al. 1989; Lancaster et al. 2000). There is an increased
presence of reverse transcriptase generated stops at nucle-
otides 603 and 604 in complexes containing cysteine-less
substituents of S8 (Fig. 2B, lane 2; Fig. 2C, lane 2);
consequently, only the distinct bands generated by Fe(II)-
C107-S8/16S rRNA and Fe(II)-C107-S8/30S directed cleav-
age (Fig. 2B, lanes 9,10; Fig. 2C, lanes 11,12) have been
classified as data. Fe(II)-C86-S8/16S rRNA generates a vari-
able cleavage pattern not observed in fully assembled par-
ticles at nucleotides 602–603 and 630–635 in half the in-
dependent probing experiments conducted (cf. Fig. 2B,
lane 7, Fig. 2C, lane 9, and Fig. 4C, lane 7). Since Lysine 86
has both hydrophobic and Van der Waals interactions with
C599, these cleavages may represent a disturbance of inter-
actions between the protein and RNA at this site (Schuwirth
et al. 2005). However, this variable cleavage pattern is re-
stricted to minimal complexes, while 30S subunits (Fig. 2B,
lane 8; Fig. 2C, lane 10; Fig. 4C, lane 10) and 70S ribosomes
formed with Fe(II)-C86-S8 (Lancaster et al. 2000) demon-
strate reproducible cleavage patterns. Overall, Fe(II)-S8 di-
rected cleavage in helix 21 is highly similar in binary com-
plexes and in fully assembled small subunits.
There are differences in cleavage patterns between binary
complexes and 30S subunits at helix 22 (Figs. 2C, 6, 7). In
the context of 30S subunits, Fe(II)-C19-S8 and Fe(II)-C67-
S8 mediate weak cleavages extending from nucleotides 745–
747 (Fig. 2D, lane 4) and 748–750 (Fig. 2D, lane 12),
respectively, while Fe(II)-C54-S8 (Fig. 2D, lane 8; Fig. 3B,
lane 7) and Fe(II)-C61-S8 (Fig. 2D, lane 10; Fig. 3A, lane 5)
cleave strongly between 748 and 750. A 39 offset relative to
cleaved nucleotides in mature subunits occurs in binary
complexes containing Fe(II)-C61-S8 (Fig. 2D, lane 9; Fig.
3A, lane 3) and Fe(II)-C67-S8 (Fig. 2D, lane 11). The
complex Fe(II)-C28-S8/16S shows no cleavage in helix 22
(Fig. 2D, lane 5). Also, Fe(II)-C54-S8 cleaves with a greater
intensity in 30S subunits than in binary complexes (Fig. 2D,
lanes 7,8). The incorporation of r-protein S15 in a minimal
RNP with Fe(II)-C19-S8, Fe(II)-C61-S8 (Fig. 3A, lane 4),
and Fe(II)-C54-S8 (Fig. 3B, lane 4) with 16S rRNA leads to
cleavage patterns similar to that in 30S subunits, indicating
that r-protein S15 influences the architecture of helix
22. These data are consistent with earlier X-ray crystal
structures and directed hydroxyl radical probing studies
(Agalarov et al. 2000; Jagannathan and Culver 2003).
The binding site of S8 at the junction of helices 25, 26,
and 26a is cleaved by Fe(II)-C19-S8, Fe(II)-C61-S8, and
Fe(II)-C67-S8 in both binary complexes and 30S subunits
(Figs. 2E, 5, 6). While cleavages at the base (nucleotides
828–830) and middle of helix 26 (nucleotides 848–852) are
similar between binary complexes and 30S subunits recon-
stituted with Fe(II)-C19-S8 (Fig. 2E, lanes 3,6; Fig. 3C,
Calidas and Culver
RNA, Vol. 17, No. 2
FIGURE 2. Directed hydroxyl radical cleavage of the central domain of 16S rRNA from Fe(II)-S8 in various RNPs detected by primer extension.
A and G are sequencing lanes. All complexes contain natural 16S rRNA and derivatized cysteine substituents of S8, with the identity of the
derivatized protein indicated at the top of each lane as Fe(II)-Cx-S8. Cys-less is the mock-treated cysteine-less control S8. The addition of
individual r-proteins and mixtures is indicated by +. Composition of mixtures: 59 dom: S4 + S5 + S12 + S16 + S17 + S20, Cent dom: S6 + S11 +
S15 + S18 + S21, 39 major dom: S2 + S3 + S7 + S9 + S10 + S13 + S14 + S19. Bars on the right indicate positions of cleavage, and the text delineates
the identity of the helices cleaved. Primers utilized are 683 (A–C), 837 (D), and 1046 (E).
FIGURE 3. Analysis of the role of individual r-proteins in small subunit assembly. A and G are sequencing lanes. All complexes contain natural
16S rRNA and derivatized cysteine substituents of S8, with the identity of the derivatized protein indicated at the top of each lane as Fe(II)-Cx-S8.
Cys-less is the mock-treated cysteine-less control S8. The addition of individual r-proteins and mixtures is indicated by +. See Figure 2 for
composition of r-protein mixtures. Primers utilized are 837 (A,B), 939 (C), 161 (D), and 480 (E).
RNA, Vol. 17, No. 2
lanes 3,4), Fe(II)-C61-S8 (Fig. 2E, lanes 7,8; Fig. 3C, lanes
8,9), and Fe(II)-C67-S8 (Fig. 2E, lanes 9,10), nucleotides
838–840 which are proximal to the loop are cleaved only in
Fe(II)-C61-S8/30S subunit (Fig. 2E, lane 8; Fig. 3C, lane 9).
Comparison of cleavage patterns at the base of helix 26a
(nucleotides 858–860) indicates a difference in intensity
between the 30S subunits (Fig. 2E, lanes 6,8,10; Fig. 3C,
lanes 4,9) and binary complexes (Fig. 2E, lanes 3,7,9; Fig.
3C, lanes 3,8) irrespective of the position at which S8 is
derivatized. Fe(II)-C19-S8 shows the greatest variation in
cleavage intensity between complexes (Fig. 2E, lanes 3,6;
Fig. 8A). Based on the aforementioned data, it appears that
the more 39 binding site for S8 is not positioned appro-
priately in the minimal complexes.
The addition of S15 results in a cleavage pattern similar,
although not identical, to that of the fully assembled
subunit (Fig. 2E, lane 4; Figs. 8B, 3C, lanes 5,10). In-
triguingly, a minimal RNP containing Fe(II)-C19-S8/S15/
S6/S18 and 16S rRNA (Fig. 2E, lane 5; Fig. 3C, lane 6)
exhibits an increased intensity of cleavage as compared to
FIGURE 4. Directed hydroxyl radical cleavage of the 59 domain of 16S rRNA from Fe(II)-S8 in various RNPs detected by primer extension. A and
G are sequencing lanes. All complexes contain natural 16S rRNA and derivatized cysteine substituents of S8, with the identity of the derivatized
protein indicated at the top of each lane as Fe(II)-Cx-S8. Cys-less is the mock-treated cysteine-less control S8. The addition of individual r-proteins
and mixtures is indicated by +. See Figure 2 for composition of r-protein mixtures. Primers utilized are 232 (A), 480 (B), and 683 (C).
Interdependencies govern 30S subunit architecture
binary complexes, 30S subunits and Fe(II)-C19-S8/S15/16S
rRNA (Fig. 2E, lanes 3,6,4; Fig. 3C, lanes 3,4,5, respec-
tively). Thus, addition of S6 and S18 to a complex of S8 and
S15 appears to induce an intermediate structure at helix 26
that is resolved by the addition of all the r-proteins that
bind the central domain (Fig. 3C, lane 7) and in fully
formed subunits. This suggests that the assembly of the
central domain may proceed through intermediate struc-
tures that are not necessarily identical to the ultimate
conformation but which are then resolved by late binding
proteins to assemble to the mature conformation.
Assembly of the 59 domain
In the 59 domain there is diminished or absent cleavage in
the presence of Fe(II)-S8 and 16S rRNA as compared to 30S
subunits (Figs. 4, 6, 7). The 59 terminus is cleaved by Fe(II)-
C73-S8 and Fe(II)-C86-S8 in the context of 30S subunits
(Fig. 4A, lanes 6,10) but not in the binary complex (Fig. 4A,
lanes 3,7). While a minimal RNP consisting of Fe(II)-C73-
S8/S12/S17 (Fig. 4A, lane 4) and Fe(II)-C86-S8/S12/S17
(Fig. 4A, lane 8) is capable of establishing a distinct cleavage
pattern between residues 5 and 9, only the addition of all
the r-proteins that bind the 59 domain results in the res-
toration of cleavage intensity to a level close to that observed
in 30S subunits (Fig. 4A, lanes 5,9). A more thorough
dissection of the relative significance of individual 59 domain
binding r-proteins in the assembly of this domain showed
that the addition of any one of three proteins, S5 (Fig. 3D,
lane 5), S16 (Fig. 3D, lane 6), or S12 (Fig. 3D, lane 7), to a
minimal complex containing all the primary proteins bind-
ing the 59 domain was not sufficient to elicit cleavage near
the 59 terminus. Cleavages were first observed only upon
assembly of the 59 domain (Fig. 3D, lane 8). This indicates
that the addition of r-protein S4 to a complex containing
S12, S17, and S8 results in the structural perturbation of that
complex. In conclusion, the addition of all the r-proteins
binding the 59 domain is necessary in order to establish
either the correct conformation or to limit the dynamicity of
the 59 terminus.
Similarly, nucleotides 298–301 within helix 12 are cleaved
in the mature subunits (Fig. 4B, lanes 6,10) but not in bi-
nary complexes containing Fe(II)-C73-S8 and Fe(II)-C86-S8
(Fig. 4B, lanes 3,7). This may indicate either an alternative
conformation at these residues or a greater dynamicity
within the loop of helix 12 in binary complexes. Previous
studies have suggested that S16 plays a significant role in
stabilizing the tertiary structure of this region (Ramaswamy
and Woodson 2009); however, a RNP consisting of S16 and
S20 did not mediate cleavage at helix 12 (Fig. 4B, lane 4)
while the addition of all the primary proteins binding the
59 domain to this particle resulted in a weak cleavage at
nucleotides 299 and 300 (Fig. 3E, lane 7). In fact, the
recapitulation of cleavage patterns requires the presence
of all the proteins that bind the 59 domain (Fig. 4B, lanes
5,9), although the cleavages may be of a reduced intensity
as compared to the mature subunit (Fig. 3E, lane 8).
From above mentioned data, it can be deduced that the
presence of all the 59 domain binding proteins results
in a conformation similar to that in the 30S subunit in
Moreover, nucleotide 566 which pairs with nucleotide 299
via purine–purine interactions is cleaved with a decreased
intensity in complexes of Fe(II)-C73-S8/16S (Fig. 4C, lane 3)
and Fe(II)-C86-S8/16S (Fig. 4C, lane 7) as compared to 30S
subunits (Fig. 4C, lanes 6,10). Indeed, the interdomain
region between the 59 and central domains at nucleotides
565–568 is cleaved with a differing intensity between the
binary complexes and 30S subunits reconstituted with
Fe(II)-C73-S8 or Fe(II)-C86-S8 (Fig. 4C, lanes 3,6,7,10).
Addition of r-proteins that bind the 59 domain results in
a cleavage pattern that closely resembles that observed in
30S subunits (Fig. 4C, lanes 5,9). Therefore, assembly of the
body leads to restoration of cleavage patterns within the
59 domain to the level observed in the 30S subunits.
Assembly of the 39 major domain
The neck (the junction of the head and body) of the 30S
subunit is cleaved by Fe(II)-C19-S8 and Fe(II)-C67-S8 at
nucleotides 1073–1078 and nucleotides 1101–1105 in 30S
subunits (Fig. 5, lanes 6,10). In contrast, no cleavages are
FIGURE 5. Directed hydroxyl radical cleavage of the 39 major domain
of 16S rRNA from Fe(II)-S8 in various RNPs detected by primer ex-
tension. A and G are sequencing lanes. All complexes contain natural
16S rRNA and derivatized cysteine substituents of S8, with the identity
of the derivatized protein indicated at the top of each lane as Fe(II)-
Cx-S8. Cys-less is the mock-treated cysteine-less control S8. The ad-
dition of individual r-proteins and mixtures is indicated by +. See
Figure 2 for composition of r-protein mixtures. Primer utilized is 1257.
Calidas and Culver
RNA, Vol. 17, No. 2
observed in binary complexes (Fig. 5, lanes 3,7) while within
the 70S ribosome, Fe(II)-C19-S8 cleaves only nucleotides
1099–1104 (Lancaster et al. 2000). Since crystal structures
(Ogle et al. 2002; Schuwirth et al. 2005) and cryo-electron
microscopy techniques (Agrawal et al. 1999; Gabashvili
et al. 1999; Stark et al. 2000) have reported conformational
changes in the neck that enable the head to swivel with
respect to the body (Ramakrishnan 2002), the change in
cleavage patterns between free 30S subunits and 30S sub-
units associated with 50S subunits in this region of the SSU
is not surprising. Reproduction of the cleavage pattern
observed in 30S subunits requires the presence of r-proteins
found in both the head and the body (Fig. 5, lanes 5,9; Fig.
8C,D). These data lend support to the hypothesis that there
is a minor conformational change in this region and/or that
this region is more dynamic in binary complexes. While the
complex of Fe(II)-C67-S8 with the 59 and 39 major domain
r-proteins and 16S rRNA recapitulates cleavage patterns at
the neck, additional cleavages at nucleotide 1148 and 1149
(Fig. 5, lane 9) are also observed. These nucleotides are
beyond cleaving distance from S8 in a particle with an
appropriately assembled 59 and 39 major domain, and thus
most likely represent a subpopulation with aberrant struc-
tures in these domains (Joseph et al. 1997; Lancaster et al.
2000). To summarize, assembly of both the head and the
body can enable the assembly or orientation of the neck,
thereby illustrating the interconnected assembly of the SSU
FIGURE 6. Directed hydroxyl radical cleavage from Fe(II)-S8 in binary complexes mapped on secondary structure of 16S rRNA (Cannone et al.
2002). The cleaved regions are expanded and the Fe(II)-derivatization site generating the cleavage is labeled. Cleavage strength is indicated as strong
(big circle), medium (medium circle), and weak (small circle). Fe(II)-C86-S8/16S rRNA exhibits variable cleavage patterns at 601–602 and 630–635.
Interdependencies govern 30S subunit architecture
Directed hydroxyl radical probing of 30S subunit assembly
as carried out using Fe(II)-S8 has shed light on the role
of cooperative interactions between r-proteins as well as
between structural domains. Outside of the well defined S8
binding site, the cleavage patterns generated by Fe(II)-S8
are altered in binary complexes of S8 and 16S rRNA alone
as compared to mature 30S subunits. Multiple equally
valid interpretations are possible for this difference, such
as more minimal complexes sampling numerous confor-
mations rapidly, or existing in two or more altered
conformations of varied stability. This hypothesis can
apply to both the r-protein and rRNA component of the
complex. At present, in common with other techniques of
generating hydroxyl radicals, it is not possible to de-
termine which of these processes result in the particular
pattern of cleavage observed (Dixon et al. 1991; Nguyenle
et al. 2006).
The total sum of the data presented above, together with
previous work (see Sykes and Williamson 2009), indicates
that the assembly of components within the subunit,
whether categorized by domain or by the binding site of
individual r-proteins, is interlinked and hierarchical.
FIGURE 7. Directed hydroxyl radical cleavage sites from Fe(II)-S8 in 30S subunits mapped on the secondary structure of 16S rRNA (Cannone
et al. 2002). Notations are as in Figure 5. Regions that exhibit differences in cleavage patterns or intensities from Figure 5 are distinguished by
Calidas and Culver
RNA, Vol. 17, No. 2
Complementary roles for r-protein S8 and S15
in central domain assembly
The data obtained by directed hydroxyl radical probing of
the central domain from Fe(II)-S8 imply that only part
of the central domain is appropriately structured in the
presence of S8 alone (Figs. 6, 7, 8A,B). The similarity of
cleavage patterns across virtually all the derivatized posi-
tions at helix 21 leads to the speculation that this helix can
assume its tertiary structure in a minimal particle during
biogenesis. The NMR structure of the binding site of S8 in
the presence and absence of the protein
also correlates thishypothesis(Kalurachchi
et al. 1997).
On the other hand, the data at the
junction of helices 25, 26, and 26a ap-
pears to be more complicated. While
there is a change in intensity of cleavage
at helix 26a and closer to the loop of
helix 26, the cleavage at the base and
middle of helix 26 appears unchanged
between the binary complex and the 30S
subunit (Figs. 6, 7, 8A,B). These data
are derived from three different probing
positions on S8: Fe(II)-C19-S8, Fe(II)-
C61-S8, and Fe(II)-C67-S8. Although
both Fe(II)-C19-S8 and Fe(II)-C67-S8
are located at similar distances from the
16S rRNA cleavage sites in the tertiary
structure, only one of these, Fe(II)-C19-
S8, shows a greater change in cleavage
intensity at helix 26a. One possible rea-
son for this difference is that the radi-
cals generated from Fe(II)-C67-S8 are
quenched by the segment of S8 that
binds to the three-helix junction. In ad-
dition, alanine 19 of S8 interacts with
U827 in the crystal structure of the 30S
subunit (Schuwirth et al. 2005), thus the
difference in patterns might represent
a change in the interaction with 16S
rRNA in the complex Fe(II)-C19-S8/
16S rRNA. Yet, Fe(II)-C19-S8/16S rRNA
cleaves reproducibly at nucleotides 828–
830 (Figs. 6, 7, 8A,B). Also, the con-
formity of changes in cleavage patterns
between complexes across all the deriv-
atized positions argues against the data
being artifactual in nature. The data
presented above imply that the archi-
tecture of this element of 16S rRNA is
influenced by other r-proteins in addi-
tion to S8.
In keeping with data from directed
hydroxyl radical probing of the central
domain mediated by Fe(II)-S15 (Jagannathan and Culver
2003), a complex of Fe(II)-S8/S15/16S rRNA was found to
restore cleavage patterns at the three-helix junction be-
tween helices 25–26a (Fig. 8A,B). Since the cleavages are
localized to the binding site of S8, three hypotheses are
possible. First, S8 could bind transiently to this region in
the binary complex and S15 could help directly or in-
directly to stabilize the interaction of S8 with 16S rRNA.
The transience of this binding could be attributed to the
possibility of a conformational change in S8 before it can
stably bind this region. Second, although S8 is bound to the
FIGURE 8. Directed hydroxyl radical cleavage sites from Fe(II)-S8 containing complexes
mapped as spheres on the three-dimensional structure of the 30S ribosomal subunit. 16S rRNA
is shown in gray and S8 is shown as a cartoon in orange. The other r-proteins are omitted or
specifically mentioned in each subpanel. The color of the sphere indicates the probing site used
(Fig. 1D), while the size corresponds to cleavage intensity. (A) Cleavages from Fe(II)-C19-S8 in
the binary complex in the central domain only. (B) Cleavages from Fe(II)-C19-S8 in a minimal
complex with S15, shown in olive green. (C) Cleavages from Fe(II)-C67-S8 in a minimal RNP
with the head and body assembled. In blue are proteins that bind the 39 major domain, in red
are the r-proteins that bind the 59 domain. (D) Same data as in panel C, except that the neck is
expanded and r-proteins omitted.
Interdependencies govern 30S subunit architecture
three-helix junction, its binding site could differ in con-
formation from that in the 30S subunit, and S15 could aid
in establishing the mature conformation. Third, the pres-
ence of a probe at the interaction site between protein and
rRNA could destabilize the interaction, and S15 might
complement the role of S8. While the last hypothesis is
a formal possibility, data from studies on the dynamics of
assembly, either by time-resolved hydroxyl radical foot-
printing (Adilakshmi et al. 2008) or by chemical probing
(Powers et al. 1993), support the first two hypotheses.
While differing in the specifics of nucleotides at which S8
dependent protections from hydroxyl radicals or chemical
probes appear, both data sets demonstrate that different
regions within the binding site of S8 at the junction of
helices 25, 26, and 26a are protected at different stages of
assembly. A study of the assembly of the central domain
of Thermus thermophilus led to the proposal of a hierarchy
of elements that bind S15, S6, and S18, i.e., helices 20–23
form a core RNP that is required for the association of
these r-proteins while helices 24, 25, and 26 were labeled
secondary binding elements that bind only the preformed
core RNP (Agalarov and Williamson 2000). Thus, based on
data presented in this and previous studies, it is possible to
extend this theory to the assembly of the central domain in
Escherichia coli, so that the junction of helices 20–22 could
be considered the core element that associates with S8 and
S15 while helices 25–26a are secondary binding elements
that need the presence of the preformed core RNP in order
to assemble. While our data are not sufficient to identify
which hypothesis or combination thereof contributes to the
results, it is clear that both S8 and S15, the early binding
proteins that associate with the central domain, aid in the
assembly of the platform. Therefore, it is possible to speculate
that compaction of the central domain during biogenesis in
vivo is a function of both S8 and S15 r-proteins. The function
of S15 in central domain assembly appears to be redundant,
as a strain bearing a deletion of rpsO is viable (Bubunenko
et al. 2006). However, S15 may increase the efficiency of
assembly in wild-type strains by aiding in the association of
S8 with 16S rRNA and central domain compaction.
Certain elements of the 59 domain assume a mature
architecture only upon assembly to the body
The data from the 59 domain reveal a general trend, in that
the 59 terminus, the loop of helix 12, and the stem of helix
19 are cleaved by Fe(II)-S8 to yield patterns resembling the
30S subunit only upon addition of r-proteins that bind the
59 domain (Figs. 6, 7). The lack or decreased intensity of
cleavages in binary complexes could be due to alternative
conformations of these particles. Another probable explana-
tion is that, in the absence of specific r-proteins, certain
regions of 16S rRNA may be more dynamic. The appropriate
orientation of the 59 terminus is functionally significant as it
is proximal to the central pseudoknot, which is a key player
in translation (Poot et al. 1998), and alterations in the
architecture of this region have been shown to affect fidelity
(Dammel and Noller 1993; Roy-Chaudhuri et al. 2010).
While recent hydroxyl radical footprinting data suggest
that S16 plays a significant role in orienting helix 12
(Ramaswamy and Woodson 2009), in our study a complex
of Fe(II)-C73-S8/S16/S20/16S was unable to cleave helix 12
(Fig. 4B, lane 4). The difference in observations may be
a result of the differing sensitivities of the techniques of
footprinting and directed hydroxyl radical probing. Further-
more, while the study by Ramaswamy and Woodson (2009)
utilized in vitro transcribed 59 domain RNA, this study
utilized full length modified 16S rRNA, consequently archi-
tectural differences may exist between the two particles.
Analysis of X-ray crystal structures reveals that among the
59 domain proteins, S5 interacts with the 59 terminus and
G568 while S12 interacts with helix 19 and the loop of helix
12 (Allers and Shamoo 2001; Schuwirth et al. 2005). Indeed,
in a previous study, the assembly of S5 caused residues at the
59 terminus and the junction of 59 and central domains to
have altered susceptibility to reactivity with chemical probes
(Stern et al. 1988). These effects were exaggerated in a
complex of S5 and S12 with 16S rRNA. Moreover, Fe(II)-S5
mediates cleavage of the 59 terminus in 30S subunits (Culver
et al. 1999). However, a minimal particle of Fe(II)-C73-S8/
S5/16S rRNA failed to recapitulate cleavages observed in
Fe(II)-S8/30S subunit (data not shown). This conundrum
may be resolved by recent studies on the dynamics of protein
incorporation. Using the technique of pulse-chase quantita-
tive mass spectrometry, it was determined that S12 and S5
are incorporated into the assembling particle at a relatively
slow rate compared to the remainder of the 59 domain
binding proteins (Talkington et al. 2005). In conclusion, it is
likely that these proteins are responsible for stabilizing or
ordering these regions of the 59 domain in the presence of all
the other 59 domain binding proteins.
Assembly of the 39 major domain alone is not sufficient
for appropriate orientation of the neck
It was found that the head is capable of rotating at the neck
(Schuwirth et al. 2005), thus the difference in cleavage
patterns between 70S ribosomes (Lancaster et al. 2000), free
30S subunits, and binary particles within this region is not
surprising (Figs. 6, 7, 8C,D). The quenching of radicals
from Fe(II)-C19-S8 or changes in conformation of this
region upon subunit association could explain the di-
minished cleavages in 70S ribosomes. The lack of cleavages
in the binary complex could be indicative of conforma-
tional changes in the neck, or due to increased motion of
the neck, or an altered alignment of the 39 domain with
respect to the 59 domain. Yet, addition of r-proteins that
bind the 39 domain to form the head did not result in the
restoration of Fe(II)-S8 cleavage patterns. Previously, a
fragment of 16S rRNA corresponding to the 39 major
Calidas and Culver
RNA, Vol. 17, No. 2
domain had been assembled with r-proteins to form the
head, and was demonstrated to possess reactivity to chemical
probes and spectinomycin binding affinity similar to the
head domain in intact 30S subunits (Samaha et al. 1994).
Taken together, these studies suggest that assembly of the
head in the presence of S8 is not sufficient to organize
the neck. The recapitulation of cleavage patterns requires the
presence of all the r-proteins binding the head and body.
While it is highly probable that the interaction of S5 with
the head and body plays a significant role in the restoration
of cleavage patterns (Schuwirth et al. 2005), a minimal RNP
of Fe(II)-C19-S8/S5/16S and Fe(II)-C67-S8/S5/16S does not
cleave the neck (data not shown). One explanation for this
is that S5 is dependent upon multiple proteins for incor-
poration (see Fig. 1A). Also, as mentioned above, S5 binds
with slower kinetics than other 59 domain binding pro-
teins with the exception of S12. It is also possible that S8 is
not appropriately aligned in particles containing only the
r-proteins binding the 39 major domain, and addition of
r-proteins that bind the body aids in appropriate organiza-
tion. Since the cleavages occur close to the functional center
of the subunit, the necessity for the appropriate assembly of
multiple domains in order to achieve mature structure may
be a quality control mechanism during in vivo biogenesis.
Moreover, previous studies have noted that the movement
of the head relative to the remainder of the subunit may play
a significant role in increasing the fidelity of translation (see
Zaher and Green 2009).
A common principle that appears to underlie the results
presented above is the interdependence of assembly between
different regions of 16S rRNA, both within a domain and
across domains. The assembly of the junction of helices 25–
26a appears to be dependent upon the establishment of ap-
propriate structure at the junction of helices 20–23. Similarly,
the orientation of the neck is dependent upon the assembly
of the head and body. Indeed, the 30S subunit can be con-
sidered a collection of ribonucleoprotein complexes that
fold through modular pathways that influence one another.
This hierarchy of folding behavior has been compared to
another large, multidomain RNA, the Tetrahymena ribozyme
(Laggerbauer et al. 1994; Zarrinkar and Williamson 1994;
Agalarov and Williamson 2000), and has also been observed
in the group II intron ribozyme ai5gD135 (Su et al. 2005).
Thus, multidomain RNAs may fold through pathways that
contain intermediates characterized by segmented folding,
with the folding of certain key segments playing a significant
role in the architecture of the entire molecule.
MATERIALS AND METHODS
Mutagenesis, expression, purification, and
derivatization of S8
Previously, a cysteine-less mutant of rpsH had been constructed in
pET 21b vector by the replacement of C126 with Alanine, and
eight positions had been substituted to cysteine residues in this
background. All nine constructs were expressed and the cells were
collected as described (Lancaster et al. 2000). The cells were lysed in
Buffer E (20 mM K+-HEPES [pH 7.6], 20 mM KCl, and 6 mM
bME) in an Avestin cell lyser. Inclusion bodies containing S8 were
pelleted by spinning at 7000 rpm for 20 min at 4°C and
resuspended in Buffer C (20 mM sodium acetate [pH 5.6], 1 M
KCl, 6 M Urea and 6 mM bME) followed by overnight dialysis into
Buffer B (20 mM sodium acetate [pH 5.6], 20 mM KCl, 6 M Urea
and 6 mM bME) with three changes. The purification protocol was
also similar to Lancaster et al. (2000) except that elution was carried
out over a 125 mL salt gradient from 20 to 130 mM KCl in Buffer B
and the proteins eluted at z22 mM KCl. Purified proteins were
dialyzed into Buffer D (80 mM K+-HEPES [pH 7.6], 1 M KCl, and
6 mM bME), aliquoted and frozen in liquid nitrogen. The
derivatization reaction was carried out as in Lancaster et al. (2000).
Reconstitution and purification of RNPs containing
16S rRNA was isolated as described (Moazed et al. 1986), heat-
activated by incubating at 42°C for 15 min in Buffer A (80 mM
K+-Hepes [pH 7.6], 330 mM KCl, 20 mM MgCl2, and 0.01%
Nikkol), and then incubated on ice for 10 min. The binary
complexes were formed by mixing 240 pmol of Fe(II)-derivatized
proteins with 40 pmol of 16S rRNA in a final volume of 100 mL
with a final KCl concentration of 330 mM. If a more complex
RNP was desired, the derivatized proteins were mixed with either
360 pmol of individual proteins or 120 pmol of domain mixes
prior to the addition of RNA. The 59 domain mix is composed of
r-proteins S4, S5, S12, S16, S17, and S20, the central domain mix
of r-proteins S6, S11, S15, S18, and S21 while the 39 major domain
mix is composed of r-proteins S2, S3, S7, S9, S10, S13, S14, S19. In
order to form the RNPs, the reactions were incubated at 42°C for 20
min. These were then cooled on ice for 10 min and purified by
passing them through spin columns with a speed of 2500 rpm for
3.5 min as described (Dutca and Culver 2008). In order to minimize
non-directed probing, 1.6 mL of 125 mM EDTA was added to yield
a final concentration of 2 mM EDTA per reaction except to the
complexes depicted in Figure 2C. The RNPs were incubated on ice
for 10 min prior to directed hydroxyl radical probing.
Directed hydroxyl radical probing
Directed hydroxyl radical probing and primer extension analysis were
carried out on various RNPs as in Culver and Noller (2000a). Each
probing experiment and the subsequent analysis were performed at
least two times and intensity of cleavage was determined visually.
All analysis was carried out on the PDB file 2AW7 (Schuwirth
et al. 2005). The structure was visualized by PyMOL (The PyMOL
Molecular Graphics System). The program ENTANGLE was
utilized to examine interactions within the structure (Allers and
The S8 cysteine substituents were a gift from the laboratory of
Dr. H.F. Noller (University of California Santa Cruz). We thank
Interdependencies govern 30S subunit architecture
all members of the Culver lab. This work was supported by NIH
grant GM62432 to G.M.C.
Received June 21, 2010; accepted November 5, 2010.
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