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
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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Interdependencies govern 30S subunit architecture