Download Studies on the structure and function of 16S ribosomal RNA using

Proc. Int. Symp. Biomol. Struct. Interactions, Suppl. J. Biosci.,
Vol. 8, Nos 3 & 4, August 1985, pp. 747–755. © Printed in India.
Studies on the structure and function of 16S ribosomal RNA using
structure-specific chemical probes
HARRY F. NOLLER, BARBARA J. VAN STOLK, DANESH
MOAZED, STEPHEN DOUTHWAITE, and ROBIN R. GUTELL
Thimann Laboratories, University of California, Santa Cruz, California 95064, USA
Abstract. Recent technological developments permit us to examine the accessibility of specific
atoms on any nucleotide in any large RNA molecule to certain chemical probes. This can
provide detailed information about the higher order structure of large RNA molecules,
including secondary and tertiary structure, protein-RNA contacts, binding sites for functional
ligands and possible biologically significant conformational changes. Here, we summarize
recent studies on (i) the conformation of naked 16S rRNA under a variety of ionic conditions,
and (ii) the behaviour of 16S rRNA in active and inactive 30S subunits, as defined by Zamir,
Elson and their colleagues. The latter study reveals a reciprocal conformational change in the
vicinity of the decoding region of 16S rRNA in 30S ribosomal subunits. This conformational
change appears to be a rearrangement of tertiary and/or quaternary structure involving several
universally conserved nucleotides. No reproducible effects are seen elsewhere in the molecule,
suggesting that the active-inactive transition is a result of the observed conformational change.
Keywords. Ribosomes; RNA; chemical probes.
Introduction
Our understanding of the role of ribosomal RNA in translation has been in a state of
rapid evolution in recent years. No longer considered a mere scaffold for the
positioning of ribosomal proteins, rRNA currently appears instead to play an active
and probably crucial part in protein synthesis (reviewed in Noller, 1984). Interest in its
structure thus originates to a large degree from a desire to understand how it works.
Perhaps this understanding will, in turn, shed light on how ribosomes (and life itself)
originated.
In this paper, we briefly review progress on the structure and function of 16S rRNA,
and summarize some recent studies in which we chemically probe this molecule in a
variety of conformational states.
Primary and secondary structures of 16S rRNA
Since the first 16S rRNA sequence appeared in 1978 (Brosius et al., 1978; Carbon et al.,
1979), over thirty additional sequences have been completed (reviewed in Gutell et al.,
1985). This list covers the phylogenetic spectrum, and includes examples from the
eubacterial, archaebacterial and eucaryotic kingdoms, as well as several chloroplasts
and mitochondria. The phylogenetic diversity of these sequences permits the use of
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comparative sequence analysis in determination of a higher order structure (Noller and
Woese, 1981; Woese et al., 1983). A given helix is considered proven when two or more
independent sets of compensating base changes are found between different 16S (or
18S) rRNAs. On this basis, the secondary structure shown in figure 1 was established
(Gutell et al., 1984; Noller and Woese, 1981; Woese et al., 1983).
There is presently general agreement between secondary structure models for 16S
rRNA derived independently by three groups of investigators (Brimacombe et al., 1983;
Ebel et al., 1983). Experimental probing of Escherichia coli 16S rRNA using a variety of
approaches strongly supports the proposed secondary structure model (reviewed in
Noller, 1984). Chemical and enzymatic studies on Bacillus brevis 16S rRNA (Kop et al.,
1984) and yeast 18S rRNA (Hogan et al., 1984) generally agree with the E. coli results,
and support our original implicit assumption that all 16S-like rRNAs are likely to have
essentially similar 3-dimensional structures. A comparison of several representative 16S
rRNA secondary structures, including a discussion of phylogenetically variable regions,
has been made recently by Gutell et al., (1985).
Probing the conformation of E. coli 16S rRNA using diethylpyrocarbonate
Peattie and Gilbert (1984) devised a method for probing the conformation of small RNA
molecules, exploiting the differential chemical reactivity of bases resulting from higher
order structure. Their method utilizes end-labelling of the RNA, and displays sites of
modification on a sequencing-type gel, made possible by aniline-induced strand
scission at these positions. In order to extend this approach to large RNAs, we sought a
means of generating RNA fragments of defined length, so that internal positions of the
molecule would be within the reach of the gel method. This was achieved by preparing
RNA-DNA hybrids using the chemically modified RNA and restriction fragments of its
gene (Van Stolk and Noller, 1984). RNA is trimmed down to the size of the DNA by
ribonuclease T1' end-labelled, and the RNA-DNA hybrids resolved by gel electrophoresis. Labelled RNA fragments are recovered by a denaturing gel, and subjected to
the Peattie-Gilbert procedure to identify the sites of modification. The low level of
modification used (on the order of 1 % of the bases are modified, typically)
poses no apparent problem for the hybridization, as shown in control experiments,
where identical modification patterns were observed whether or not the RNA had
undergone prior hybridization (Van Stolk and Noller, 1984).
An example of the use of this approach is shown in figure 2, in which an internal
region (around position 1150) of E. coli 16S rRNA is probed. Chemical modification of
naked intact 16S rRNA was carried out under three sets of conditions, which we term
denatured (90°C, 80 mM potassium cacodylate, pH 7·0, 1 mM EDTA), quasisecondary (37°C, 80 mM potassium cacodylate, 1 mM EDTA), and native (37°C,
80 mM potassium cacodylate, 20 mM MgCl2, 300 mM KCl). Readily apparent in
figure 2 are several positions whose relative reactivities with diethylpyrocarbonate are
affected either by salt or temperature. (Also seen is the salt-dependent modification of
certain cytidine residues, the significance of which is not entirely clear). The results of
probing nearly the entire 16S rRNA by this method are shown in figure 1. A number
of interesting findings emerge from this study.
Structure and function of 16S ribosomal RNA
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Figure 1. Secondary structure model for E. coli 16S rRNA (Woese et al., 1983). Helices
considered to be proven (two or more sets of compensating base changes) are shaded. Sites of
reactivity toward diethylpyrocarbonate (Van Stolk and Noller, 1984) under native conditions
are shown by circles: О, unreactive; ●, weakly reactive; z, strongly reactive. Reactivity under
quasi-secondary conditions is shown by triangles: ∆, unreactive; ▲, weakly reactive; ▲, strongly
reactive.
(i) Numerous unpaired adenines are unreactive toward diethylpyrocarbonate under
native conditions. This indicates that they are either stacked, base-paired, involved in
tertiary contacts or shielded specifically by salt (probably Κ or Mg). Yet other adenines
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Figure 2. Autoradiograph of an 10 % Polyacrylamide gel showing reactivity of nucleotides
between positions 1117 and 1191 of E. coli 16S rRNA (Van Stolk and Noller, 1984). RNA
fragments from this region were prepared by hybridization with DNA from a Hha I digest of
the 16S rRNA gene.
Structure and function of 16S ribosomal RNA
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are quite reactive under native conditions, showing that they are unstacked. Only four
out of the 309 adenines probed were reactive under native conditions and shown as
base-paired, providing strong general support for the secondary structure model.
(ii) Candidates for possible tertiary interactions are identified as adenine residues that
are unpaired in the model and unreactive under native conditions but reactive under
quasi-secondary conditions. We find 57 such residues that show this behaviour.
(iii) An unexpectedly stable structure has been identified in the region between
positions 109 and 279, where many residues remain unreactive even at 90°C in EDTAcontaining buffer. This region may correspond to a structural 'core' that is important
for early events in ribosome assembly (Garrett et al., 1973).
Probing the active-inactive transition in 50S ribosomal subunits
Zamir, Elson and their coworkers discovered a cation-dependent conformational
transition in which 30S subunits can be reversibly inactivated. Reactivation depends on
the presence of critical concentrations of Mg2+ and NH4+ or K+ and requires a brief
heating step (Zamir et al., 1969, 1971; Vogel et al., 1970). Inactivation is characterized by
loss of non-enzymatic tRNA binding, dihydrostreptomycin binding, and formation of
70S couples, among other things. Reactivities of certain ribosomal proteins toward
N-ethylmaleimide are altered by the transition (Ginzburg et al., 1973), but no clearly
demonstrable changes in the overall structure of the 30S subunit are evident (see, for
example, Kearny and Moore, 1983).
Previously, we reported studies on the kethoxal reactivity of active and inactive 30S
subunits (Hogan and Noller, 1978), in which they were probed under their two different
sets of ionic conditions. On the basis of the strong dependence of reactivity on Mg2+
and K+ concentrations observed in the experiments summarized above (figures 1, 2), we
were led to re-examine our earlier studies (Hogan and Noller, 1978). Thus, we sought to
find conditions where both active and inactive subunits could be probed under identical
ionic and temperature conditions. By carrying out the reactions at 0°C, we could study
both active and inactive subunits in "active" buffer, because activation of inactive
subunits requires heating. The two kinds of subunits were probed at adenine with
diethylpyrocarbonate, and at guanine and cytosine with dimethyl sulphate, as described
by Peattie and Gilbert (1984). The entire E. coli 16S rRNA was examined, using the
RNA-DNA hybridization method (Van Stolk and Noller, 1984), or direct 3'-end
labelling of the intact 16S rRNA.
Using this strategy, our findings (Moazed et al., 1985) differed dramatically from our
previous results. Whereas previously we had reported large differences between active
and inactive subunits at numerous positions (Hogan and Noller, 1978), the present
experiments (Moazed, D., Van Stolk, B. J., Douthwaite, S., Noller, Η. F., unpublished
results) show almost identical patterns of reactivity throughout the molecule, with the
exception of a single region. Figure 3 shows most of the observed differences in
reactivity, which are clustered in the 1400 and 1500 regions of the 16S rRNA chain.
These two regions are of exceptional interest because (a) the sequences in these two
regions are universally conserved in all organisms sequenced to date, including
eubacteria, archaebacteria, and higher and lower eucaryotes (Gutell et al., 1985; Woese
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Figure 3. Differential reactivity of sites in the 1400 and 1500 regions of E. coli 16S rRNA in
active and inactive subunits (Moazed, D., Van Stolk, B. J., Douthwaite, S., Noller, Η. F.,
unpublished results). RNA from modified subunits was labelled with [32P] -pCp and subjected
to aniline-induced cleavage and gel electrophoresis. Lanes C and U were treated according to
Peattie under C and U sequencing conditions (Peattie, 1979). In and Ac indicate inactive and
active subunits, respectively, and show differences in modification of (left to right) G, C and A,
respectively. Reproducibly observed differences are indicated by position, and summarized in
table 1.
Structure and function of 16S ribosomal RNA
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Figure 4. Schematic representation of the secondary structure of 16S rRNA (Gutell et al.,
1985). Universally conserved nucleotides are shown as large letters, highly conserved as small
letters, and less conserved or variable nucleotides as large or small dots, respectively. Binding
sites for proteins S8 and S15 are shaded. Kethoxal-reactive sites are indicated by Κ; z, sites
protected by 50S subunits; , sites selected against in damage/selection experiments ▲; sites
protected in polysomes but not in 70S ribosomes. Sites of mutations affecting resistance to the
antibiotics paramomycin (Pmr), Hygromycin (Hmr), Kasugamycin (Ksg) and Spectinomycin
(Spc) are indicated. Col E3 shows the site of strand scission by colicin E3, mRNA indicates the
region used for mRNA selection and tRNA shows the site of photoreaction of the tRNA
anticodon with 16S rRNA (summarized in Gutell et al., 1985).
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et al., 1983), (b) codon-anticodon interaction must take place within a few Angstroms
of C 1400, because the tRNA anticodon has been directly crosslinked to this base (Prince
et al., 1982), (c) the site of attack of colicin E3 as well as sites of mutations affecting
sensitivity to several antibiotics are all in this vicinity (reviewed in Noller, 1984; Gutell
et al., 1985). These points are summarized in figure 4, which shows the positions of
universal and highly conserved nucleotides.
Table 1 shows the relative reactivities of the relevant nucleotides in active and
inactive subunits. Of particular interest is the fact that conversion to inactive subunits
cannot be simply an unfolding of some structure that is present in active subunits; some
positions become more structured (i.e., less reactive) in the inactive state, while others
appear to be more structured in the active state. Thus, the active-inactive transition
seems to involve interconversion between two different structured states in this region
of 16S rRNA.
Table 1. Reactivity differences in 16S rRNA
from active and inactive 30S ribosomal subunitsa
a
DMS, dimethyl sulphate; DEP, diethylpyrocarbonate, In, inactive subunits; Ac, active subunits.
The nature of either of these two structured states is not clear. No supportable
secondary structure has been found for this region of the molecule, favouring the
possibility that the observed changes involve tertiary and/or quaternary structure. The
alterations in reactivity observed by Zamir and co-workers for protons S18 and S21
lend support to the latter (Ginzburg et al., 1973).
Do these conformational changes reflect physiologically significant "switch" mechanisms? Zamir and her colleagues have argued that the active-inactive transition may in
fact be related to some normal translational event (Zamir et al., 1969, 1971), because
certain translational factors appear to mimic the heat activation step. It should now be
possible to test this hypothesis by probing 16S rRNA in its various functional states
during translation.
Structure and function of 16S ribosomal RNA
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Acknowledgement
These studies were supported by NIH grant No. GM 17129.
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