From the Department of Chemistry and Program in Biological Chemistry, Bates College, Lewiston, Maine 04240
Received for publication, February 18, 2003, and in revised form, February 21, 2003
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ABSTRACT |
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Expression of Bacteria employ many different molecular strategies for coping
with environmental stresses. To initiate the process of transcribing a
specific gene, the multisubunit bacterial RNA polymerase must select
and bind to the promoter sequence of the gene or operon (1). There are
several different classes of promoter sequences, each recognized by
specific RNA polymerase holoenzymes containing appropriate Following exposure to high osmolarity (osmotic upshock), the cytoplasm
of Escherichia coli undergoes dramatic changes in the concentrations and composition of its solutes (4-6) resulting in
differential expression of approximately 70 genes (5). During osmotic
shock, transcription of many of these genes is controlled by RNA
polymerase holoenzyme containing the Exposure of E. coli to high external osmolarity induces an
increase in translation of RpoS mRNA by a signaling pathway likely to involve changes in RpoS mRNA structure (8-11). It is predicted that under normal growth conditions, the ribosome-binding site of RpoS
mRNA is trapped in a secondary structure that results in repression
of translational initiation (see Fig.
1A) (11, 12). Osmotic shock
may induce conformational changes in the mRNA that influence the
interactions of the mRNA with regulatory factors (3). During
osmotic shock, the regulatory factors are expected to increase the
single-stranded nature of the ribosome-binding site (the
"accessibility" of the ribosome-binding site) and therefore to
increase the ability of the 30 S subunit to bind to the mRNA and
the fraction of mRNA participating in translational initiation (3,
13, 14).
s, the gene
product of rpoS, is controlled translationally in response
to many environmental stresses. DsrA, a small 87-nucleotide non-coding
RNA molecule, acts to increase translational efficiency of RpoS
mRNA under some growth conditions. In this work, we demonstrate
that DsrA binds directly to the 30 S ribosomal subunit with an
observed equilibrium affinity of 2.8 × 107
M
1. DsrA does not compete with RpoS mRNA
or tRNA
1, indicating that the full affinity of the
interaction requires the entire DsrA sequence. In order to investigate
translational efficiency of RpoS mRNA, we examined both
ribosome-binding site accessibility and the binding of RpoS mRNA to
30 S ribosomal subunits. We find that that ribosome-binding site
accessibility is modulated as a function of divalent cation
concentration during mRNA renaturation and by the presence of an
antisense sequence that binds to nucleotides 1-16 of the RpoS mRNA
fragment. The ribosome-binding site accessibility correlates with the
amount of RpoS mRNA participating in 30 S-mRNA "pre-initiation" translational complex formation and provides evidence that regulation follows a competitive model of regulation.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
subunits
(2). The bacteria have evolved so that expression of entire families of
proteins under specific growth conditions is initiated by transcription
from a shared class of promoter sequences (2). In response to a range
of external stimuli, the core RNA polymerase exchanges one type of
factor for another, shutting off transcription of one family
of proteins while initiating expression of another family (2, 3). As a
result, the expression of entire gene families can be controlled by
affecting the rate and extent of
factor production (3).
s subunit, the gene
product of rpoS (3, 7, 8).
View larger version (18K):
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Fig. 1.
A, predicted secondary structure of the
RpoS mRNA sequence. Secondary structure was predicted using
the computer folding algorithm Mfold (45, 46) and has a
G =
49.3 kcal/mol. This structure is supported in
part by mutational analysis of the helix containing the
ribosome-binding site. Positions 17 and 97 are predicted to base pair
in this structure. Disruption of this base pair lessens regulation of
expression and compensatory mutations at these positions restores
regulation of translation (11). B, secondary structure of
DsrA mRNA (18).
Three non-coding RNAs (ncRNAs),1 DsrA, RprA, and OxyS, influence translational initiation of RpoS mRNA. Both DsrA and RprA non-coding RNAs activate translation during osmotic shock, whereas OxyS RNA represses translation during oxidative stress. DsrA is a small, 87-nucleotide, untranslated RNA that stimulates RpoS mRNA translation at low temperatures (15). Its secondary structure is shown in Fig. 1B. DsrA ncRNA has been proposed to enhance translational initiation by base pairing to the 5'-untranslated region of RpoS mRNA (including nucleotides 1-16 shown in Fig. 1A) thereby preventing intramolecular base pairing of the ribosome-binding site (16-18) with other cis-acting mRNA sequences. In this work, we perform the first in vitro studies characterizing the relationship between the accessibility of the ribosome-binding site of RpoS mRNA and formation of translational initiation complexes in vitro.
The mechanism for DsrA translational stimulation is unknown. It has
been shown that the effects of DsrA ncRNA on rpoS expression are much greater in the presence of the protein Hfq (11, 19-23). The
role of Hfq in the global regulation of gene expression increasingly appears to be that of a facilitator of RNA-RNA interactions that influence rates of mRNA translation and degradation (23-25). The 3' domain of DsrA interacts specifically with Hfq, and it has been
proposed that Hfq assists in forming the DsrA:RpoS mRNA hybrid postulated to stimulate translation of RpoS mRNA (21). In this work
we demonstrate that DsrA specifically interacts with 30 S ribosomal
subunits in vitro. This interaction may be important in the
mechanism of rpoS regulation by DsrA.
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EXPERIMENTAL PROCEDURES |
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RNA Transcripts--
RpoS mRNA, DsrA RNA, and transcripts
containing the nucleotides 1-34 (DsrA-(1-34)) or nucleotides 23-87
of DsrA (DsrA-(23-87)) were synthesized from PCR products containing
T7 promoters using the Ribomax T7 Transcription kit from Promega
(Madison, WI) following the transcription kit protocol. The
rpoS PCR products were synthesized from pTE661 (11) (the
kind gift of Tom Elliott) using the following primer set: 5'-CCC GGT
ACC TAA TAC GAC TCA CTA TAC GGG TTA CAA GGG GAA ATC CGT AAA CCC GCT-3';
5'-CCG CAT CTT CAT TTA AAT CAT GAA CTT TCA GCG TAT TCT GAC TCA TAA GGT
GGC TCC TAC-3'. DsrA, DsrA-(1-34), and DsrA-(23-87) PCR products were
generated from pDDS164 (21, 26) (the kind gift of Darren Sledjeski)
using the following primer sets: DsrA, 5'-GGT AAT ACG ACT CAC TAT AGG AAC ACA TCA GAT TTC CTG G-3' and 5'-AAA TCC CGA CCC TGA-3';
DsrA-(1-34), 5'-GGT AAT ACG ACT CAC TAT AGG AAC ACA TCA GAT TTC CTG
G-3' and 5'-AAA AAA TTC GTT ACA CC-3'; DsrA-(23-87), 5'-GGT AAT
ACG ACT CAC TAT AGG AAC GAA TTT TTT AAG TG-3' and 5'-AAA TCC CGA CCC
TGA-3'. RNA was purified using Nensorb columns (PerkinElmer Life
Sciences) or Ultrafree-0.5 Centrifugal Filter Devices (Millipore Corp., Bedford, MA) according to the manufacturers' instructions.
Radiolabeling of transcripts was accomplished by incorporating
[-32P]ATP (Amersham Biosciences) during
in vitro transcription (27).
RNA Renaturation-- RNA was denatured in standard renaturation buffer (30 mM Tris-Cl (pH 7.3) and 100 mM potassium acetate) at 65 °C for 5 min. The RNA was then placed in a 42 °C bath and renatured in the renaturation buffer with varying concentrations of Mg2+ or putrescine2+ as appropriate (see figure legends) for at least 20 min. In the antisense oligonucleotide-dependent experiments, appropriate concentrations of RpoS mRNA and antisense oligonucleotide were renatured in standard renaturation buffer. The renatured RNA was immediately placed on ice, and buffer composition was normalized so that all samples had identical concentrations of divalent cation.
DNA Oligonucleotide--
AS-(1-16) (5'-GAT TTC CCC TTG TAA
C-3') and AS-(101-115) (5'-CAT AAG GTG GCT CCT-3') were purchased from
MWG Biotech (High Point, NC). Oligonucleotides were 5' end-radiolabeled
with [-32P]ATP (Amersham Biosciences) using T4
polynucleotide kinase (Fisher) and purified with Microspin G-25 columns
(Amersham Biosciences) following the manufacturers' instructions.
Ribosome-binding Site Accessibility-- Renatured RNA (under appropriate buffer conditions with or without AS-(1-16)) was diluted in 1× renaturation buffer and added to a fixed amount of radiolabeled AS-(101-115) oligonucleotide probe. The reactions were incubated at 0 °C for 30 min and loaded into a pre-chilled, 8% non-denaturing polyacrylamide gel. Gels were run in 0.5× TBE buffer at 4 °C and 100 V for ~2.5 h. Gels were dried and subjected to autoradiography. Subsequently, radioactive bands were excised and counted using liquid scintillation. "Fraction bound" refers to the fraction of radiolabeled oligonucleotide in the binary (or ternary) complex band divided by the sum of all the radioactivity in the free and bound bands. At least three independent gels were run for each set of renaturation conditions. Binding constants were determined by fitting the fraction bound to a single site binding isotherm (27) using the software program Sigma Plot (SPSS Science, Chicago).
30 S ribosomal subunits were purified from MRE600 cells grown to A600 of 1 (grown at The Johns Hopkins University) (27). Briefly, frozen cells were thawed on ice, resuspended in Buffer A (20 mM Tris-Cl (pH 7.5 at 4 °C), 100 mM NH4Cl, 10.5 mM magnesium acetate, 0.5 mM EDTA, 3 mM 2-mercaptoethanol; 2 ml/g cells), and lysed by passage through a pre-chilled French press (12,000 pounds/square inch) one or two times. Lysed cells were immediately centrifuged at 30,000 × g for 30 min, and the supernatant was retained. The fraction precipitating between 35 and 70% ammonium sulfate (first addition, 0.196 g of ammonium sulfate per 100 ml of supernatant; second addition, 0.22 g/100 ml) was redissolved in 3.3 ml of Buffer A for each 1 g of cells. The resuspended ribosomes were centrifuged at 52,000 rpm (Beckman Ti55.2 rotor) for 140 min, and the pellet material was resuspended overnight at 0 °C in Buffer A. Ribosomes were salt-washed one time by centrifugation through an equal volume of Buffer A plus 15% (w/v) sucrose and 500 mM NH4Cl. 30 S subunits were prepared by sucrose gradient sedimentation of ribosomes in Buffer A with 1.1 mM magnesium acetate. 30 S subunits were renatured in Buffer A at 37 °C for 30 min before use.
Binding Assays--
Equilibrium titrations were performed by
incubating varying concentrations of 30 S ribosomal subunits with a
fixed amount of radiolabeled, renatured RNA at 0 °C in Buffer A
(27). In competition assays, fixed concentrations of 30 S subunits and radiolabeled RNA were added to increasing concentrations of competitor RNA and incubated in Buffer A at 0 °C. 20-µl reactions were
allowed to equilibrate for 30 min and were then filtered onto prewetted nitrocellulose BA85 filters (Schleicher & Schuell). Filters were washed
with 200 µl of Buffer A and then counted by liquid scintillation. Binding constants were determined from (minimally) duplicate binding assays assuming a single site binding isotherm (27) and fit with the
software program Sigma Plot (SPSS Science, Chicago). Fractional
retention values were determined as the counts retained on the filter
at saturating 30 S subunit concentrations divided by the total number
of counts of radioactive RNA applied to the filter.
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RESULTS |
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DsrA Binds to 30 S Ribosomal Subunits--
We examined whether
DsrA ncRNA interacts specifically with the 30 S subunit by performing
a direct binding assay of radiolabeled DsrA ncRNA transcript with 30 S
subunits in binding buffer at 0 °C. The affinity of the 30 S
subunit for the DsrA ncRNA was determined from fits to a single site
binding isotherm with an equilibrium affinity to the 30 S subunit,
K30 S, equal to 2.8 ± 1.4 × 107 M1 (Fig.
2).
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The origin of the interaction between DsrA ncRNA and the 30 S subunit was the focus of our next set of investigations. DsrA binds to the protein Hfq (21, 26), which is associated with ribosomes in E. coli cells (28). Hfq interacts with a variety of RNA and DNA sequences (29, 30). Western blotting analysis using Hfq antisera (the kind gift of Gisela Storz) demonstrated no detectable levels of Hfq in either 30 S ribosomal subunit or 70 S ribosome preparations (data not shown), strongly suggesting that binding of the DsrA to the 30 S subunit resulted from an interaction of the ncRNA with 16 S rRNA or small subunit ribosomal proteins.
To determine which part of the DsrA ncRNA was involved in binding to
the 30 S subunit, we constructed two different DsrA fragments. DsrA-(1-34) includes the sequence that forms the first stem loop (nucleotide 1-34) and contains the hybridization sequence to RpoS mRNA (14). DsrA-(23-87) contains the sequences (nucleotides 23-87) that form the second and third stem loops and the Hfq binding domain of DsrA (26). DsrA-(1-34) can bind to 30 S subunits
(K30 S 0.2 × 107
M1); however, it has weaker affinity than
full-length DsrA (Table I). DsrA-(23-87)
shows some affinity to the 30 S subunit, but with our renaturation
conditions less than 5% of the total RNA participated in 30 S subunit
binding leading to significant error in the quantitative determination
of the binding affinity.
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RpoS mRNA-30 S Ribosomal Subunit Interactions--
We
measured the affinity of the 30 S subunit to RpoS mRNA using
standard nitrocellulose filter binding assays. Binding curves were fit
to a single site binding isotherm, and we found that K30 S, the equilibrium constant for 30 S
subunit-mRNA complex formation, was similar to that measured for
other mRNAs and was ~2 × 107
M1 for mRNA renatured in standard
renaturation buffer, see Table I for data and Fig.
3 for a typical binding isotherm. The
affinity for this mRNA fragment to the 30 S subunit was the
same as the affinity measured for longer fragments (data not
shown).
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The affinities of RpoS mRNA and DsrA noncoding RNA for 30 S
subunits were similar to each other. To determine whether they compete
with each other for binding to the 30 S subunit, radiolabeled RpoS
mRNA was incubated with 30 S subunits and increasing
concentrations of either unlabeled RpoS or DsrA competitor RNA. Both
the RpoS mRNA and the competitor RNAs were renatured independently
as described under "Experimental Procedures." The labeled RpoS
mRNA and 30 S subunits were allowed to equilibrate with competitor
RNA and then were filtered. No competition for 30 S subunit binding
was observed with the DsrA competitor (Fig.
4A), although, as expected, unlabeled RpoS mRNA was an effective competitor for 30 S subunit binding (Fig. 4B). Similar competition experiments with
labeled DsrA, 30 S subunits, and unlabeled tRNA
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Regulation by Increasing Ribosome-binding Site Accessibility-- Hybridization of DsrA to RpoS mRNA has been postulated to stabilize mRNA conformations that are likely to be more translationally active, thereby inducing translation during osmotic stress (16-18). In order to examine the effect of DsrA hybridization on the extent of 30 S-RpoS mRNA complex formation, we examined whether DsrA RNA could form a complex with RpoS mRNA in vitro. We found only a low extent of hybridization of DsrA ncRNA to RpoS mRNA renatured under standard conditions (data not shown). The RNA-binding protein Hfq is necessary for DsrA to affect rpoS translational efficiency in vivo (21). Hfq has been shown to facilitate RNA-RNA interactions and may be required to reduce the effects of competing secondary structures in the DsrA ncRNA and in the RpoS mRNA to allow effective base pairing of the molecules to each other (23, 25, 31).
We designed a DNA antisense oligonucleotide to
simulate the effects of hybridizing a smaller region of DsrA to RpoS
mRNA. In designing the AS-(1-16) oligonucleotide, we desired to
maximize the interaction of the oligonucleotide with the mRNA to
maximize possible effects on regulation. AS-(1-16) is perfectly
complementary to nucleotides 1-16 of the RpoS mRNA structure
(shown in Fig. 1A), which is known to hybridize with DsrA
in vivo (14). By using these gel mobility shifts, we
observed that AS-(1-16) forms a hybrid complex with renatured RpoS
mRNA (Fig. 5A, lanes
1 and 2) with K-(1-16) ~1 × 107 M1 (Table
II). We examined whether this antisense
oligonucleotide would influence the accessibility of the
ribosome-binding site to 30 S subunit binding. Accessibility of the
ribosome-binding site was measured by the ability of the RpoS mRNA
to hybridize to a radiolabeled DNA oligonucleotide, AS-(101-115),
complementary to nucleotides 101-115 of the ribosome-binding site. We
renatured the RpoS mRNA in the presence (or absence) of AS-(1-16),
and we then probed the accessibility of the
ribosome-binding site with AS-(101-115). We used non-denaturing
gel electrophoresis to detect hybrid complexes containing
AS-(101-115). By kinetically trapping the population of renatured
mRNA molecules, we ensured that the differences in the extent of
hybridization with the DNA probe resulted from differences in the
distribution of structures formed during renaturation. We observed
formation of a band that migrates more slowly than the band formed from
the RpoS mRNA·AS-(101-115) binary complex (Fig. 5A,
compare lanes 5 and 8). This slower migrating band provides evidence for formation of a ternary complex involving RpoS mRNA simultaneously bound to both AS-(101-115) and AS-(1-16) (Fig. 5A, lanes 3-5). Renaturation of
RpoS mRNA with AS-(1-16) resulted in increased binding
(1.2-1.8-fold increase) of AS-(101-115) to the mRNA (Fig.
5B), demonstrating that the disruption of intra-molecular mRNA-mRNA base pairs involving nucleotides 1-16 of the
mRNA increased accessibility of the ribosome-binding site. The
affinity of the ribosome-binding site probe AS-(101-115) to RpoS
mRNA in the absence of AS-(1-16) was K-(101-115) = 2.5 ± 0.6 × 106 M
1
and 5.9 ± 2.1 × 106
M
1 for RNA renatured in the presence of
AS-(1-16) (see Table II). Hybridization of RpoS mRNA to AS-(1-16)
increased the RpoS mRNA ribosome-binding site accessibility.
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Effects of an Antisense DNA Oligonucleotide Mimicking DsrA Binding
on the Interaction of RpoS mRNA 30 S Ribosomal Subunit
Interactions--
The increased accessibility of the ribosome-binding
site resulting from hybridization of the RpoS mRNA to AS-(1-16)
suggested a mechanism of translational induction. In a competitive
model of translational regulation by secondary structure, the
ribosome-binding site must be unfolded to interact with the 30 S
subunit (32). If hybridization of DsrA shifts the stability of the
mRNA to favor a larger fraction of translationally active
molecules, then we would expect to observe that a larger fraction of
our total mRNA interacts with saturating concentrations of 30 S
subunits when the mRNA is hybridized to the DsrA-like AS-(1-16)
oligonucleotide than when the mRNA is renatured in its absence. We
postulated that the increased accessibility of the ribosome-binding
site correlates with an increased fraction of translationally active mRNA. To investigate this possibility, we renatured radiolabeled RpoS mRNA with excess AS-(1-16) (0.2-2.0 µM) and
used the hybrid complexes in 30 S equilibrium binding assays. We
observed that hybridization of RpoS mRNA with an excess of the
AS-(1-16) oligonucleotide resulted in an increase in the amount of
total mRNA retained on the nitrocellulose filters at saturating
30 S subunit concentrations (Fig. 6,
A and B). These results suggest that AS-(1-16)
hybridization to nucleotides 1-16 of the RpoS mRNA increases the
fraction of mRNA that is translationally active. This effect may be
very significant in translational induction.
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Divalent Cations Affect Ribosome-binding Site Accessibility and
Fractional Retention of RpoS mRNA--
To understand better the
effects of changing RNA structure on translational regulation, we
examined the effects of altering the concentrations of divalent cations
present during RpoS mRNA renaturation on the ribosome
accessibility, on the equilibrium affinity constant for 30 S subunit
binding, and on the fraction of mRNA participating in translational
initiation. In all of these experiments, the concentration of divalent
cation is varied only during renaturation of the mRNA and is
subsequently normalized to identical standard concentrations at 0 °C
prior to hybridization or binding assays. We have observed that
renaturation of the mRNA in the presence of 40 mM
Mg2+ alters the extent of AS-(101-115) hybridization to
the mRNA relative to the levels measured for renaturation in the
absence of Mg2+ (Fig. 7,
A and B). Results of several sets of experiments
indicate that K-(101-115) = 2.5 ± 0.6 × 106 M1 for RNA renatured without
Mg2+ and K-(101-115) <0.6 × 106 M
1 for RNA renatured with
Mg2+ (see Table II). These results strongly indicate the
formation of different final folded structures for the RpoS mRNA
when renatured under different conditions, and that the
ribosome-binding site is accessible for ribosome binding in a larger
fraction of RpoS mRNA when it is renatured in the absence of
Mg2+.
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Increasing the amount (from 0 to 40 mM) of Mg2+ cations or physiologically relevant putrescine2+ cations present during RpoS mRNA renaturation results in a slight (~20%) reduction in the binding affinity of the 30 S subunits for the RpoS mRNA (see Table I). We observed in our binding assays that when the mRNA is renatured in the presence of increasing concentrations of Mg2+ (or putrescine2+ (not shown)), the fraction of the total mRNA that is retained on the filters at saturating 30 S subunit concentrations decreases. A plot of fractional retention of total mRNA versus Mg2+ concentration present during mRNA renaturation buffer is shown in Fig. 6B.
Fractional retention depends upon the fraction of mRNA participating in binding and the efficiency of retaining complexes on the nitrocellulose filters. Because the solution conditions present during 30 S subunit-RpoS mRNA complex formation and filtration are identical for all reactions, these differences in filter retention must reflect structural/stability differences in the mRNA induced during renaturation. The divalent cation concentration in the renaturation buffer could affect the partitioning of the mRNA into structures that are either translationally active, participating in 30 S binding, or translationally inactive. In this model, the percentage of the total mRNA that binds the 30 S subunit is different for mRNA renatured under different conditions. Alternatively, different renaturation conditions may allow the same percentage of the RpoS mRNA to bind 30 S subunits but alter some subtle aspect of the mRNA structure that affects the ability of a 30 S-mRNA complex to "stick" to the filter.
We used competition assays to distinguish between these possibilities.
If the renaturation conditions affect the ability of a complex to
stick to the filter, then the effectiveness of the competition
for binding to 30 S subunits with the same concentration of unlabeled
RpoS mRNA will be independent of the renaturation conditions of the
unlabeled RpoS mRNA. However, if different renaturation conditions
yield different percentages of RpoS mRNA competent to bind 30 S
subunits, then the renaturation condition of the unlabeled RpoS
mRNA will determine the extent of competition of a given
concentration of unlabeled RpoS mRNA with radiolabeled mRNA for
30 S subunit binding. When renaturation affects the partitioning of
active and inactive structures, renaturation conditions that increase
the concentration of active RpoS mRNA structures (and yield the
highest filter retention) also yield the most effective competition.
Unlabeled mRNA renatured in standard renaturation buffer more
effectively competes with radiolabeled mRNA for binding to 30 S
subunits than mRNA renatured in renaturation buffer containing 40 mM Mg2+ (Fig. 4B), supporting the
idea that increasing the concentration of divalent cation in the
renaturation buffer decreases the fraction of translationally active
mRNA molecules.
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DISCUSSION |
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Early models of translational regulation of rpoS predicted that changes in mRNA structure resulting from bacterial exposure to stress would influence the accessibility of the ribosome-binding site to ribosomes and therefore translational efficiency (3, 19). The non-coding RNA molecule DsrA has been implicated in translational regulation of rpoS expression (15, 33) as a factor influencing the ribosome-binding site accessibility. In this work, we describe our observation that the non-coding RNA DsrA binds to 30 S subunits. We demonstrate that the ribosome-binding site accessibility is modulated through changes in divalent cation concentration during mRNA renaturation as well as the presence of a DsrA-like antisense oligonucleotide that binds to nucleotides 1-16 of RpoS mRNA. The accessibility of the ribosome-binding site correlates with levels of RpoS mRNA participating in forming 30 S-mRNA "pre-initiation" complexes and provides evidence for a competitive model of regulation.
30 S Subunits Bind the Non-coding RNA DsrA--
30 S ribosomal
subunits bind to the ncRNA DsrA specifically, with an affinity
comparable with that of many mRNA molecules for 30 S subunits.
DsrA contains three stem loops (14, 16, 34). The (5') stem loop
contains the sequences that hybridize to RpoS mRNA (14, 16, 34).
The 3' sequences are necessary for the interaction of DsrA with the
protein Hfq (26). Hfq is also involved in regulating translation of
RpoS mRNA. We investigated which part of the DsrA molecule was
responsible for interacting with 30 S subunits. A weak interaction
(K30 S ~2 × 106
M1) is observed between the 30 S subunit and
the first 34 nucleotides of the DsrA sequence that forms the first stem
loop. However, low filter retention of 30 S subunit-RNA complexes
prevented us from accurately measuring the affinity of 30 S subunits
to an RNA transcript containing the 3' end sequences (nucleotides
23-87) of DsrA (data not shown). These results indicate that the full affinity of DsrA for the 30 S subunit (K30 S
~2.8 × 107 M
1) may depend
upon proper folding of secondary and tertiary structure of the entire
DsrA molecule.
The translational regulator protein Hfq has been shown to associate with 70 S ribosomes (28) and interacts with DsrA (21, 26). We expected that Hfq might be associated with our 30 S subunits and be involved in DsrA ncRNA binding. Hfq specifically binds a 3' domain in DsrA (26) and has been shown to interact strongly with DNA sequences (29, 30). Western blot analysis using Hfq antisera demonstrates it is not present at detectable levels in our 30 S subunit preparations and therefore is not responsible for the interaction of the molecules with the 30 S subunit in our in vitro assays.
DsrA binds to 30 S subunits with an affinity comparable with that of
RpoS mRNA to the ribosomal subunit. We propose that RpoS mRNA
and DsrA ncRNA bind to different sites on the 30 S subunit. In
competition experiments between unlabeled DsrA and radiolabeled RpoS
mRNA, 30 S subunits and DsrA ncRNA are both in excess over the
radiolabeled RpoS mRNA. Because DsrA can bind to both 30 S subunits and to the 5' end of the RpoS mRNA, excess DsrA is
required to allow a fraction of DsrA to hybridize the RpoS mRNA and
the rest to be available for 30 S subunit binding. If DsrA and RpoS mRNA bind to the same site of the ribosomal subunit, then at high concentrations of DsrA, RpoS mRNA will be displaced from the 30 S
subunit. We do not observe this. The extent of RpoS mRNA bound to
the 30 S subunit does not decrease even at 400 nM DsrA,
suggesting that molecules are not competing for binding at the same
site on the 30 S subunit. In similar competition experiments, we
observed that tRNA
The interaction of the mRNA with the 30 S subunit is stabilized in large part by interactions between the Shine-Dalgarno sequence on the mRNA and the anti-Shine-Dalgarno sequence on the 3' end of the 16 S rRNA (32). Additional stabilizing interactions arise through contacts with ribosomal protein S1. We examined whether the DsrA ncRNA had any short regions of complementarity to the 16 S rRNA and found no complementary regions where both the ncRNA and the 16 S rRNA were predicted to be single-stranded. Examples of the regions predicted to have perfect Watson-Crick base pairing involve nucleotides 81-87 of DsrA with a conserved single-stranded bulge in the 16 S rRNA (nucleotides 606-612) and nucleotides 6-12 of DsrA with nucleotides 634-640 of the of the 16 S rRNA. The extent of proposed base pairing between DsrA and the 16 S rRNA in these and several other regions is comparable with the base pairing between Shine-Dalgarno sequences with other mRNA sequences, which may account for the similarity in affinity between 30 S subunits for DsrA and for typical mRNA messages. Although a sequence (or sequences) of the rRNA have not been ruled out in DsrA binding, it is possible that ribosomal proteins specifically interact with DsrA.
Hybridization of RpoS mRNA to an Antisense DNA Oligonucleotide Increases Accessibility of the Ribosome-binding Site-- At low temperatures, the presence of the non-coding RNA DsrA increases translational efficiency of RpoS mRNA and is required for full induction of rpoS expression following osmotic shock (15). Hybridization of the ncRNA has been proposed to stabilize a structure of RpoS mRNA that is efficiently translated (14, 16, 18). In the translational induction model, DsrA binds to an upstream RpoS mRNA sequence, preventing specific intra-molecular base pairing interactions in a helix containing the ribosome-binding site. Loss of the intra-molecular base pairs is expected to result in a locally single-stranded ribosome-binding site that is more readily translated than the normal repressed structure.
In vivo work has demonstrated interactions between the
non-coding RNA DsrA and a region of the RpoS mRNA that overlaps
nucleotides 1-16 in our transcript (14). We observe that the affinity
of DsrA for RpoS mRNA was low at 0 °C (data not shown). In
vivo it has been shown that regulation by DsrA requires the
presence of the protein Hfq. Hfq has been shown to facilitate RNA-RNA
interactions and has been implicated in unfolding RNA structures that
prevent formation of binary RNA complexes (11, 20-23, 25, 35). Both DsrA and RpoS mRNA are proposed to have extensive secondary
structures in the regions that complement each other (14, 16, 34). The
interaction of DsrA with RpoS mRNA requires melting out secondary structure in both DsrA and RpoS and, therefore, may be
thermodynamically or kinetically unfavorable in the absence of other
factors (such as the protein Hfq). Even if DsrA had no secondary
structure of its own, it is not perfectly complementary to the RpoS
mRNA leader sequence and therefore has a lower affinity for the
mRNA than a perfectly complementary sequence would have. In order
to make a stronger hybrid complex and to eliminate the requirement for denaturing the DsrA structure, we designed an oligonucleotide, AS-(1-16), that perfectly complements the RpoS mRNA sequence. The
effect of increasing intermolecular complementarity and decreasing self-hybridization is that we have made a molecule that hybridizes to
renatured RpoS mRNA with an affinity of ~9.5 × 106 M1 at 0 °C. We used
this synthetic DNA oligonucleotide, AS-(1-16), to determine whether
its hybridization to RpoS mRNA increases the accessibility of the
ribosome-binding site. As expected, we observed an increase in the
ability of the AS-(101-115) probe to interact with the
ribosome-binding site when RpoS mRNA is hybridized to the
AS-(1-16) DNA oligonucleotide.
Ribosome-binding Site Accessibility Correlates Well with the Fraction of mRNA Participating in 30 S Subunit Binding-- In determinations of the equilibrium affinity of 30 S subunits for RpoS mRNA, we find that less than 50% of the total RNA interacts with 30 S subunits at saturating concentrations. These data provide evidence for two functionally distinct fractions of RpoS mRNA. One fraction of the mRNA (up to about 50% of the total mRNA in our assays) can bind 30 S subunits to form complexes retained on a nitrocellulose filter or can compete with radiolabeled mRNA to bind 30 S subunits. We designate this fraction of the mRNA to be functionally "translationally active." The other mRNA fraction is designated "translationally inactive." It either does not bind to 30 S subunits in the concentration range (0-1.0 µM) we examined or it forms 30 S complexes that rapidly dissociate during the filtration and wash steps of our assay. Controlling the composition of the renaturation buffer of the mRNA can modulate the equilibrium distribution of the two fractions. In the experiments shown here, we have allowed the renaturing mRNA to equilibrate at 42 °C. The rates of interconversion between the "active" and "inactive" forms are greatly reduced by placing the reactions on ice. Differences in the fraction of total mRNA retained on the filter as well as differences in the accessibility to the ribosome-binding site suggest that there are differences in the mRNA structure that persist from renaturation until binding over varied incubation times (15-90 min) indicating that interconversion between different structures is significantly reduced at 0 °C. When the reactions are incubated at 42 °C rather than at 0 °C, we observe a greater extent of hybridization of the RpoS mRNA to AS-(101-116) (data not shown), indicating that at high temperatures, the interaction of AS-(101-115) with the renatured mRNA can shift the equilibrium distribution of mRNA structures. At low temperatures, the mRNA is kinetically trapped so that the inactive form cannot be converted to the active form. Two (or more) populations of RNA structures with different chemical and functional properties is not unusual and has been reported for RNAs as diverse as mRNAs (36) and ribozymes (37).
We observe that renaturation conditions resulting in high levels of AS-(101-115) hybridization to the RpoS mRNA also increase fractional retention of RpoS-mRNA-30 S subunit complexes. These renaturation conditions result in mRNA that is a more effective competitor with radiolabeled RpoS mRNA for 30 S subunits. Renaturation with AS-(1-16) results in a 30-50% increase in the accessibility of the ribosome-binding site and up to an additional 30% of the mRNA participating in binding 30 S subunits determined by fractional retention data. These results are consistent with an interpretation that a ribosome-binding site that more easily interacts with single-stranded DNA is also efficient at 30 S subunit binding and translational initiation.
Divalent Cation Concentration Influences Structure of the mRNA-- We have explored whether the accessibility of the ribosome-binding site is modulated by exposure of the mRNA to different divalent cation concentrations. By using the antisense DNA oligonucleotide AS-(101-115) as a probe, we observed that the accessibility of the RpoS mRNA ribosome-binding site is greater when the mRNA is renatured in standard renaturation buffer than when the mRNA is renatured in buffer containing 40 mM Mg2+. A small reduction in the affinity of 30 S subunits for RpoS mRNA renatured in the presence of 40 mM Mg2+ or 40 mM putrescine2+ was observed. Analysis of filter retention data and competition data indicates that renaturing the mRNA in the presence of 40 mM Mg2+ results in a 30-35% decrease in the amount of the mRNA that is binding at saturating 30 S subunit concentrations relative to that for mRNA renatured in standard buffer. These data again support our conclusion that increased ribosome-binding site accessibility correlates with an increased fraction of mRNA participating in translational initiation events.
Translational Repression and Induction-- Translational regulation of rpoS appears to be consistent with a competitive (also called "displacement" (38, 39)) model of regulation. Although small differences are detected in the affinity of both 30 S subunits and AS-(101-115) for the differentially renatured mRNA forms (see Table I), the primary effect of altering renaturation conditions appears to be to change the fraction of the mRNA participating in translational initiation events.
Translational efficiency is evaluated from the rate of protein
synthesis and may be proportional to the concentration of a stable
ternary "Initiation Complex" composed of initiator
tRNA
|
In a competitive model of translational repression, an mRNA
structure forms that prevents the 30 S subunit from interacting with
the ribosome-binding site of the mRNA (32, 38, 39). In this
mechanism, only a fraction of the total mRNA molecules, those with
an accessible ribosome-binding site, participate in the formation of a
pre-initiation complex, and therefore in translational initiation.
Under conditions of thermodynamic control, the distribution between
mRNA molecules available to productively participate in initiation
is determined by the equilibrium constant between these active and
inactive structures (32, 38). We have no evidence that translational
regulation is under kinetic control (39), and therefore we are assuming
that the simpler model of thermodynamic control applies to repression
of rpoS expression. In this model, the level of gene
expression, E (measured in vivo as the relative rate of protein synthesis and in vitro as the fraction of
mRNA binding 30 S subunits), can be described by Equation 1,
![]() |
(Eq. 1) |
We applied the thermodynamic competitive model to our in
vitro fractional retention data using the value of
K30 S we measured (2 × 107
M1) and a 30 S subunit concentration (8 µM) expected to be physiologically relevant (32). The
analysis suggests that the difference between fractional retention for
mRNA renatured in the presence and absence of 40 mM
Mg2+ is explained by a value of Kf in
renaturation buffer containing 40 mM Mg2+ that
is 1.6-fold larger than Kf value in standard
renaturation buffer and a free energy difference of 300 cal/mol between
the RNA samples. (Small differences in our estimates of
K30 S (over the range of values observed at low
and high Mg2+ concentrations) did not significantly change
the differences in Kf observed.) Hybridization of
0.2 µM or 2.0 µM AS-(1-16) to RpoS
mRNA results in a destabilization of the mRNA structure by
another 470 or 960 cal/mol, respectively, assuming that the affinity of
30 S subunits to the mRNA is not significantly altered by the
hybridization (see Table III).
|
We also used this approach to analyze in vivo expression
data. Again we assumed that K30 S = 2 × 107 M1 and that the 30 S subunit
concentration is 8 µM, and that maximal expression,
E = 1, corresponded to the most highly expressed
variant of rpoS, SD2, as reported in the work of Cunning
et al. (11). In vivo, expression of the wild-type
sequence was reduced to 17% of maximal (i.e. SD2)
expression under normal growth conditions and to 41% under osmotic
shock conditions (11). Sledjeski and co-workers (21) found that a DsrA
mutant decreased expression from wild-type cells grown under normal
growth conditions almost 5-fold (to ~4% of SD2 expression).
By using the in vivo expression data, we predict that the
RpoS mRNA structure (in the absence of Hfq) formed during osmotic stress is more open (less stably folded). We predict that the value of
G is ~760 cal/mol higher than for the mRNA
under normal growth conditions (see Table III). Osmotic stress reduces
the intracellular concentration of divalent cations and leads to a
change in
G that is in the same direction as that
predicted from in vitro data for RpoS mRNA folded in the
presence and absence of Mg2+ in the absence of any
trans-acting factors. In the absence of functional DsrA, where
rpoS expression is reduced, we predict an increase in the
stability of the translationally inactive mRNA conformation of 960 cal/mol from in vivo data, similar to our estimates from the
in vitro data. Although our in vitro conditions are quite dissimilar to the in vivo cytoplasm, our data
suggest that at least part of the osmotic induction of RpoS mRNA
translation may be regulated simply by changes in cytoplasmic ion
concentration. Binding of DsrA to the mRNA clearly has a similar
enhancing effect on the fraction of mRNA participating in
translational initiation both in vivo and in
vitro. The effects (in addition to the effects of other regulatory
factors) result in induction of the rpoS expression.
Both Hfq and DsrA have been implicated in regulating osmotic induction of RpoS mRNA translation. However, in the absence of the trans-acting regulatory factors Hfq and DsrA, in vivo translation of RpoS mRNA can be partially induced by osmotic shock (11, 13). During osmotic upshock, the composition of ions in the cytoplasm of E. coli is greatly altered: K+ concentration increases 4-fold from 0.2 to 0.8 M and concentrations of the divalent cation putrescine decrease from 0.05 to 0.01 M (4, 6). Although we are not attempting to reproduce physiological conditions, our experiments demonstrate that reduction in the concentration of the divalent cations putrescine2+ or Mg2+ increases the ribosome-binding site accessibility and increases the fraction of mRNA participating in translational initiation. It is plausible that some of the initial translational induction by osmotic shock results from mRNA conformational changes. The effects that we have observed are in the same direction as those predicted to occur during osmotic induction in vivo. Divalent cations have been shown, in vitro, to affect the secondary and tertiary structures of RNAs over the salt concentration ranges predicted to be relevant in vivo (40-43). These conformational changes (affecting both structure and thermodynamic stability of the mRNA) are likely to influence the ability of the mRNA to participate in translational initiation. Although changes in the composition of ions in the cell may affect the structure and stability of other mRNAs in vivo, only genes where the rate-limiting step in gene expression is translational initiation caused by an inhibitory mRNA structure will be significantly affected by osmotic shock. Additionally, rpoS may be particularly suited to osmotic induction since specific regulatory factors may amplify the effects of mRNA structural and stability changes caused by osmotic stress. van Duin and co-workers have elegantly, and repeatedly, shown that stabilization of secondary structure which sequesters the ribosome-binding site results in decreased translational efficiency (36, 38). Our data support the proposal that the stability and structure of the RNA itself is likely to be modulated as a result of osmotic stress (3) directly influencing translational induction of the gene.
Why Does DsrA Interact with 30 S Subunits?-- The function of the interaction of DsrA with the 30 S ribosomal subunit is presently unknown. At 25 °C, full-length DsrA has an extraordinarily long half-life (~23 min at 25 °C), but a truncated form of DsrA isolated from in vivo cultures has a half-life of only 5 min (44). The truncated form of DsrA lacks the 5' sequence that we found binds to 30 S subunits, and we propose that association with the ribosome is responsible for stabilizing DsrA in vivo.
RpoS mRNA is tightly folded and requires the action of several trans-acting factors for full translational induction. Base pairing between DsrA and RpoS mRNA is necessary for translational activation by DsrA (14). It has been shown that the effects of RprA, OxyS, and DsrA non-coding RNAs on rpoS expression are much greater in the presence of the protein Hfq (9, 11, 20-23). The role of Hfq in global regulation of gene expression increasingly appears to be a facilitator of RNA-RNA interactions that influence rates of translation and degradation of a given mRNA (23-25). Hfq may form a ternary complex with both the ncRNA and mRNA to facilitate their interaction (23), or may transiently alter an RNA structure to promote ncRNA-mRNA interactions (21). The ability of the Hfq protein and an ncRNA to interact with RpoS mRNA, either simultaneously or sequentially, appears to be required in regulating the translation of the wild-type RpoS mRNA sequence.
Although one role of the interaction of DsrA with the ribosomal subunit may be to increase the half-life of the ncRNA, we propose that the association of DsrA with the 30 S subunit is mechanistically important in the translational regulation of RpoS mRNA. The interaction between 30 S subunits and DsrA may serve to increase the local concentration of DsrA with Hfq and/or RpoS mRNA. Localizing DsrA to the 30 S subunit may accelerate the interaction of DsrA with ribosome-associated Hfq protein and/or RpoS mRNA, increasing the rate of interactions required for translational induction of RpoS mRNA.
The interaction between DsrA and 30 S subunits may serve to lock the DsrA ncRNA or the 30 S subunit in a conformation that prevents it from prematurely interacting with either Hfq or the RpoS mRNA. The 3' end of DsrA is required for interactions with Hfq (21). The 5' end of DsrA binds both RpoS mRNA (14, 17) and weakly to the 30 S subunit. Because Hfq is associated with the ribosome, we speculate that Hfq binds to the 3' end of the ncRNA and assists in the transfer of the 5' end of the ncRNA from a binding site on the 30 S subunit to its binding site on the mRNA. DsrA structural changes induced by binding Hfq could decrease the affinity of DsrA to the 30 S subunit and increase the probability of a DsrA-RpoS mRNA interaction. As the ncRNA anneals to the RpoS mRNA, the mRNA unfolds and the ribosome-binding site becomes single-stranded and is immediately stabilized by an interaction with the anti-Shine-Dalgarno sequence of the poised 30 S subunit. As the 30 S subunit clears the ribosome-binding site during initiation, DsrA may re-associate with the 30 S subunit to allow the ncRNA to serve as a catalyst for translational initiation of RpoS mRNA.
Both Hfq and DsrA are required to regulate expression of H-NS protein (14). DsrA binding to hns mRNA decreases the half-life of the hns mRNA significantly (18), possibly as a result of translational repression or through inducing exposure of RNase-sensitive sites. The DsrA sequences involved in binding to the hns mRNA are downstream from the sequences involved in binding RpoS mRNA, suggesting that the interaction of DsrA with the 30 S subunit may have different effects on hns and rpoS regulation.
The molecules regulating translational initiation of RpoS mRNA are
known, but the choreography of their interactions is just beginning to
be characterized. The interactions of DsrA with 30 S subunits must be
considered in understanding regulation of translation. Further studies,
characterizing the timing and strengths of association of appropriate
complexes, may provide insight into the mechanism by which a stress is
sensed, and s levels are increased.
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ACKNOWLEDGEMENTS |
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Maine Medical Research Institute provided use of the French press, and Bowdoin College allowed the use of centrifuge rotors for 30 S subunit purification. Scott Morrow at The Johns Hopkins University grew the MRE600 cells used in 30 S subunit preparation. We thank Anne Barlow, Amanda Colby, Steve Hallas, Mike Mroz, Adam Rives, and Chuck Tucker for their laboratory assistance. We also thank Dr. Katherine Covert, Dr. David Draper, Dr. Carol Gross, Dr. T. Glen Lawson, and Dr. Peter E. Schlax, Jr., for support and extremely useful discussions and comments.
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FOOTNOTES |
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* This work was supported in part by Research Corporation Cotrell College Science Award CC5038 and by grants from the Howard Hughes Medical Foundation and the SURDNA Foundation, which were awarded to Bates College.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence should be addressed. Tel.: 207-786-6290;
Fax: 207-786-8336; E-mail: pschlax@bates.edu.
Published, JBC Papers in Press, February 24, 2003, DOI 10.1074/jbc.M301684200
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ABBREVIATIONS |
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The abbreviation used is: ncRNAs, non-coding RNAs.
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