| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
(Received for publication, September 22, 1995; and in revised form, December 1, 1995) From the
We have investigated the highly conserved GAUCA sequence of
small subunit ribosomal RNA. Within this region, the invariant
nucleotides G1530 and A1531 of Escherichia coli 16 S rRNA were
mutagenized to A1530/G1531. These base changes caused a lethal
phenotype when expressed from a high copy number plasmid. In low copy
number plasmids, the mutant ribosomes had limited effects when
expressed in vivo but caused significant deficiencies in
translation in vitro, affecting enzymatic tRNA binding,
non-enzymatic tRNA binding, subunit association, and initiation factor
3 (IF3) binding. Mutant 30 S ribosomal subunits showed a 10-fold
decrease in affinity for IF3 as compared to wild-type subunits but
showed an increased affinity for IF3 when in 70 S ribosomes.
Additionally, IF3 did not promote dissociation of 70 S ribosomes, which
had mutated subunits as monitored by light-scattering experiments.
However, extension inhibition experiments (toeprinting) showed that IF3
retained its ability to discriminate between initiator and elongator
tRNAs on mutated subunits. The results indicate that the two functions
of IF3, tRNA discrimination and subunit dissociation, are separable and
that the invariant nucleotides are important for correct subunit
function during initiation.
Ribosomal RNA (rRNA) plays a significant role in the process of
translation, and specific rRNA regions have been implicated in several
translational functions(1, 2) . The best evidence for
direct involvement of rRNA is the base-paired interaction between the
polypurine Shine-Dalgarno (SD) (
Figure 1:
Location of the
A1530/G1531 mutations. Secondary structure map of E. coli 16 S
rRNA (7) with the G to A base change at position 1530 and the A
to G base change at position 1531.
The conserved
nature of the GAUCA sequence implies a functional significance for
these nucleotides. However, little is known about the role of this
site. In experiments done with E. coli ribosomes, kethoxal
modification of nucleotide G1530 moderately inhibited subunit
association (10) while nucleotide A1531 displayed enhanced
reactivity toward chemical modification upon 50 S subunit
binding(11) . Nucleotide A1531 also displayed enhanced
modification upon subunit inactivation(12) , a reversible
conformation change associated with monovalent or divalent cation
depletion. The site is protected from nuclease attack by initiation
factor 3 (IF3) binding (13) , and the adjacent stem structure
has been cross-linked to both IF3 (14) and 23 S
rRNA(15) . Recently, nucleotide G1530 has been cross-linked to
mRNA between the Shine-Dalgarno region and the AUG start
codon(16) . Based partially on the conserved nature of the
region, Kössel et al.(17) proposed that an interaction occurs between the GAUCA
sequence and the 5`-end of 16 S rRNA as a discrete functional state
during elongation, whereas Thanaraj and Pandit (18) have
proposed that the GAUCA sequence functions as a translational enhancer
by base pairing with a complementary sequence in mRNA upstream of the
start codon. The conserved nature of the GAUCA sequence and its
proximity to the anti-SD region, the decoding site, and the terminal
helix place it in the center of a very important functional region of
the 30 S subunit. Here, we describe experiments designed to investigate
the function of the GAUCA sequence, especially the invariant G1530 and
A1531 residues. We have switched the order of these two purines on a
plasmid-borne copy of the rrnB operon and assayed the effects of the
mutations in vivo and in vitro. We found that
expression of mutant 16 S rRNA affected several subunit functions in vitro, including initiation complex formation, subunit
association, and IF3 binding.
Figure 2:
Primer extension analysis of A1530/G1531
subunit re-association gradient fractions. Ribosomal RNA was isolated
from the 30 and 70 S fractions of subunit re-association gradients
containing 1.5, 5, 10, or 15 mM MgCl
The ability of 30 S
subunits containing mutant rRNA to associate with 50 S subunits to form
70 S ribosomes was analyzed as described (27) except that
MgCl
Fluorescence and light-scattering
measurements were performed on a Spex Fluorolog The equilibrium binding constants for IF3/ribosome
interactions were determined by monitoring the change in fluorescence
intensity as FITC-labeled IF3 (FITC/IF3) bound to ribosomes. IF3 (1
µM) was titrated with increasing amounts of ribosomes in
buffer 1 containing 1 mM MgCl
where [A] is the concentration of the
uncomplexed FITC/IF3, [B] represents the free
ribosomal subunits, and [C] is the FITC-IF3-ribosome
complex. Normalized fluorescence is defined by :
where F
where K is the equilibrium binding constant, [A]
where R
where K = K`(òn/òc) To remove dust, all
samples (1.2 ml of a 0.19 µM solution of 70 S ribosomes)
were filtered through a sterile 0.22-µm Millex-GV filter unit
(Millipore). Excitation and emission monochromators were set to 560 nm.
Intensity of the light was monitored as ribosomes were titrated with
increasing amounts of IF3.
The
distribution of plasmid-encoded rRNA in 30 S subunits, 70 S ribosomes,
and polyribosomes was determined by primer extension analysis (24) utilizing the identity of nucleotide 1192 in 16 S rRNA to
distinguish between plasmid-encoded (U1192) and chromosomally (C1192)
encoded rRNA. The results of primer extension analysis of rRNA
recovered from polysome gradients is shown in Table 1. The
distribution of rRNA was similar from both wild-type and mutant
plasmids. The presence of pMFM161-encoded rRNA in the polysome fraction
indicated that mutant rRNA was assembled into 30 S subunits and
actively participated in translation. The fact that mutant rRNA was
present in polysomes at near wild-type levels and that growth rates
were only slightly decreased by the mutant plasmid indicated that
mutant ribosomes were not interfering with translation by wild-type
ribosomes.
Figure 3:
Enzymatic f[
Figure 4:
Monitoring subunit dissociation function
of IF3. Wild-type and mutant tight couple 70 S ribosomes were subjected
to increasing concentrations of IF3. As ribosomes are dissociated into
free subunits, light-scattering intensity decreases.
Another function of IF3 is to discriminate
between an elongator tRNA and an initiator tRNA binding on the 30 S
subunit during initiation complex formation. We used extension
inhibition analysis (toeprinting) to analyze this function of IF3 on
mutant subunits. In this system, the complex between T4 gene 32 mRNA,
tRNA, and 30 S subunits halts the extension of a primer annealed
downstream on the mRNA and results in a characteristic band (the
toeprint) when the assay is examined by gel
electrophoresis(28, 29) . Under the conditions used in
this experiment, the toeprint was strictly dependent on bound tRNA. In
the presence of tRNA
Figure 5:
Toeprint analysis of the IF3-dependent
tRNA discrimination function. Toeprint reactions were carried out as
described (28, 29) using a constant amount of 30 S
subunits (1 pmol) containing either wild-type (wild type 30S) or
A1530/G1531 mutant (mutant 30S) rRNA, excess
tRNA
Expression of the A1530/G1531 mutant rRNA produced severe
effects on translation. It was lethal when expressed from a high copy
number plasmid, and even at low copy it had significant, yet subtle,
effects on growth rate. In vitro, the mutant subunits
displayed explicit functional defects, the most dramatic involving IF3
binding and function. IF3 is bifunctional. It promotes proper selection
of the initiator tRNA by recognizing specific determinants on
tRNA Tapprich and co-workers (27) showed that the 10-fold decrease in IF3 binding seen for
the mutation at 791 was entirely due to a 10-fold increase in off-rate (k The decrease in enzymatic poly(A,G,U) f[ Finally, the conserved nucleotides between
1529-1534 have been proposed to base pair with mRNA and function
as a translational enhancer (the TP interaction, (18) ). Using
compensatory base change analysis, we have compared the in vivo expression of a lacZ reporter gene containing a TP site upstream
of the SD sequence, which was complementary to either the wild-type or
A1530/G1531 mutant 16 S rRNA. No significant change in expression was
observed (data not shown). Furthermore, if the region was functioning
as a translational enhancer, we might expect differences in spot
intensities on two-dimensional protein gels corresponding to increased
or decreased complementarity to specific mRNAs, much as was seen in
experiments involving Shine-Dalgarno mutants(41) . The fact
that we see no differences in relative intensities for proteins in
two-dimensional gels (data not shown) together with the results of
compensatory base change analysis indicates that expression of
different proteins was not altered and does not support the
translational enhancer model.
Volume 271,
Number 9,
Issue of March 1, 1996 pp. 4693-4698
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)sequence in mRNA and the
polypyrimidine anti-SD region at the extreme 3`-end of 16 S rRNA during
translational initiation (3) and
elongation(4, 5) . The SD interaction does not occur
in eucarya, and the anti-SD region is conserved only in archaeal and
bacterial rRNA(6) . However, the GAUCA sequence (nucleotides
1530-1534 in Escherichia coli 16 S rRNA, see Fig. 1) immediately upstream of the anti-SD region is highly
conserved in all three domains (8) and includes invariant
nucleotides at positions 1530 and 1531(9) .
Bacterial Strains and Plasmids
Strain SU1675
((F` lacI
Tn::5 kan
) recA 
(lac-pro) thi ara), a derivative
of CSH26 (19) was used routinely as host in this study. Strain
MDA6646 (FilvB1202 ilvH2202 rbs221 ara thi
(lac-pro) gpt pcnB recA)
(a gift from Dr. E. J. Murgola) was used during the initial cloning to
reduce plasmid copy number. Strain XL-1 Blue (Stratagene) was used to
propagate M13 phage, and the ung dut strain CJ236 (Bio-Rad)
was used to prepare uracil containing M13 DNA. The intact rrnB operon was carried in pKK3535, a pBR322-derived high copy
plasmid(20) , and in pMO11, a derivative of pSC101, a low copy
plasmid(21) . A single C to U nucleotide change at position
1192 in the 16 S rRNA gene in both pKK3535 and pMO11 confers
spectinomycin resistance yielding pKK1192 and pMM1192, respectively.
The A1530/G1531 16 S rRNA mutations were carried on plasmids pMFK161 (a
derivative of pKK1192) and pMFM161 (a derivative of pMM1192). The rrnB operon in all plasmids was transcribed from the
constitutive PP
promoters.Mutagenesis and Ribosome Preparation
The
A1530/G1531 mutation was constructed and ribosomes and ribosomal
subunits were prepared basically as described(22) .
Polyribosomes were prepared as described (23) and separated by
sucrose density gradient centrifugation using polysome buffer (20
mM Tris-HCl, pH 7.6, 100 mM KCl, 15 mM MgCl, 1 mM dithiothreitol). Fractions were
collected and precipitated with ethanol. The proportion of
plasmid-encoded rRNA in total cellular RNA, 30 S subunits, 70 S
ribosomes, and polysome preparations was determined by the method of
Sigmund et al. (24) . A P 5`-end-labeled
primer complementary to 16 S nucleotides 1193-1216 was used as
described in the legend of Fig. 2. Autoradiograms were scanned
using an LKB Ultroscan XL laser densitometer.
. The samples
were analyzed by the primer extension method of Sigmund et al. (24) using the entire amount of rRNA isolated from each peak,
an excess of labeled primer, and dCTP, dGTP, dTTP, and ddATP. A
one-base extension corresponded to U1192 (plasmid-encoded rRNA). If a
cytidine residue was encountered by the polymerase at position 1192
(chromosomally encoded rRNA), extension continued to the next uridine
residue, at position 1189, resulting in a four-base
extension.
In Vitro Assays
Aminoacylation and formylation of
tRNA (Sigma) were carried out as
described (25) except that a commercial synthetase mix was used
(Sigma). Binding of f[
H]Met-tRNA
to 30 S subunits was carried out as described (26) except that
the mRNA used was a random copolymer of adenosine, guanosine, and
uracil (poly(A,G,U), Sigma, 50 pmol), which contained a random
distribution of AUG start codons as well as SD sequences. IF1, IF2, and
IF3 were gifts from Dr. Claudio Gualerzi.
concentrations indicated in the Fig. 2were
used. Toeprint analysis was carried out as described(28, 29) using free 30 S subunits (93% mutant rRNA) and
bacteriophage T4 gene 32 mRNA.IF3 Binding Experiments
IF3 was labeled with
5`-fluorescein isothiocyanate (FITC) (Molecular Probes). IF3 (1
µg/µl), dissolved in 0.01 N NaHCO, was
incubated with a 5-fold molar excess of FITC solution in freshly
distilled MeSO for 2 h at 4 °C. The total volume was 50
µl. The sample was applied to a 1-ml Sephadex G-25 column
equilibrated with 50 mM Tris-HCl, pH 7.6, 250 mM KCl,
0.5 M NH
Cl, 5% glycerol. The molar ratio was
determined as described previously(30) . Experiments were
performed in 10 mM Tris-HCl, pH 7.8, 50 mM KCl, 6
mM 2-mercaptoethanol, and MgCl as described under
``Results.''2 fluorometer,
which employed a 450-watt mercury-xenon lamp. In steady-state mode, the
excitation monochromator entrance and exit slits were 1.4 mm, while the
emission monochromator entrance and exit slits were 2.0 mm. A 1.25-mm
variable slit in the path of the light was used to diffuse the incoming
light into the sample (preventing photobleaching) and reference
detectors.
. Fluorescence
excitation was 488 nm, and emission was monitored at 520 nm. The
binding of FITC/IF3 to ribosomes was assumed to proceed through the
mechanism A + B &lrarr2; C, where A is
the FITC/IF3, B is the ribosomal subunit, and C is
FITC-IF3-ribosome complex. defines the binding equilibrium
constant:
is the fluorescence end point
and F
is the initial fluorescence point. The
following conservation equations were used: [A] = [C] + [A] and [B]
= [C] + [B]. By making the appropriate substitutions into and and converting the solved equation into
quadratic form, results. Data were fit by ENZFITTER
(Elsevier Science Publishers BV) using :
is the total FITC/IF3, and [B]
is the total ribosome concentration after
each addition. The data were fit by allowing K, F
, and [A]
to
vary. The large errors in this experiment result partly from allowing
the concentration of IF3 to vary. Small differences in [A]
change K
. As IF3 is
a notoriously sticky protein, it is difficult to work with, and the
concentration was known to be a relatively imprecise ±5%.
Light Scattering
gives the expression
for light scattering(31) :
is Rayleigh's ratio (a
measure of the intensity of the scattered light), c is the
concentration in g/ml, M is the molecular weight, n is the refractive index, and K` is an instrumental
constant. is valid as long as the diameter of the
molecules to be studied is smaller than a tenth of the wavelength of
the incident light, the second virial coefficient can be neglected, and
no significant depolarization occurs. These assumptions were previously
discussed with regard to the ribosome system(31) . can therefore be rewritten as follows:
and
is valid as long as no significant depolarization occurs; c` represents concentration (moles/liter).
Expression of the 16 S rRNA Mutation
G1530A/A1531G
Two invariant nucleotides (G1530/A1531)
immediately 5` to the anti-Shine-Dalgarno sequence in 16 S rRNA were
changed to A1530 and G1531 (Fig. 1) by oligonucleotide-directed
mutagenesis. The mutant DNA from M13 was first subcloned into the high
copy plasmid pKK3535. Ligations were transformed into both strain
SU1675 and strain MDA6646 (pcnB), which reduces the copy
number of pBR322-derived plasmids. Transformants were isolated from the pcnB strain, but no clones were obtained in strain SU1675,
suggesting that the mutant rRNA was lethal at high gene dosage. Because
the mutant plasmid was stably maintained in a low copy system, the
mutations were subcloned into a pSC101-derived vector, pMM1192. This
low copy plasmid contains the complete E. coli rrnB operon
with the wild-type PP promoters. The resulting
plasmid carrying the A1530/G1531 mutations was called pMFM161. Both
pMFM161 and pMM1192 carried a second mutation at position 1192 (C to U)
in 16 S rRNA as a marker for plasmid-encoded rRNA. The doubling time
for SU1675 pMFM161 was 54.2 ± 2.3 min compared to 47.7 ±
0.5 min for SU1675 pMM1192 (wild type). Thus, expression of the
A1530/G1531 mutations, which were lethal at high copy, caused a small
but detectable deleterious effect on translation at low copy.
Subunit Association of A1530/G1531 Ribosomes
To
investigate the effect of the A1530/G1531 mutation on subunit
association, salt-washed 30 and 50 S subunits were prepared and
re-associated by incubating at 37 °C in the presence of 1.5, 5, 10,
or 15 mM MgCl. Free subunits and 70 S ribosomes
were then separated by sucrose density gradient centrifugation in the
same buffer conditions. Primer extension analysis of the rRNA from each
30 and 70 S fraction is shown in Fig. 2. The data indicate that
the 30 S peaks were enriched with mutant rRNA. Thus, subunits
containing the A1530/G1531 mutations were deficient in the ability to
form 70 S ribosomes. It is important to note, however, that these
subunit re-association assays were carried out in vitro,
performed in the absence of translation factors, mRNA, or tRNA. Sucrose
density gradients of polysome samples (see Table 1) showed mutant
rRNA was present in ribosomes and polysomes. Apparently, during
initiation of translation in vivo, the subunit association
defect of the mutant subunits was suppressed.In Vitro Initiation Complex Formation on A1530/G1531
Subunits
Given the proximity of the mutant nucleotides to the
anti-Shine-Dalgarno region and the IF3 cross-link site(14) , it
was possible that the A1530/G1531 mutations would affect events of
translational initiation. We therefore analyzed the ability of mutant
subunits to form the initiation complex in vitro. Activated
salt-washed 30 S subunits were used in poly(A,G,U)-directed f[H]Met-tRNA
binding assays in the presence of the three initiation factors. The
mutant subunit preparation used in this experiment contained 65%
plasmid-encoded (mutant) rRNA. As can be seen in Fig. 3, tRNA
binding to mutant subunits was severely reduced relative to wild-type
subunits. Indeed, the overall reduction in tRNA binding was almost
equal to the amount of mutant rRNA present in the subunit preparation,
suggesting that the A1530/G1531 mutations resulted in a complete
inability to bind f[
H]Met-tRNAin vitro.
H]Met-tRNA
binding to wild-type and A1530/G1531 mutant subunits. Increasing
amounts of f[
H]Met-tRNA
were bound to a constant amount of activated 30 S subunits from cells
containing wild-type (
) or mutant (+) rRNA plasmids in the
presence of excess IF1, IF2, IF3, and poly(A,G,U) mRNA. Reaction
mixtures were filtered through nitrocellulose, and the radioactivity
retained on the filters was determined by scintillation counting.
Approximately 65% of the subunits from cells containing the mutant
plasmid were the A1530/G1531 mutant subunits as determined by primer
extension.
Initiation Factor 3 Binding to A1530/G1531 30 S Subunits
and 70 S Ribosomes
Because IF3 has a central role in
translational initiation and has been cross-linked very near
(nucleotides 1506-1529) to the mutagenized
nucleotides(14) , the binding of IF3 was examined in more
detail. Steady-state experiments were carried out in which the change
in fluorescence intensity as FITC-labeled IF3 bound to ribosomes and 30
S subunits was monitored to determine equilibrium binding constants. In
these experiments, we took advantage of the propensity of mutant
ribosomes to dissociate at lower magnesium ion concentration to obtain
a subunit fraction enriched in mutant rRNA. Samples of dissociated
mutant ribosomes, which contained both chromosomally encoded
(wild-type) rRNA and plasmid encoded (mutant) rRNA, were re-associated
at 10 mM MgCl. Under these conditions,
chromosomally encoded 30 S subunits readily associated with 50 S
subunits to form 70 S ribosomes leaving the 30 S fraction enriched in
mutant subunits. The resulting mutant 30 S samples contained 95%
plasmid-encoded (mutant) rRNA. However, tight couple 70 S ribosomes
used in steady-state experiments contained only 50% mutant rRNA. The
FITC-IF3-ribosome binding constants are summarized in Table 2.
FITC-IF3 bound to wild-type 30 S subunits with a K of 27
10
M and to
wild-type 70 S ribosomes with a K
of 0.30
10M. Both of these equilibrium
constants agree with previously reported
values(27, 31) . FITC-IF3 bound to mutant 30 S
subunits (95% mutant rRNA) with an equilibrium association constant of
7
10M, a 3-fold
decrease in equilibrium affinity compared to wild-type 30 S subunits.
Surprisingly, the affinity of FITC-IF3 for mutant 70 S particles was
increased dramatically, approximately 30-fold relative to wild-type
ribosomes (K
= 10
10M, 50% mutant rRNA). The affinity of
IF3 for wild-type 30 S subunits is 100-fold greater than for wild-type
70 S ribosomes; however, we saw essentially no difference in the
affinity of FITC-IF3 for mutant 30 S subunits or mutant 70 S ribosomes, i.e. IF3 bound equally well to both mutant 30 S and mutant 70
S particles.
Analysis of IF3 Function on A1530/G1531
Ribosomes
IF3 shifts the equilibrium toward dissociation of 70 S
ribosomes into free subunits(32) . This phenomenon can be
visualized by observing the change in light scattering after the
addition of increasing amounts of IF3 to 70 S ribosomes. As the 70 S
dissociates into 30 and 50 S subunits, the intensity of light
decreases. Light-scattering experiments using wild-type and A1530/G1531
mutant 70 S ribosomes are shown in Fig. 4. As expected, at
increasing concentrations of IF3 the dissociation of wild-type tight
couple 70 S ribosomes into 30 and 50 S components occurs. However, upon
addition of IF3 to mutant tight couple 70 S ribosomes (50% mutant
rRNA), little change in the intensity of the light was observed,
indicating that little or no dissociation of mutant 70 S ribosomes
occurred and suggesting that the IF3 dissociative function was
compromised. Because of the 30-fold higher affinity of mutant ribosomes
for IF3, the IF3 was effectively sequestered, and dissociation of
wild-type ribosomes was not observed until much higher concentrations
of IF3 were added.
represents
light-scattering intensity of wild-type 70 S ribosomes, and 
represents mutant 70 S light-scattering
intensity.
and tRNA
(which decodes the second codon), two stops were observed. A
toeprint band corresponding to position +16 of T4 gene 32 mRNA
(numbering the A of the AUG start codon as +1) was seen
corresponding to tRNA
bound in the
P-site, and a second stop was at +19 corresponding to tRNA
bound at the P-site. As increasing amounts of IF3 were added to
mutant subunits in the presence of both tRNAs, the tRNA
toeprint band decreased, leaving only the
tRNA
toeprint (Fig. 5). This
demonstrates that the tRNA discrimination function of IF3 was not
disrupted by mutant subunits.
and tRNA
(2.5 pmol
each), and increasing amounts of IF3 (0.03, 0.19, 0.48, and 1.2) in a
final volume of 10 µl. MET, toeprint bands corresponding
to tRNA
bound in the P-site. PHE, toeprint bands corresponding to tRNA
bound
in the P-site. EXT, control toeprint reaction without tRNA or
IF3.
and promotes subunit dissociation by
preferential binding to free 30 S
subunits(32, 33, 34) . IF3 is an elongated
protein consisting of two separate domains (35) and has been
shown by footprinting (13, 36) and cross-linking (14) studies to interact with the 30 S subunit at both the
central and the 3`-minor domains of 16 S rRNA (see (2) and
references therein). The central domain binding site includes the 700
region, the 790 loop, and the 840 stem while the 3`-binding site
includes the 1500 region and the 3`-terminal helix. These sites of IF3
interaction with 16 S rRNA correlate well with IF3 function as the 700
region, the 790 loop, and the terminal helix have all been implicated
in subunit association and are clustered in the vicinity of the P-site (2) . Additionally, a mutation at 791 not only resulted in a
10-fold decrease in IF3 binding, an effect remarkably similar to the
effect seen in the A1530/G1531 mutant, but also resulted in decreased
subunit association(27) , further linking the two functions to
the central domain. The 3`-end of 16 S rRNA has also been implicated in
subunit
association(2, 9, 14, 37, 38) .
In the present study, the toeprint data (Fig. 5) indicate that
the A1530/G1531 mutation does not alter IF3's ability to
discriminate between initiator and elongator tRNAs. However, subunit
dissociation is affected. The binding of IF3 does not cause
dissociation of mutant 70 S ribosomes, a particularly interesting
result since mutant ribosomes have a propensity for dissociation. Thus,
it appears that the mutations at 1530 and 1531 uncouple the two
functions of IF3 on the 30 S subunit.
), with on-rate (k
)
remaining unchanged. This led to their proposal that IF3 can interact
with either binding site but requires a specific set of interactions,
including G791, to form a stable complex. This model is supported by
studies in which IF3 was bound to 30 S subunits lacking the 3`-colicin
E3 fragment (nucleotides 1493-1542) in which only weak IF3
interactions were seen(39) . Our data are consistent with a
model in which IF3 first interacts with the 3`-domain binding site and
is then stabilized by cooperative interactions with the central domain
binding site. The loss of the cooperative interactions in subunits
containing the A1530/G1531 mutant rRNA could account for the 10-fold
decrease in IF3 binding. This model also provides a mechanism for IF3
release upon 50 S binding to the 30 S initiation complex. After proper
initiation occurs, 50 S subunits can bind the central domain,
eliminating the cooperative interactions and reversing the process of
IF3 binding.H]Met-tRNA
binding may be due, in part, to the inability of mutant subunits to
reform the active conformation. Activation, defined by the ability of
the subunit to bind N-acetyl-Phe-tRNA
in the
presence of poly(U)(40) , is associated with a conformational
change in the 3`-minor domain(12) . In fact, one of the mutated
sites, A1531, has been shown to have enhanced reactivity in inactive
subunits(12) .
)
-We thank Anna La Teana, Steve Ringquist, and
Ruth Van Bogelen for procedural assistance and Claudio Gualerzi for
providing initiation factors. We also thank Michael O'Connor,
William Tapprich, and Kathy Lieberman for helpful suggestions and
critical reading of the manuscript and Mary Sue Purzycki, Stephen
Lodmell, Steven Gregory, Don Van Ryk, Carleen Brunelli, and George Q.
Pennabble for numerous discussions.
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
CiteULike 
Complore 
Connotea 
Del.icio.us 
Digg 
Reddit 
Technorati What's this?
This article has been cited by other articles:
|
|
 |
|
  Md. E. Haque, D. Grasso, and L. L. Spremulli The interaction of mammalian mitochondrial translational initiation factor 3 with ribosomes: evolution of terminal extensions in IF3mt Nucleic Acids Res., February 2, 2008; 36(2): 589 - 597. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. A. Khan, H. Miyoshi, D. R. Gallie, and D. J. Goss Potyvirus Genome-linked Protein, VPg, Directly Affects Wheat Germ in Vitro Translation: INTERACTIONS WITH TRANSLATION INITIATION FACTORS eIF4F AND eIFiso4F J. Biol. Chem., January 18, 2008; 283(3): 1340 - 1349. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Le, R. L. Tanguay, M. L. Balasta, C.-C. Wei, K. S. Browning, A. M. Metz, D. J. Goss, and D. R. Gallie Translation Initiation Factors eIF-iso4G and eIF-4B Interact with the Poly(A)-binding Protein and Increase Its RNA Binding Activity J. Biol. Chem., June 27, 1997; 272(26): 16247 - 16255. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| All ASBMB Journals | Molecular and Cellular Proteomics |
| Journal of Lipid Research | ASBMB Today |
