(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) ()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) .
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 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.
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 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.
Fluorescence and light-scattering
measurements were performed on a Spex Fluorolog 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.
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. 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:
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 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 :
where K is the equilibrium binding constant, [A] 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%.
where R 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:
where K = K`(òn/òc) and
is valid as long as no significant depolarization occurs; c` represents concentration (moles/liter).
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[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.
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. represents
light-scattering intensity of wild-type 70 S ribosomes, and
represents mutant 70 S light-scattering
intensity.
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 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.
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 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.
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 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.
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), 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.
The decrease in enzymatic poly(A,G,U) f[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) .
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.