From the Department of Biochemistry and Molecular Biology,
University of Massachusetts, Amherst, Massachusetts 01003-4505
Protein L27 has been implicated as a constituent
of the peptidyl transferase center of the Escherichia coli
50 S ribosomal subunit by a variety of experimental observations. To
define better the functional role of this protein, we constructed a
strain in which the rpmA gene, which encodes L27, was
replaced by a kanamycin resistance marker. The deletion mutant grows
five to six times slower than the wild-type parent and is both cold-
and temperature-sensitive. This phenotype is reversed when L27 is
expressed from a plasmid-borne copy of the rpmA gene.
Analysis of ribosomes from the L27-lacking strain revealed deficiencies
in both the assembly and activity of the 50 S ribosomal subunits.
Although functional 50 S subunits are formed in the mutant, an assembly
"bottleneck" was evidenced by the accumulation of a prominent 40 S
precursor to the 50 S subunit which was deficient in proteins L16, L20,
and L21, as well as L27. In addition, the peptidyl transferase activity
of 70 S ribosomes containing mutant 50 S subunits was determined to be
three to four times lower than for wild-type ribosomes. Ribosomes
lacking L27 were found to be impaired in the enzymatic binding of
Phe-tRNAPhe to the A site, although the interaction of
N-acetyl-Phe-tRNAPhe with the P site was
largely unperturbed. We therefore infer that L27 contributes to peptide
bond formation by facilitating the proper placement of the acceptor end
of the A-site tRNA at the peptidyl transferase center.
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INTRODUCTION |
Protein L27 is one of the smallest and the most basic polypeptides
in the Escherichia coli ribosome (1). A combination of
immune electron microscopy and protein-protein cross-linking results
places L27 at the base of the central protuberance on the interface
side of the 50 S subunit (2). According to in vitro studies,
L27 is a late assembly protein (3) and does not have an identifiable
binding site on the 23 S rRNA. Chemical and UV cross-linking studies,
however, demonstrated that L27 is associated closely with domain V of
the 23 S rRNA (4-6), a region that comprises part of the peptidyl
transferase center (for review, see Ref. 7). The proximity of L27 to
the peptidyl transferase center was also supported by affinity labeling
studies with inhibitors of peptidyl transferase activity, such as
chloramphenicol, carbomycin, tylosin, and spiramycin (8-11), as well
as with puromycin (12), an antibiotic that mimics the
aminoacyl-adenosine moiety of aminoacyl-tRNA and is a substrate for
peptidyl transferase (13). Direct evidence for the presence of L27 at
the peptidyl transferase center was obtained through the use of
derivatives of tRNAPhe containing photoreactive
azidonucleotides within the 3'-terminal ACCAOH sequence
(14, 15). When bound to the ribosomal A or P site and irradiated, these
probes became cross-linked predominantly to protein L27 and domain V of
23 S rRNA (15, 16).
The contribution of protein L27 to the peptidyl transferase center has
been addressed by a number of studies. It has been found, for instance,
that the omission of L27 during in vitro reconstitution of
the 50 S subunit significantly reduces the peptidyl transferase
activity of 70 S ribosomes (17, 18) and that the growth of a bacterial
strain that fails to express the protein is impaired greatly (19).
These findings led to the conclusion that protein L27 does not itself
constitute the peptidyl transferase but that it is part of the peptidyl
transferase center and is necessary for efficient peptide bond
formation. To assess further the role of L27 in the bacterial ribosome,
we have substituted rpmA gene, which encodes this protein,
with a kanamycin resistance (Kanr) marker. The resulting
strain exhibited a severe growth defect and accumulated a precursor to
the 50 S particle which was deficient in several ribosomal proteins,
demonstrating that L27 is important for subunit assembly in
vivo. Ribosomes lacking protein L27 were also impaired in peptidyl
transferase activity. Our analysis suggests that this defect is caused
by the reduced affinity of aminoacyl-tRNA for the ribosomal A site.
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EXPERIMENTAL PROCEDURES |
Materials--
All restriction enzymes were purchased from New
England Biolabs. Plasmid pEE, plasmid pMT101, and E. coli
strain MS367 were gifts of Drs. K. Isono, P. Schimmel, and S. Brown,
respectively. [14C]tRNAPhe,
[14C]Phe-tRNAPhe,
Ac[14C]Phe-tRNAPhe,1
and fractionated poly(U) were kindly provided by Dr. S. V. Kirillov. The sources of other enzymes, radioactive compounds, and biological materials were as described (14, 20).
Construction of the
rpmA::kan Strain--
Bacterial
strains and plasmids used in this study are listed in Table
I. E. coli strain XL1-B was
the host for the purification and maintenance of newly constructed
plasmids. Liquid cultures and plates consisted of 2 × YT medium
containing antibiotics as needed: 50 µg/ml kanamycin, 200 µg/ml
ampicillin, and 30 µg/ml chloramphenicol. All recombinant DNA and
gene transfer procedures were performed by standard techniques (25,
26). Replacement of the rpmA gene with the kanamycin
resistance marker was carried out by the method of Hamilton et
al. (27). E. coli strain LG90 was first transformed
with pTSkan and pUCrpmA plasmids. Logarithmically growing recipients were spread on prewarmed plates containing kanamycin, ampicillin, and 1 mM
isopropyl-1-thio-
-D-galactopyranoside (IPTG) and
incubated at 43 °C for 2 days. Colonies resistant to both
antibiotics were then subjected to three cycles of growth at 30 °C
in liquid medium with ampicillin and IPTG, but without kanamycin, to
promote the resolution of cointegrates. Log phase cells from the final
30 °C culture were diluted 1,000-fold and incubated for 8 h at
43 °C in the absence of kanamycin to destroy the resolved pTS
plasmid. The cells were then spread on plates containing kanamycin,
ampicillin, and IPTG and incubated for 2-3 days at 43 °C. Colonies
resistant to kanamycin but susceptible to chloramphenicol at both 30 and 43 °C were tested by Southern blot analysis for the replacement
of the chromosomal rpmA gene by kan (see Fig. 1).
Transduction with bacteriophage P1 was used to transfer the
kan construct into LG90 cells lacking plasmid pUCrpmA. The resulting strain, IW312 (rec+), was
mated with MS367 to create strain IW326 (rec
).
Analysis of Ribosomes and Ribosomal Proteins--
The
distribution of polysomes and ribosomal particles from logarithmically
growing cells was analyzed by sucrose gradient centrifugation (14).
Proteins were isolated from ribosomes or ribosomal particles by acetic
acid extraction and fractionated by two-dimensional PAGE (28).
Enzymatic Binding of Phe-tRNAPhe to the Ribosomal A
Site--
To block the ribosomal P site, 10 pmol of 70 S ribosomes was
incubated with 13 µg of poly(U) and 13 pmol of tRNAPhe
for 10 min at 37 °C in 10 µl of TMA buffer (50 mM
Tris-Cl, pH 7.6, 50 mM NH4Cl, 10 mM
MgCl2) containing 1 mM dithiothreitol (TMA/SH).
Ternary complexes were then prepared by adding 10 pmol of EF-Tu and 10 pmol of [14C]Phe-tRNAPhe (1,400 dpm/pmol) to
40 µl of a solution containing 0.58 mM GMP-PNP, 0.5 mM phosphoenol pyruvate, and 20 µg/ml pyruvate kinase in
TMA/SH buffer. After 10-fold dilution, the ternary complexes were added to the tRNA-poly(U)-ribosome complexes, and the mixtures were incubated
on ice. Aliquots were withdrawn at different time intervals, and the
amount of [14C]Phe-tRNAPhe bound was measured
by filter retention (29).
Puromycin
Reaction--
Ac[14C]Phe-tRNAPhe was bound
to the P site of poly(U)-programmed ribosomes (30). Reaction mixtures
contained 1 µM 70 S ribosomes, 0.5 µM
Ac[14C]Phe-tRNAPhe (1,400 dpm/pmol), and 1 mg/ml poly(U) in dithiothreitol (TMA/SH) buffer and were incubated for
15 min at 37 °C. After measuring the amount of tRNA bound to the
ribosomes, aliquots of the complex were incubated in the presence of
0.1-1 mM puromycin for various times at 0 or 37 °C, and
the initial rates of Ac[14C]Phe-puromycin formation were
determined (31).
Cross-linking of Photoreactive tRNAPhe
Derivatives to Ribosomes--
32P-Labeled
[2N3A76]tRNAPhe was prepared, aminoacylated,
and acetylated (13, 32). Non-aminoacylated
[32P][2N3A76]tRNAPhe and
Ac[14C]Phe-[32P][2N3A76]tRNAPhe
were purified by high performance liquid chromatography on a C4 reverse
phase column (33). Photoreactive tRNAPhe dervatives were
bound to ribosomes by incubating 0.5 µM
tRNAPhe derivative with 1 µM 70 S ribosomes
and 1.3 mg/ml poly(U) in TMA buffer for 15 min at 37 °C. Complexes
were then irradiated for 2 min at 4 °C with 300-nm UV light (14).
Labeled ribosomal components were analyzed as described (14, 15).
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RESULTS |
Deletion of the rpmA Gene from the E. coli Chromosome--
The
rpmA gene, which encodes ribosomal protein L27, was deleted
from the chromosome of E. coli strain LG90 and replaced by a
kanamycin resistance marker as described under "Experimental Procedures." Southern blots demonstrated that the 6.1-kilobase BglII fragment encompassing the rplU-rpmA operon
was substituted by a 7.5-kilobase fragment that lacked the
KpnI restriction site present in the rpmA gene
(Fig. 1). In addition, the latter
fragment hybridized with a probe for kan but not
rpmA. The kan construct was used to generate
mutant strains IW312 and IW326, which carry the L27 deletion in
rec+ and rec
backgrounds, respectively (Table
I). Two-dimensional PAGE of ribosomal proteins from LG90 and IW326
showed that the mutant ribosomes lack only protein L27 (Fig.
2), and an immunoprecipitation test with
anti-L27 antibodies confirmed the absence of L27 from the mutant cell
extract (not shown).

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Fig. 1.
Replacement of the rpmA gene by
kan. Panel A, strategy of gene replacement.
The Bsu36I-AgeI fragment of the E. coli chromosome encoding rpmA was replaced with the
BamHI-NheI fragment of pACYC177 carrying
kan (see "Experimental Procedures"). The E. coli map is from Ref. 23. Restriction sites are: A,
AgeI; Ba, BamHI; Bg,
BglII; Bs, Bsu36I; H,
HindIII; K, KpnI; Nh,
NheI; P, PstI. Panel B,
Southern blot analysis of chromosomal DNA from wild-type (LG90) and
mutant (IW312) strains. Hybridization probes, shown as black
bars in panel A, correspond to the
PstI-Bsu36I fragment upstream from the
rpmA gene (probe A), the
HindII-XhoI fragment of pACYC177 including most
of the kan gene (probe B), and the
Bsu36I-AgeI fragment spanning the rpmA
gene (probe C). The following restriction digests were used:
lane 1, BglII; lane 2,
BglII and PstI; lane 3,
BglII and KpnI.
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Fig. 2.
Two-dimensional polyacrylamide gel analysis
of ribosomal proteins from wild-type and L27-lacking E. coli. Proteins were extracted from 10 A260 units of purified 70 S ribosomes from
strains LG90 (left) and IW326 (right) and
separated by acidic two-dimensional PAGE (28).
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Phenotype of the L27-deletion Strain--
The growth of strains
LG90, IW312 and IW326 was compared at different temperatures (Table
II). The wild-type strain, LG90, grew
rapidly under all conditions tested. In contrast, strains IW312 and
IW326 were impaired severely; at 37 °C, small colonies appeared on
solid medium only after 2-3 days, and their doubling times in liquid
culture were five to six times greater than that of LG90. When strains
IW312 and IW326 were streaked on agar plates and incubated at 25 °C
or 43 °C for up to 2 weeks, only a trace of growth was visible at
the beginning of each streak. Similarly, there was no measurable growth
in liquid culture at these temperatures. Thus, deletion of the
rpmA gene renders the cells both cold- and heat-sensitive.
However, because cells that failed to grow at 25 and 43 °C
reinitiated growth upon transfer to 37 °C, the absence of L27
appears to be bacteriostatic rather than bacteriocidal at the two
temperature extremes.
The impaired growth of strains IW312 and IW326 was largely alleviated
when plasmids carrying a functional rpmA gene were
introduced into the mutant strain. The presence of plasmids pEE and
pUCrpmA both led to a significant increase in cell growth
rate at 37 °C and abolished cold and temperature sensitivity,
whereas the corresponding parental plasmids lacking rpmA,
pBR322, and pUC119, as well as a plasmid lacking the 5'-terminal half
of the rpmA gene, pEE
BK, did not (Table II). The faster
growth of cells harboring pEE as opposed to pUCrpmA is
attributable to the more efficient expression of L27 in the former
strain as demonstrated by specific immunoprecipitation of the protein
from cell extracts (not shown).
Assembly of Ribosomal Particles Lacking L27 in Vivo--
The
distribution of ribosomes in lysates of log phase LG90, IW326, and
IW326/pEE cells was examined by sucrose gradient centrifugation. As
shown in Fig. 3A, panel
a, there is little variation in the polysome profiles of the three
strains, although there is a significant difference in the ratio of 70 S monosomes to 50 S and 30 S subunits. Lysates of the wild-type strain
contained a large 70 S peak with very few 50 S and 30 S particles,
whereas lysates of the L27-deletion strain contained more ribosomal
subunits than 70 S monosomes. In the latter case, longer centrifugation
times revealed the presence of particles sedimenting at approximately
40 S in addition to the 30 S and 50 S subunits (not shown). In
contrast, lysates obtained from strain IW326/pEE, in which the
chromosomal deletion was complemented by a plasmid-borne copy of the
rpmA gene, exhibited a ratio of 70 S ribosomes to 50 S and
30 S subunits resembling that of the wild-type strain. This observation
demonstrates that protein L27 is required for efficient assembly of the
50 S subunit.

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Fig. 3.
Distribution of ribosomes, ribosomal
subunits, and ribosomal RNA in wild-type and mutant bacteria.
A, sucrose gradient analysis of ribosomes from strains LG90
(wild type), IW326 ( L27), and IW326/pEE. Panel
a, crude cell lysates were fractionated on 14-40% sucrose
gradients containing 10 mM Mg2+.
Panel b, ribosomal subunits were resuspended from
100,000 × g ribosome pellets and centrifuged on
10-30% sucrose gradients containing 0.3 mM
Mg2+. B, electrophoretic analysis of rRNA from
ribosomal subunits fractionated on sucrose gradients as in panel
b above. RNA was extracted with phenol/chloroform from 0.5 to 1.0 A260 units of ribosomal particles from LG90
(left) and IW326 (right) and separated by
electrophoresis on a 0.7% agarose gel in TBE buffer. Lanes
1, 2, and 3 correspond to 50, 40, and 30 S
fractions, respectively.
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To characterize the 40 S particles present in lysates of strain IW326,
crude ribosomes prepared in 10 mM Mg2+ were
analyzed on sucrose gradients containing 0.3 mM
Mg2+ (Fig. 3A, panel b). Whereas the
sample isolated from the wild-type strain contained 50 S and 30 S
subunits in the expected 1:1 ratio, the L27-deletion strain yielded
three components sedimenting at approximately 50, 40, and 30 S. As
evident from the figure, the 30 S peak was considerably more prominent
than the 50 S peak. In the sample obtained from IW326/pEE, the presence
of extrachromosomal copies of the rpmA gene led to a
restoration of the typical 50 S to 30 S ratio of 2:1.
Peak fractions from the IW326 lysate were analyzed for RNA and protein
content (Figs. 3B and 4). The
50 S fraction contained 23 S rRNA and all of the 50 S subunit proteins
except L27. The 40 S fraction also contained 23 S rRNA, suggesting that
it is a precursor to the large subunit. Two-dimensional PAGE analysis showed that large subunit proteins L16, L20, and L21 were missing from
the 40 S particle in addition to protein L27. A pulse-chase experiment
established that the 40 S particles mature to 50 S subunits and are
therefore true intermediates in 50 S subunit assembly (not shown).
Although the 30 S subunit fraction from the wild-type lysate contained
mostly 16 S rRNA and was contaminated by only a small amount of 23 S
rRNA, the corresponding fraction from the mutant lysate contained 23 S
and 16 S rRNA in a 1:1 ratio (Fig. 3B). Therefore, the 30 S
fraction appeared to contain another, smaller precursor to the 50 S
subunit in addition to the 40 S particle. We conclude that protein L27
strongly affects the kinetics of 50 S subunit assembly in
vivo as the absence of L27 leads to the accumulation of partially
assembled precursor particles in addition to active 50 S subunits. This
bottleneck in 50 S subunit maturation is very likely to contribute to
the slow growth phenotype of the L27-deletion strains.

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Fig. 4.
Two-dimensional polyacrylamide gel analysis
of ribosomal proteins extracted from 50 S subunits and 40 S precursor
particles isolated from strain IW326. Ribosomal particles were
purified on sucrose gradients in 0.3 mM Mg2+ as
in Fig. 3A, panel b. Protein samples were
separated by two-dimensional PAGE as in Fig. 2.
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Activity of Ribosomes Lacking L27 in Vitro--
To determine the
effects of L27 deletion on ribosome function, we compared wild-type and
mutant ribosomes with respect to their ability to interact with tRNA
and to carry out the puromycin reaction in vitro. Both types
of ribosomes were first assayed for their capacity to bind
AcPhe-tRNAPhe and Phe-tRNAPhe to the ribosomal
P and A sites, respectively. Approximately 60% of the ribosomes from
the two strains were active in binding AcPhe-tRNAPhe to the
P site, and no differences in the kinetics of binding were noted (not
shown). On the other hand, the rate of enzymatic binding of
Phe-tRNAPhe to the A site of mutant ribosomes was slower
than that to wild-type ribosomes at both 0 and 37 °C (Fig.
5). In addition, when the P site was
filled with AcPhe-tRNAPhe, the L27-lacking ribosomes bound
only 0.6 pmol of Phe-tRNAPhe/pmol of active ribosomes,
whereas wild-type particles could bind more than 0.8 pmol of
Phe-tRNAPhe/pmol of active ribosomes.

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Fig. 5.
Kinetics of EF-Tu-directed binding of
Phe-tRNAPhe to wild-type and L27-lacking ribosomes.
Enzymatic binding of [14C]Phe-tRNAPhe to
poly(U)-programmed ribosomes whose P site had been filled previously
with tRNAPhe is shown (see "Experimental Procedures").
Results are expressed as pmol of
[14C]Phe-tRNAPhe bound per pmol of 70 S
ribosomes from strains LG90 ( ) and IW326 ( ).
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The peptidyl transferase activity of wild-type and L27-lacking
ribosomes was assessed by monitoring the puromycin reaction at 0 and
37 °C as shown in Fig. 6. The maximum
reaction velocities for wild-type ribosomes determined from the plot
are 0.625 min
1 at 37 °C and 0.033 min
1
at 0 °C. The corresponding values for the mutant ribosomes are 0.192 min
1 at 37 °C and 0.008 min
1 at 0 °C,
or three to four times less than for the wild-type ribosomes. Because
the puromycin reaction is a direct measure of peptidyl transferase
activity, the difference in reaction kinetics between the two kinds of
ribosomes indicates that the L27-lacking ribosomes are defective in
peptidyl transferase activity as well as in 50 S subunit assembly.

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Fig. 6.
Peptidyl transferase activity of wild-type
and L27-lacking ribosomes. Double-reciprocal plots of the velocity
of the puromycin reaction were constructed from data obtained as
described under "Experimental Procedures." Ribosomes were from
strains LG90 ( ) and IW326 ( ). Although the Km
values for the puromycin reaction are the same for LG 90 and IW326
ribosomes at 37 °C, they differ markedly at 0 °C, suggesting a
weaker interaction between the antibiotic and the A site of the mutant
ribosomes at the lower temperature.
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Cross-linking of Photoreactive tRNA Derivatives to Ribosomes
Lacking L27--
Our previous studies have shown that a photoreactive
derivative of tRNAPhe containing 2-azidoadenosine at
the 3' or acceptor end labels both protein L27 and 23 S rRNA when bound
and cross-linked from the ribosomal A and P sites (15, 16). In the
present work, we have used the same tRNA derivative to determine how
the absence of L27 affects the way in which the 3' end of tRNA
interacts with the ribosome. [2N3A76]tRNAPhe
and its peptidyl analog AcPhe-[2N3A76]tRNAPhe
were bound to the P site of poly(U)-programmed wild-type and mutant
ribosomes. P site binding was confirmed in each case through the use of
edeine, an antibiotic that specifically interferes with tRNA-ribosome
interaction at the P site (34, 35), and by the reaction of
AcPhe-[2N3A76]tRNAPhe with puromycin (29,
36). Upon irradiation of the tRNA-ribosome complexes, 15-30% of the
noncovalently bound tRNA became covalently linked to both types of
ribosomes, although the pattern of labeling differed markedly (Table
III). In wild-type ribosomes,
AcPhe-[2N3A76]tRNAPhe was cross-linked to
protein L27 and two specific sequences within domain V of the 23 S rRNA
as reported earlier (14, 16). In ribosomes lacking L27, however, all of
the label was associated with the 23 S rRNA, although the cross-linked
sequences were identical to those in wild-type ribosomes. These results
indicate that the 3' ends of the peptidyl-tRNA analog adopt a similar
orientation relative to the 23 S rRNA regardless of the presence or
absence of L27. A somewhat different distribution of cross-links was
observed in experiments with non-aminoacylated
[2N3A76]tRNAPhe; in wild-type ribosomes,
the label was again associated with L27 and 23 S rRNA, whereas in
mutant ribosomes, L33 was labeled in addition to 23 S rRNA. Because the
labeling of L33 is primarily attributable to E site-bound
[2N3A76]tRNAPhe in wild-type ribosomes
(37), the present findings suggest that the 3' end of deacylated tRNA
is not fixed to a unique site in the mutant ribosomes.
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Table III
Cross-linking of AcPhe-[2N3A76]tRNAPhe and
[2N3A76]tRNAPhe to the P site of wild-type and mutant
ribosomes
Irradiated [32P]tRNA-ribosome complexes were dissociated into
subunits by centrifugation through 10-30% sucrose gradients, and
covalent tRNA-50 S subunit cross-linking was estimated from
radioactivity cosedimenting with the 50 S peak. The percentage of tRNA
cross-linked to 50 S ribosomal proteins and rRNA was measured from the
distribution of 32P-labeled material on SDS-urea polyacrylamide
gels. Covalent [32P]tRNA-50 S subunit complexes were digested
with RNase T1 and analyzed by SDS-PAGE. The percentage of tRNA
cross-linking to ribosomal proteins L27 and L33 was estimated from the
distribution of 32P label between these two proteins.
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DISCUSSION |
Protein L27 has been identified as a component of the peptidyl
transferase center of the E. coli ribosome by numerous
biochemical and genetic studies (for reviews, see Refs. 38 and 39). We have shown, for instance, that L27 is the main protein cross-linked from 2- or 8-azidoadenosine at positions 73 and 76 of
tRNAPhe at the P and A sites (14-16). Because the
azidoadenosines give rise to very short cross-links, on the order
of 3-4 Å, specific labeling of L27 by both P and A site-bound tRNAs
is in accord with the expected juxtaposition of their 3'-terminal
adenosines during peptidyl transfer. To characterize further the
functional role of L27 in translation, we constructed a strain of
E. coli from which the L27 gene was deleted. We show here
that the mutant bacteria grow five times more slowly than wild-type
E. coli and are both cold- and temperature-sensitive. The
mutant phenotype is reversed upon expression of protein L27 from a
plasmid, however, demonstrating that the impaired growth of the
deletion strain results from the lack of protein L27. We have traced
the molecular basis of growth impairment to interrelated defects in the
assembly and function of the 50 S ribosomal subunit.
Logarithmically growing cells of the rpmA deletion strain
accumulate 40 S precursors to the 50 S subunit which lack proteins L16,
L20, and L21 in addition to L27. Pulse-chase experiments showed that
the 40 S particles eventually mature to 50 S subunits even in the
absence of L27 and subsequently are incorporated into 70 S monosomes
and polysomes. This observation shows for the first time that L27 plays
a role in the assembly of E. coli ribosomes within the cell
and indicates that there are significant differences between 50 S
subunit assembly in vivo and in vitro. The
absence of L16, L20, and L21 from the 40 S particles implies that the assembly of these proteins is dependent on L27 in vivo. By
contrast, in vitro reconstitution experiments indicate that
L27 is a late assembly protein and therefore not expected to influence
assembly of the 50 S particles. In particular, the in vitro
assembly map does not predict any assembly linkage between protein L27
and proteins L16, L20, and L21, although L27 has been cross-linked to
L16 in the mature 50 S subunit (40, 41) and is a neighbor of L16
according to immunoelectron microscopy studies (42). Furthermore,
although L20 promotes the incorporation of L21, neither one depends
upon nor stimulates L27 binding in vitro (3). Moreover, protein L20 is considered as a primary assembly protein with a binding
site in the 5' third of the 23 S RNA (43), whereas protein L27, which
appears to lack an independent RNA binding site, has been cross-linked
to three sites in domain V of the 23 S rRNA (4-6). The presence of
protein L30 in 40 S precursor particles is also unexpected because this
protein does not assemble into the 50 S subunit in vitro
without proteins L20 and L21. However, the early assembly of L27
in vivo is supported by the observation that a 32 S
precursor found in wild-type cells contains protein L27 along with 16 other ribosomal proteins, including L20 and L21 (44). It is of interest
in this connection that protein L21 is encoded by the same operon as
L27 (45). Although the mechanism by which this operon is regulated has
not been elucidated, deletion of the L27 gene does not appear to reduce
the synthesis of L21.
It has been found recently that a 371-amino acid protein from the large
ribosomal subunit of yeast mitochondria, Rml27p, contains an
amino-terminal domain of 84 residues which is homologous to E. coli L27 as well as a carboxyl-terminal domain unrelated to any
other known ribosomal protein (46). Deletion analysis revealed that
this protein is essential for the structural and functional integrity
of the yeast mitochondrial ribosome. Several amino acid substitutions
in conserved positions of the L27-like domain led to cold- and
temperature-sensitive defects in the assembly of the mitochondrial
large ribosomal subunit and the accumulation of more slowly sedimenting
particles containing large subunit rRNA. These striking parallels to
the properties of L27-lacking E. coli provide further
evidence that this protein contributes to the assembly of the large
ribosomal subunit, a role that has apparently been conserved in the
evolution of diverse genetic systems.
In addition to defects in 50 S subunit assembly, we have found that the
absence of protein L27 leads to the impairment of peptidyl transferase
activity. This defect appears to result from a decrease in the ability
of the mutant ribosomes to facilitate EF-Tu-dependent
binding of aminoacyl-tRNA to the A site. Earlier photochemical
cross-linking studies have shown that the 3'-terminal adenosine of A
site-bound Phe-[2N3A76]tRNAPhe is in
close proximity to protein L27 (15). Our present findings suggest that
L27 is not only in proximity to the 3' end of the A site tRNA but that
there is a functional interaction between them which contributes to
tRNA binding. This proposal is supported by our observation that
removal of L27 weakens the interaction of puromycin, an analog of the
3' end of aminoacyl-tRNA (13), with the ribosomal A site. In contrast,
the rate and extent of binding of a peptidyl-tRNA analog,
AcPhe-tRNAPhe, to the P site were not perturbed.
We conclude from the results reported here that protein L27 plays a
role in mediating the proper placement of the 3' end of A site tRNA at
the peptidyl transferase center and that it may also influence the
interaction of the 3' end of deacylated tRNA with the ribosome after
peptidyl transfer. These inferences are consistent with several
properties of the mutant ribosomes, including the lower rate of
peptidyl transfer, the reduced enzymatic binding of aminoacyl-tRNA to
the A site, and the observation that the 3' end of deacylated tRNA can
be cross-linked to L33, a protein component of the ribosomal E site.
Because L27 is an unusually basic protein (1), its function may be to
screen the negative charge of the tRNA molecules from that of the 23 S
rRNA during the peptidyl transferase reaction.
We are indebted to Drs. K. Isono, P. Schimmel, S. Brown, and S. V. Kirillov for providing bacterial
strains, plasmids, and materials used in this work, and to Dr. S. V.
Kirillov for much useful advice and discussion.