From the Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, Florida 33101
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ABSTRACT |
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Escherichia coli ribosomal RNA contains 10 pseudouridines, one in the 16 S RNA and nine in the 23 S RNA. Previously, the gene for the synthase responsible for the 16 S RNA pseudouridine was identified and cloned, as was a gene for a synthase that makes a single pseudouridine in 23 S RNA. The yceC open reading frame of E. coli is one of a set of genes homologous to these previously identified ribosomal RNA pseudouridine synthases. In this work, the gene was cloned, overexpressed, and shown to code for a pseudouridine synthase able to react with in vitro transcripts of 23 S ribosomal RNA. Deletion of the gene and analysis of the 23 S RNA from the deletion strain for the presence of pseudouridine at its nine known sites revealed that this synthase is solely responsible in vivo for the synthesis of three of the nine pseudouridine residues, at positions 955, 2504, and 2580. Therefore, this gene has been renamed rluC. Despite the absence of one-third of the normal complement of pseudouridines, there was no change in the exponential growth rate in either LB or M-9 medium at temperatures ranging from 24 to 42 °C. From this work and our previous studies, we have now identified three synthases that account for 50% of the pseudouridines in the E. coli ribosome.
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INTRODUCTION |
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Pseudouridine (),1
the 5-ribosyl isomer of uridine, occurs in rRNA (1), tRNA (2), and
small nuclear and nucleolar RNA (3, 4) but not in mRNA or viral
genomic RNAs. All the RNAs in which
is found share a common
characteristic, namely a tertiary structure that must be maintained for
proper function.
is made after the polynucleotide chain has been
formed by an enzyme-catalyzed but energy-independent isomerization of
uridine (reviewed in Ref. 5). A considerable amount of
is found in
ribosomal RNA approaching 8% of the uridines in mammals (6). The
number and distribution of
is different between the two large
rRNAs. In small subunit (SSU) RNA, the number varies from 0 or 1 (yeast
mitochondria) to 1 (Escherichia coli) to ~40 (mammals),
and
are deployed throughout the molecule, whereas in the large
subunit (LSU) RNA, although there is also a wide variation in the
number of
from 1 (yeast mitochondria) to 4-9 (prokaryotes) to
55-57 (mammals), the distribution is conserved in all organisms to
three defined secondary structural regions at or near the peptidyl
transferase center (reviewed in Ref. 5). In E. coli, the
organism studied in this work, there are 10
residues, one in the
SSU RNA at position 516 (7) and nine in the LSU RNA at positions 746, 955, 1911, 1915, 1917, 2457, 2504, 2580, and 2605 (8, 9).
1915 is
further modified by methylation at N3 (10).
Despite the specificity implicit in the conservation of geographic
localization in the LSU to the functionally important peptidyl transferase center, there is no known role for in the ribosome. To
address this issue, we have embarked on a program to identify all of
the synthases responsible for formation of the 10
in E. coli rRNA with the aim of deleting specific
residues by
inactivating the genes for the corresponding synthases. So far,
rsuA, the gene for the synthase responsible for forming
516 in 16 S RNA (11), and rluA, the gene for the synthase
that makes
746 in LSU RNA (12), have been identified. In this work
we show that the gene yceC, renamed rluC, makes
the synthase responsible for formation of
at positions 955, 2504, and 2580. Deletion of this gene and thus the absence of these three
residues has no detectable effect on the exponential growth rate in
either rich or minimal glucose medium at temperatures ranging from 24 to 42 °C.
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EXPERIMENTAL PROCEDURES |
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Gene Deletion-- The yceC gene was deleted by the method of Hamilton et al. (13). The insert cloned into the KpnI and XbaI sites of pMAK705 was prepared by PCR as described by Nelson and co-workers (see Fig. 2 in Ref. 14). It contained 841 bases 5' to the AUG start and 870 bases 3' to the UAA termination codon. 39 bases of the N-terminal portion of the gene and 49 bases of the C terminus were retained with the remainder being replaced by the kanamycin resistance gene, obtained by PCR amplification from pUC4K (Amersham Pharmacia Biotech, catalog number 27-4958-01). The host strain for pMAK705 was MC1061 as described by Hamilton et al. (13). The deleted yceC gene was moved into strain SJ134 (Ref. 15; a gift of Dr. Barry Hall, University of Rochester, Rochester, NY) and from there into strain MG1655 (Ref. 16; a gift of Dr. Kenneth Rudd, this department) by bacteriophage P1 transduction (17).
Rescue Plasmid-- This plasmid (pLG338/yceC) was constructed by insertion into the SmaI site of pLG338 (18) of a PCR-amplified fragment of DNA starting 116 bases 5' to the AUG initiator of yceC and ending 124 nucleotides 3' to the termination codon. This insertion site inactivates the KanR gene of the plasmid. The construct includes the 71-base promoter, 45-base spacer, 960-base gene, 77-base spacer, 44-base termination sequence, and 3 bases beyond. pLG338 also carries a tetracycline resistance gene. Putative promoter and terminator sequences were identified by examination of the upstream and downstream regions. For the promoter, a web site was used.2 The terminator sequence was located visually and verified by folding using M-fold version 3.0 (19).3
Other Methods and Materials--
Transformants of
yceC-deleted SJ134 and of wild type and
yceC-deleted MG1655 with pLG338 and pLG338/yceC
were selected by tetracycline resistance. All growth media contained 10 µg/ml tetracycline to retain the plasmid in the
tetracycline-sensitive host cells. sequencing was performed as
described previously (8, 20). For the growth experiments, overnight
cultures at 37 °C in the medium to be tested were diluted 50-fold
(minimal medium) or 100-fold (rich medium) and placed at the testing
temperature. Cell density was monitored at 600 nm.
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RESULTS |
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Identification of Putative Synthase Genes in E. coli--
From
the amino acid sequences of RsuA and RluA, as well as those for two
tRNA
synthases, TruB (21) and TruA (22), Koonin (23) and Gustafsson
et al. (24) were able to identify putative
synthase ORFs
by searching for sequence motifs. Five ORFs were identified in E. coli (Table I). A sixth ORF,
ymfC, was found subsequently when the entire E. coli genome sequence became available. The ORFs could be divided
into four subfamilies based on their sequence motifs (23, 24) as
indicated in Table I. By chance, the sequences of the four initially
identified genes happened to define each of the four subclasses. Note,
however, that the six putative synthase genes subsequently identified
are all in class A or B. The predicted protein properties and the
site(s) of
formation recognized by the synthases are also indicated in Table I when known.
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yceC--
Because homologs to yceC are common in other
species (23, 24), we selected this ORF for our initial study. The gene
was cloned, the protein was overexpressed, and formation activity was detected using 5-[3H]uridine-labeled 23 S RNA as an
in vitro substrate, confirming that this gene product was
indeed a
synthase. To determine the specificity of the synthase
under in vivo conditions, the gene was deleted by insertion
of the kanamycin resistance gene (13). Verification of gene deletion
was done by PCR amplification from the N and C termini of the
yceC gene in the chromosomal DNA of the deletion mutant. The
wild type control gave the expected 1.0-kb band, whereas the mutant
supposed to contain the KanR insert was, as expected, 1.4 kb in size. Further evidence was obtained by amplification from the N
and C termini of the KanR gene. The mutant gave the
expected 1.3-kb band, but nothing was obtained from the wild type.
Preliminary
sequence analysis showed the absence of
955, 2504, and 2580 as a result of this single gene deletion.
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Growth Rate of the Mutant Strain--
Deletion of these three residues from the ribosome was not lethal. To assess any more subtle
metabolic defects, growth rates were measured at different temperatures
in both rich and minimal glucose media (Table
II). For this purpose, the
rluC deletion was moved by P1 transduction into strain
MG1655, the same strain whose genome was sequenced by Blattner et
al. (16), to provide a well defined background. The MG1655
deletion strain was selected by its kanamycin resistance, and PCR
amplification from the yceC termini confirmed the deletion
(the expected 1.4-kb band was found instead of a 1.0-kb band for the
wild type). Wild type and mutant MG1655 were transformed with both the
rescue plasmid and its control. Exponential growth rates for all four
strains are shown in Table II. Even though both rich and minimal media
were tested over a range of temperature from 24 to 42 °C, no
significant change in growth rate was observed. Moreover, no instance
was found where the maximum cell density at stationary phase was
limited by the deletion mutation. We conclude that at least under the
described conditions, there is no effect of the loss of three of the
nine
present in the large subunit of the ribosome.
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DISCUSSION |
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Specificity--
In this work, we have shown that yceC
is indeed a gene for a synthase and that its gene product is the
only synthase which in vivo is capable of forming
955,
2504, and
2580 in E. coli 23 S RNA. In view of its
specificity, the gene has been renamed rluC. Because this
work was done in vivo in the presence of all of the other
synthases of the cell, it is clear that none of the other synthases
share the specificity for positions 955, 2504, and 2580 with RluC.
However, the reverse is not necessarily the case. RluC might share the
ability with another synthase for recognition of one or more of the
remaining
sites. This can only be determined with certainty when
all of the other rRNA
synthases are identified and deleted. RluC
does not share the ability to form
516 in 16 S RNA with RsuA (Table
I), because deletion of the rsuA gene blocks formation of
516 in vivo.4 A
possible dual specificity of recognition with one of the sites in tRNA,
like that found for RluA (12), has not yet been tested.
RNA Recognition Site--
Although all three sites for formation are well separated in the primary structure of 23 S RNA, 2504 and 2580, but not 955, approach each other when the RNA is folded into
its secondary structure (Fig. 2A). However, in the tertiary
structure existing in the ribosome,
955 is probably not far away
either, because cross-linking results place A960 next to
C2475 (28). These results suggest a possible mode of
recognition of these three sites, which otherwise appear dissimilar in
both sequence and secondary structure. Indeed, the only common
structural element is that all three Us destined to be isomerized to
are followed by a G residue. We postulate that any UG sequence
within a given short distance of the catalytic center will be
recognized. Thus if there is a tertiary structure of the 23 S RNA at
some stage of ribosome biogenesis in which U955 is
sufficiently close to the recognition site of the synthase, which must
also be near to U2504 and U2580, it might be
possible for the synthase to catalyze
formation at all three sites.
In this regard it should be noted that the closest other UG sequence to
any of the three sites is 11 residues away at U2493.
Function of --
No effect on exponential phase growth rate
was found when
955, 2504, and 2580 were absent, even when both
medium and temperature were varied. Other growth parameters such as
survival in stationary phase and the length of the lag phase were not
examined in detail, but preliminary observations suggest that there is
little or no effect (data not shown). In the absence of any such clues
as to
function, the ribosomes from
-deficient cells will need to be examined for their ability to support the partial reactions of
protein synthesis in vitro. This has been done previously to study the effects of rRNA mutations on ribosome function (29-31).
Comparison with Other Known Synthases--
Table
III summarizes the properties of all
known cloned
synthases and their genes. Table III includes
synthases for E. coli and Bacillus subtilis rRNA
and for E. coli and yeast tRNA. It is clear that the
specificity of each synthase varies from being limited to one kind of
RNA at one site to multiple sites in one class of RNA or even sites in
different kinds of RNA. To help in categorizing these synthases and
those still to be discovered, four specificity classes have been
defined. Class I specificity is most stringent, in that only a single
specific site in one kind of RNA is recognized. RsuA, RluB, TruB, and
its homolog in yeast, Pus4p, are examples of this class. Class II
defines those synthases that modify any U residue within a span of 5-6
nucleotides, but only in one kind of RNA. TruA and its yeast homolog,
Pus3p, are examples. In class III, the specificity is relaxed even
more, and distant sites become recognizable, although again only in the
same class of RNA. Pus1p in yeast is an example of this type because it
can recognize up to eight different sites in tRNA, and RluC is another
example. Although both synthases recognize sites spread some distance
apart, the maximum distance in the case of Pus1p is 41 residues,
whereas for RluC it is 1625 nucleotides. Class IV specificity is
reserved for those synthases that recognize specific single sites in
more than one class of RNA, a property we have termed "dual
specificity" (12). So far, RluA is the only member of this class, but
this may be at least in part because of the fact that testing for dual
specificity is not straightforward because it requires a
predetermination of which site in which RNA to test. The dual
specificity of RluA was only discovered serendipitously.
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Further Studies--
The intriguing RNA recognition properties of
the RluC synthase coupled with the ease of overexpression of the
recombinant enzyme with demonstrable formation activity of
appropriate specificity make this protein a logical candidate for x-ray
crystallographic analysis. Major questions are how RNA recognition at
three such disparate sites is accomplished and what is the mechanism of
U isomerization to
. Such studies, in collaboration with R. Fenna of
this department, are underway.
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ACKNOWLEDGEMENTS |
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We thank Ferez Nallaseth for participation in the preliminary sequence analysis of the rluC-disrupted strain, Daanish Kazi for assistance in constructing the rescue plasmid, and K. Rudd for assistance with sequence analysis and for enthusiastic moral support of this project.
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FOOTNOTES |
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* This work was supported by a Markey Foundation grant to the Department of Biochemistry and Molecular Biology, University of Miami School of Medicine.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.
Present address: RMS International, 12020 Sunrise Valley Dr.,
Suite 250, Reston, VA 20191.
§ To whom correspondence should be addressed. Tel.: 305-243-3677; Fax: 305-243-3955; E-mail: jofengan{at}mednet.med.miami.edu.
1
The abbreviations used are: ,
pseudouridine(s); rRNA, ribosomal RNA; SSU, small subunit; LSU, large
subunit; ORF, open reading frame; PCR, polymerase chain reaction; kb,
kilobase pair(s).
2 A. M. Huerta, H. Salgado, and J. Collado-Vides, www.cifn.unam.mx/Computational_Biology/E. coli-predictions/.
3 www.ibc.wustl.edu/~zuker/rna.
4 L. Niu and J. Ofengand, unpublished results.
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REFERENCES |
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