Received 3 July 2001/Returned for modification 2 August
2001/Accepted 17 September 2001
rRNAs are the central players in the reactions catalyzed by
ribosomes, and the individual rRNAs are actively involved in different ribosome functions. Our previous demonstration that yeast 5S rRNA mutants (called mof9) can impact translational reading
frame maintenance showed an unexpected function for this ubiquitous
biomolecule. At the time, however, the highly repetitive nature of the
genes encoding rRNAs precluded more detailed genetic and molecular
analyses. A new genetic system allows all 5S rRNAs in the cell to be
transcribed from a small, easily manipulated plasmid. The system is
also amenable for the study of the other rRNAs, and provides an ideal
genetic platform for detailed structural and functional studies.
Saturation mutagenesis reveals regions of 5S rRNA that are required for
cell viability, translational accuracy, and virus propagation.
Unexpectedly, very few lethal alleles were identified, demonstrating
the resilience of this molecule. Superimposition of genetic phenotypes
on a physical map of 5S rRNA reveals the existence of phenotypic
clusters of mutants, suggesting that specific regions of 5S rRNA are
important for specific functions. Mapping these mutants onto the
Haloarcula marismortui large subunit reveals that these
clusters occur at important points of physical interaction between 5S
rRNA and the different functional centers of the ribosome. Our analyses
lead us to propose that one of the major functions of 5S rRNA may be to
enhance translational fidelity by acting as a physical transducer of
information between all of the different functional centers of the ribosome.
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INTRODUCTION |
The ribosome is the central
component of an extremely accurate cellular protein synthesis
apparatus. Its function is to efficiently and accurately decode mRNAs.
Eukaryotic ribosomes contain four rRNAs: three large-subunit-associated
rRNAs (28S-25S in eukaryotes and 23S in prokaryotes, plus 5.8S and
5S) and the small-subunit rRNA (18S in eukaryotes and 16S in
prokaryotes). Although these rRNAs were initially thought to provide
the scaffolding for the enzymatic ribosomal proteins, early
reconstitution and depletion experiments hinted at broader roles for
these molecules (reviewed in references 37 and 39), and it
is now clear that the rRNAs are the central players in the reactions
catalyzed by ribosomes and that the individual rRNAs are actively
involved in different ribosomal functions (reviewed in references
9, 30, 38, 41, and 63). Thus, understanding the molecular
basis of rRNA structure and function is central to furthering our
comprehension of the translational apparatus.
The 5S rRNA is a component of the large ribosomal subunit in all living
organisms (with the exception of mitochondrial ribosomes) (see
reference 27 for a review). In eukaryotic cells, 5S rRNA is synthesized in the nucleolus by RNA polymerase III, processed into
its mature form, and then imported into the nucleus, where it
associates with ribosomal protein L5. The 5S-L5 ribonuclear particle is
reimported into the nucleolus, where it is assembled into the central
protuberance as one of the last steps in the biogenesis of the 60S
subunit (1, 3, 11, 12). The central protuberance lies
opposite the head of the small subunit, and chemical probing and X-ray
crystallographic analyses provide evidence to support the notion that
5S rRNA is in close contact with this subunit (56, 66).
The crystal structures of the Haloarcula marismortui and
Thermus thermophilus large subunits show that 5S rRNA
also extends along the posterior face of the large subunit (2,
35). Chemical cross-linking, nucleolytic protection, pharmacological, and X-ray crystallographic studies show that 5S rRNA
interacts with multiple functional regions of the large rRNA, including
the A site region of the peptidyltransferase center and the
GTPase-associated region of 23S rRNA (2, 18, 20, 36, 45, 52, 53,
56, 66). Numerous functions have been hypothesized for 5S rRNA,
e.g., that it helps enhance aminoacyl-tRNA (aa-tRNA) binding to the
ribosome (18, 21), that it assists in defining the
topology of the peptidyltransferase center (25), and that
it enhances peptidyltransferase activity (18, 20, 52).
Although 5S rRNA has been the subject of literally thousands of
studies, how it works to ensure the proper function of the ribosome in
vivo is still not clearly understood. Programmed ribosomal frameshifting, i.e., the ability of specific cis-acting
viral signals to program ribosomes to shift translational reading frame by one base in either the 5' (
1) or 3' (+1) direction, provides a
convenient probe of ribosome structure and function relationships in
intact cells. A series of genetic screens of the yeast
Saccharomyces cerevisiae that were designed to
identify mutations that increased 1 programmed ribosomal
frameshifting efficiencies revealed the presence of a family of genes
called MOF, for maintenance of frame (reviewed in reference
13). The mof9 alleles were of particular interest because they were shown to be allelic to 5S rRNA, representing the first genetic phenotype associated with 5S rRNA (17).
However, the presence of 100 to 200 tandemly arranged copies of rRNA
genes on chromosome XII presented the major barrier to further genetic and functional studies of the function of 5S rRNA at the time. In the
interim, in vivo systems for yeast which have progressively improved
the prospects for this line of research have been developed (4,
10, 40, 60). Recently developed yeast strains called rdn1
, in which the entire RDN1 locus,
including all of the flanking 5S ribosomal DNA (rDNA) clusters have
been deleted (40), are ideally suited for further studies
on 5S rRNA because they allow examination of the effects of mutant 5S
rRNAs without interference from wild-type sequences. Here we show that
the rdn1 strain background allows for both global and
targeted mutagenesis of 5S rRNA in yeast. A total of 247 5S rRNA
alleles were generated in the rdn1 strain background
and tested with regard to a series of translation-related phenotypes.
The paucity of lethal 5S rRNA alleles and those responsible for
ts
and cs phenotypes
supports other studies demonstrating the resiliency of the rDNAs
(32, 43, 47, 58). Mapping the mutant 5S rRNA alleles with
regard to termination suppression, nonsense-mediated mRNA decay, and
maintenance of the killer virus phenotypes revealed functional regions
of the molecule. The strongest global effects were seen in the most
conserved regions of the molecule: the loop B
loop C region at the
top of the central protuberance; the helix IV-loop E interface region,
where 5S rRNA interacts with the A site finger (ASF); and the highly
conserved G91 base in loop D, contacting helix 39 of the large subunit
(25S) rRNA. Our findings lead us to propose an allosteric signaling
role for 5S rRNA. We posit that, by providing a physical link between
all of the different functional centers of the ribosome, it acts as a
transducer of information, facilitating communication between the
different functional centers and coordination of the multiple events
catalyzed by the ribosome.
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MATERIALS AND METHODS |
Media, genetic methods, and enzymes.
Escherichia
coli strains DH5
, CJ236, and MV1190 were used to amplify
plasmids, and E. coli transformations were performed using
the standard calcium chloride method as described previously (50). Yeasts were transformed using the alkali cation
method (24). YPAD, YPG, SD, synthetic complete medium
(H-), and 4.7-MB plates for testing the killer phenotype were used as
described previously (62). Cytoduction of L-A and
M1 from strain JD759 into rho-o strains was
performed as previously described (16). Plasmid shuffle
techniques using 5-flouroorotic acid (5-FOA) were as previously
described (48). The sequences of the 5S rDNA mutants were
determined using modified T7 DNA polymerase (57)
(Sequenase, version 2.0; U.S. Biochemicals) using standard 20
and reverse primers (IDT).
Assays for killer virus maintenance and programmed ribosomal
frameshifting followed previously described protocols
(15). Briefly, strain 5X47
(MATa/MAT his1/+ trp1/+
ura3/+ K R) was
used as the indicator strain to score for the presence of the killer
virus. Yeast colonies containing either mutant or wild-type pJD209.TRP
(control) plasmids were replica plated onto 4.7-MB plates seeded with a
lawn of 5X47 cells and grown at 20°C for 2 to 3 days. Killer
phenotypes were scored by either the lack of a halo of growth
inhibition surrounding cells harboring 5S rDNA mutants
(K) or the lessening (Kw)
of the diameter of the halo compared to the halo surrounding cells
harboring the wild-type gene. Frameshifting efficiencies were
calculated by dividing the beta-galactosidase activities obtained from
cells harboring a 1 ribosomal frameshift reporter by those from cells
harboring a 0-frameshift control. All assays were performed in
triplicate, and standard errors were calculated as previously described
(46).
Assays for cold and heat sensitivity and assays to monitor suppression
of the ade2-1 and can1-100
alleles (at 60 µg of canavanine/ml) were performed as previously
described (22). To monitor ade2-1 status, yeast colonies carrying either mutant or wild-type 5S rDNA
plasmids were cultured overnight in synthetic liquid medium lacking
leucine and tryptophan (H-Leu-Trp medium) and then used for the
dilution spot assay on standard YPD plates. Serial dilutions of 2 × 105 to 2 × 10 CFU/5 µl were prepared
in water, spotted onto the plates, and incubated at 30°C. Colonies
harboring the wild-type gene were pink. Lack of color (white colonies)
was interpreted as indicative of suppression of the
ade2-1 nonsense mutation. Conversely, colonies that were bright red were indicative of high-fidelity mutants. A second
serial dilution spot assay was used to score the nonsense suppression
phenotypes of the 5S rRNA mutants with regard to the can1-100 allele, which confers resistance to
canavanine in wild-type cells. Canavanine sensitivity was indicative of
the ability of the mutant 5S rRNAs to act as nonsense suppressors.
Scoring used a +/ system in which was indicative of no
growth, + indicated growth of the spot containing 2 × 105 CFU only, ++ indicated growth of the two
densest spots, etc. Assays for heat and cold sensitivities were
similarly performed and scored.
Plasmids.
pNOY290 is a 2µm plasmid containing both the
URA3 and leu2d selectable markers and a complete
copy of an rDNA repeat that carries the hygromycin resistance
(hygr) allele of 25S rRNA
(40). pNOY353 is a 2µm plasmid containing the
TRP1 and leu2d markers and a complete copy of an
rDNA repeat in which transcription of the 35S operon is driven from a
GAL7 promoter (40). The pJD180 series of
plasmids (pJD180.URA and pJD180.TRP) are pRS400-series 2µm vectors
(5) with a 9,082-bp DNA fragment that contains a complete
copy of an rDNA repeat from pRDN1-wt (kindly provided by Y. O. Chernoff) (4) inserted into the SmaI
restriction site. Digestion of pJD180.TRP with BstEI removed
a 0.8-kb fragment containing 5S rDNA, and subsequent self-ligation was
used to produce pJD210.TRP. Digestion of this plasmid with SalI and NotI produced a 7.5-kb fragment
containing the complete 35S rDNA operon, which was inserted into
similarly restricted pRS425 to create pJD211.LEU.
Plasmids expressing 5S rRNA alone were constructed as follows.
High-copy-number URA3-selectable vector pJD106.URA was made by inserting the 2.1-kb EcoRI fragment containing a copy of
the 5S rDNA gene into pRS426, and pJD106.TRP was constructed by cloning the insert from pJD106.URA into pRS424 (17). pJD209.TRP
was prepared by excising the 424-bp SmaI/SalI
fragment containing 5S rDNA from pJD116Y5 (17) and
subcloning it into SmaI/SalI-restricted pRS424.
Yeast strains.
HFY870 (MATa
ade2-1 his3-11,15
leu2-3,112 trp1-1
ura3-1 can1-100 upf1::HIS3
UPF2 UPF3) was a kind gift from the laboratory of A. Jacobson. The
rdn1 strain NOY891 (MATa
ade2-1 ura3-1 leu2-3
his3-11 trp1 can1-100
rdn1::HIS3 plus pNOY353) was kindly provided by M. Nomura (40). This strain is an adaptation
of previous rdn1 deletion strains in which the entire
RDN1 locus and flanking regions of chromosome XII, including
an unspecified number of 5S rDNA genes, have been deleted. In this
strain background, all of the cellular rRNAs are produced from pNOY353.
To obtain strains that were more amenable to genetic analyses, a series
of strains was constructed so that the final
killer+ strain harbored the 35S rDNA operon on a
high-copy-number LEU2 vector and the 5S rDNA gene on a 2µm
URA3 vector, thus allowing us to transform cells with mutant
5S rDNA alleles on a high-copy-number TRP1-based vector and
screen for loss of the wild-type plasmid using 5-FOA. The genealogies
of the strains are as follows. JD1089 was constructed by introducing
pJD180.URA into NOY891 by transformation, and strains that had lost
pNOY353 were identified by their Ura+
Trp Leu phenotypes. The
killer virus was introduced into the resulting strain by cytoduction
from strain JD759 (MAT kar1-1 arg1
thr[i,x] [L-A HN
M1]), and the killer+
phenotype was confirmed as described below. Transformation of JD1089
with pJD180.TRP and subsequent identification of colonies that had lost
pJD180.URA by their ability to grow in the presence of 5-FOA were used
to construct strain JD1110. Plasmids pJD106.TRP and pJD211.LEU were
subsequently cotransformed into JD1110, and cells that had lost
pJD180.TRP were identified by their Ura+
Leu+ Trp
killer+ phenotypes. A single colony having this
phenotype was selected to be JD1111. The lack of chromosomal copies of
rDNA genes was confirmed by DNA blot analysis using 5S and 25S
rDNA-specific probes.
Mutagenesis of 5S rDNA. pJD209.TRP was used to make a systematic
collection of mutants covering the entire 121-bp sequence of 5S rDNA.
pJD209.TRP was introduced into E. coli strain CJ236, and uracil-containing single-stranded DNA was obtained by infection with the R408 helper phage (Promega, Madison, Wis.). Site-directed oligonucleotide mutagenesis using T4 DNA polymerase and subsequent transformation into MV1190 were performed in accordance with standard methods (28). To mutate 5S rDNA to saturation, a series of
121 mutagenic antisense 31-mers were designed to "walk along" the (plus strand) single-stranded DNA (ssDNA) 5S rDNA sequence of uracil containing ssDNA of pJD209.TRP. The identity of the 16th base of each synthetic oligonucleotide was randomized so that it
contained a mixture of all three possible mutant bases for each of the
121 different positions. The 5' and 3' 15 nucleotides flanking the
mutagenic base perfectly complemented the 5S rDNA sequences present on
the plasmid. The identity of each mutant was confirmed by DNA sequence
analysis. On a technical note, since the position but not the identity
of a particular mutant was known beforehand, we were able to multiplex
sequence using up to five different clones (mutated at different
positions) per sequencing reaction.
Generation of the yeast mutant collection.
Strain JD1111 was
grown in H-Leu-Ura synthetic complete medium, transformed with mutant
pJD209.TRP plasmids, and plated onto H-Leu-Trp. After 5 to 6 days of
growth at 30°C, the yeast colonies were replica plated onto H-Leu-Trp
medium supplemented with 1 g of 5-FOA/liter and incubated for 6 days at 30°C. The resultant yeast colonies were replica plated onto
H-Ura medium to test for the absence of the initial pJD106.URA plasmid
containing wild-type 5S rDNA. For each variant, at least three colonies
were selected and stored at 80°C as stocks for all further work. To
confirm that the process was not selecting for reversion or second-site mutations, mutant 5S rRNAs were amplified from cell lysates from 10 selected strains by direct sequencing of reverse transcription products
in accordance with previously described protocols (65) using primer 5' AGGTTGCGGCCATATG 3' (complementary to the 3'
end of 5S rRNA) and the products were analyzed by sequencing.
RNA blot analyses.
RNA blot analyses to monitor for the
presence of the L-A and M1 double-stranded RNAs
(dsRNAs) and to monitor the endogenous Cyh2 pre-mRNA and Cyh2 mRNA
species were performed as previously described (7, 16).
The L-A and M1 blots were normalized for genomic
DNA concentrations by using standardized samples. For the Cyh2
blots, equal units of optical density at 260 nm, indicative of cellular
RNA, were used. The images were visualized and quantitated by
phosphorimagery using a Typhoon 8600 phosphorimager and ImageQuant, version 5.2, software (Molecular Dynamics).
| |
RESULTS |
Construction of a system for mutagenesis of 5S rRNA.
The
present study was made possible by the initial development of a yeast
strain lacking the entire RDN1 locus, as well as flanking
sequences that contain an unknown number of 5S rDNA genes (40). The rRNAs were originally supplied to this
rdn1 strain by a large plasmid (pNOY353) that contains
a single rDNA repeat encoding all four rRNAs. Due to its size, this
plasmid is rather unstable in E. coli making it difficult to
manipulate. To solve this problem, we replaced pNOY353 with two
plasmids: pJD106.URA, which carried only the 5S rRNA gene, and
pJD211.LEU, which carried the 35S rRNA operon (the product of which is
processed into the 25S, 18S, and 5.8S rRNAs). The resultant strain,
JD1111, had no discernible growth defects compared to the parental
strain and was able to stably support the yeast killer virus. This
strain served as the basis for the subsequent phenotypic
characterization of a saturation library of mutant 5S rRNAs.
Oligonucleotide site-directed mutagenesis was used to create the
library of 5S rRNA mutants because it allowed the location of each
mutation to be known in advance and also because it ensured against
biases for or against specific nucleotides or regions of the molecule.
The 5S rRNA-encoding plasmid pJD209.TRP was the target for mutagenesis.
This plasmid contains a short (424-bp) fragment of rDNA harboring the
121-bp 5S rDNA plus 5' and 3' flanking regions of 147 and 156 bp,
respectively. Each position of the 5S rDNA was mutated using a specific
synthetic oligonucleotide reagent consisting of a mixture of three
oligonucleotides that were identical except for a mutation of the
corresponding wild-type base into any of the three possible
substitutions. Mutagenesis reactions were performed separately for all
121 positions of 5S rDNA. Mutant plasmids were isolated from E. coli clones and checked by sequencing for the presence of desired
mutations. In total, 1,024 plasmid samples were sequenced, yielding 246 unique point mutants at each of the 121 positions of 5S rDNA.
These are shown in Fig. 1. Only one
allele was generated for 24 positions, two alleles were generated for
69 positions, and all three possible mutants were generated for 28 positions. Two mutant 5S rDNA clones harbored deletions (marked ),
and one contained an additional A residue between positions 103 and 104 (marked +A104). Substitutions of pyrimidines greatly outnumbered those
of purines (especially G residues), presumably because the smaller size
of the pyrimidines favored their incorporation during the chemical
synthesis of the mutagenic oligonucleotides. Standard plasmid shuffle
methods were used to replace the wild-type 5S rDNA plasmid with those
harboring mutants. Direct reverse transcription sequencing of
cellular 5S rRNAs harvested from 10 different mutants was used to
confirm that all 5S rRNAs in the cells corresponded to single clonal
mutants.
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FIG. 1.
Map of yeast 5S rRNA. The wild-type Saccharomyces
cerevisiae 5S rRNA sequence is shown. Arrows indicate
the 246 mutants with point mutations. , deletion allele (e.g.,
deletion of one nucleotide at positions 21 and 36); +A, insertion of an
extra A residue between positions 103 and 104.
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Phenotypic characterization of the 5S rRNA mutants.
Endogenous
genetic markers were used to assess the effects of the 5S rRNA alleles
on ribosome-related functions. These are summarized in Table
1. In general, transversions (Pu
Py)
tended to be twice as likely to produce mutant phenotypes as
transitions (PuPu and PyPy). This was especially apparent in
replacements of pyrimidines by purines, indicating that the phenotype
might be more affected by the size of a substituting nucleotide rather than by the precise nature of the substitution. The effects of the 5S
rRNA mutants on specific phenotypes are discussed in greater detail
below.
Effects of the 5S rRNA mutants on cell growth and viability.
Although 5S rRNA is highly conserved, only four of the mutants were
lethal: C98G, U114C, and C116A alleles and the +A104 allele (Fig.
2). To test whether a compensatory
mutation would rescue the C116A allele, we constructed a 5S rDNA clone
harboring the C116A plus G5U allele. Expression of this 5S rRNA species
had no discernible effect on cell growth, nor did it produce a result in any other obvious mutant phenotype.
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FIG. 2.
Growth phenotypes of the 5S rRNA mutants. The sequence
of wild-type S. cerevisiae 5S rRNA is in the center.
Alleles conferring specific growth-related phenotypes are in boldface.
Diamonds, lethal alleles; circles and squares, alleles
conferring lethality due to temperature sensitivity (37°C) and cold
sensitivity, respectively.
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Since many mutant alleles in yeast confer sensitivity to extremes in
temperature, we assayed the effects of the library of 5S rRNA mutants
at both 13 and 37°C (Fig. 2). Because cold sensitivity tends to be
associated with macromolecule assembly (e.g., ribosome biogenesis)
defects, we monitored cell growth at 13°C. Only one cs-conferring allele was identified, the
C10A allele. This was the only allele in the highly conserved loop A
region conferring any detectable phenotype, and the C10U allele had no
such effect. The paucity of cs-conferring
alleles is consistent with the fact that the 5S-L5 particle is
assembled into the ribosome late in the ribosomal biogenesis program,
i.e., that 5S rRNA does not provide a critical foundation for assembly
of subsequent essential ribosomal structural features. Four
ts-conferring alleles were also identified: the
A24C, C36U, C47G, and G99A alleles.
Nonsense suppression and nonsense-mediated mRNA decay-associated
phenotypes.
The presence of endogenous nonsense-containing alleles
(ade2-1 and can1-100)
provided inbuilt monitors of the effects of the 5S rRNA mutants on the
ability of ribosomes to recognize termination codons. The
ade2-1 allele of the phosphoribosylaminoimidazole carboxylase (AIR decarboxylase) gene harbors a nonsense (ochre) codon,
and cells harboring this allele that are wild type with respect to most
other genes are pink when grown in the absence of exogenous adenine as
a result of the accumulation of a red pigment (AIR). Cells that
were able to suppress this mutation grew as either light pink or white
colonies, indicating increased readthrough of the nonsense codon. In
contrast, cells in which a mutation causes the translational apparatus
to be hyperaccurate generated bright red colonies in this strain
background. Of the entire collection of 242 viable mutants, 239 were
assayed regarding their Ade phenotypes (Fig.
3A). A total of 44 (19%) of the alleles were strong suppressors. There was almost a threefold-greater frequency
of this class of mutants occurring at a conserved nucleotide than at a
nonconserved one (32 to 12). Of the five alleles (2% of the total)
that conferred hyperaccurate phenotypes, there was an even split
between mutations at conserved versus nonconserved positions (three to
two).
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FIG. 3.
Nonsense-suppression phenotypes of the 5S rRNA mutants.
The endogenous ade2-1 (A) and
can1-100 (B) nonsense-containing alleles
were used to monitor the effects of all of the 5S rRNA mutants on the
ability of the ribosomes to recognize termination codons. Scoring for
degree of accuracy, e.g., hyperaccurate, wild-type, and weak and strong
suppression, is described in Materials and Methods. (C) The C19A, A23G,
U86A and U48A alleles of 5S rRNA can suppress
ade2-1 but not
can1-100. Serial dilutions of cells
harboring wild-type 5S rRNA or the indicated 5S rRNA alleles were
spotted onto H-Arg medium containing 60 µg of canavanine/ml or onto
YPD medium lacking adenine and incubated at 30°C for 3 days.
Suppression of can1-100 is indicated by
the inability of cells to grow in the presence of canavanine.
Suppression of ade2-1 is indicated by the
formation of white colonies. (D) Mutations in 5S rRNA do not affect the
nonsense-mediated mRNA decay pathway. Total RNAs (10 µg) extracted
from JD1111 cells harboring wild-type (WT) 5S rRNA or selected 5S rRNA
mutant alleles or from the upf1 strain HFY870 were
separated through a 1% denaturing agarose gel, transferred to
nitrocellulose, and probed with a 32P-labeled Cyh2 fragment
as previously described (6). The abundances of the mature
Cyh2 mRNA and the Cyh2 pre-mRNA were determined using a Typhoon 8600 phosphorimager and ImageQuant, version 5.2, software (Molecular
Dynamics). The pre-Cyh2/Cyh2 ratios for all of the mutants were
normalized to that from cells harboring wild-type 5S rRNA.
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Similarly, the can1-100 allele harbors an ochre
mutation in the CAN1-encoded arginine permease that
prevents transport of canavanine, a toxic arginine homolog, into the
cell. Thus, the parental JD1111 strain is Canr,
whereas suppression of this allele renders cells
CanS. We tested 223 mutants for growth in the
presence of 60 µg of canavanine/ml (Fig. 3B). The results closely
paralleled the ade2-1 tests: 47 total alleles
conferred canavanine sensitivity, with a threefold greater
chance of this occurring at a conserved nucleotide. Interestingly,
though four of the 5S rRNA mutants were able to suppress the
ade2-1 nonsense-containing allele, they were unable to
suppress the can1-100 allele (C19A, A23G, U86A
and U48A alleles; Fig. 3C). We suggest that this is reflective of
quantitative differences in their abilities of suppress nonsense alleles.
The presence of premature termination codons in mRNAs, as a
consequence of point mutations or frameshift mutations, or of those
contained in the introns of unspliced mRNAs that escape to the
cytoplasm could result in production of toxic truncated protein
products. It is therefore important that cells be able to identify and
rapidly rid themselves of nonsense-containing mRNAs. A complex, yet
evolutionarily conserved, apparatus involving trans-acting
factors and cis-acting signals called the nonsense-mediated mRNA decay (NMD) pathway has evolved to handle this problem (reviewed in references 23 and 49). Since nonsense suppression
defects have been linked to defects in the NMD pathway, we surveyed the NMD phenotypes in a selected subset of mutants. The inefficiently spliced endogenous Cyh2 precursor mRNA was used to monitor the status
of the NMD pathway in cells expressing both wild-type and mutant 5S
rRNAs. The results from this partial survey of the mutants show that
none of the strong ade2-1 or
can1-100 suppressors confer defects in NMD (Fig.
3D). Probing these blots for the Can1 transcript revealed that this was
also not stabilized by any of the 5S rRNA mutants (data not shown). The
fact that none of the nonsense suppressors had NMD defects is
consistent with previous work demonstrating that, although the two
mechanisms are linked, they are also separable (reviewed in reference
8).
Killer virus maintenance and programmed 1 ribosomal
frameshifting.
Propagation of the yeast killer virus is extremely
sensitive to the status of the host translational apparatus, providing an inbuilt indicator of defects in ribosome function (44).
Figure 4A shows the summary results for
the mutants for the killer phenotype. Of 229 alleles examined, over
one-third had discernible effects on the killer. Virus-infected yeast
cells can be further divided into those that completely lost the
killer phenotype (K) and those around which the
zone of growth inhibition was reduced, i.e., weak killers
(Kw). Examples of these different phenotypes are
shown in Fig. 4B. The 5S rRNA alleles tended to favor production of the
K over the Kw phenotype
by a ratio of approximately 3:2 (Table 1).
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FIG. 4.
Killer phenotypes of the 5S rRNA mutants. (A) Effects of
each of the mutants on the killer virus phenotype. The map of 5S rRNA
and mutants is as described for Fig. 1, and killer phenotypes are
shown. (B) Representative killer plate assay. Colonies of JD1111 cells
harboring various 5S rRNA alleles were replica plated onto a lawn of
cells that are sensitive to the secreted killer toxin produced by the
M1 satellite virus of L-A. Killer activity was observed as
a zone of growth inhibition around the colonies. (Left) Photograph of
the killer plate assay. (Right) Key indicating 5S rRNA genotypes. WT,
wild type. (C) Northern blot probing for L-A and M1 viral
RNAs. Total RNAs were isolated from the indicated strains and analyzed
by Northern blotting for the presence of the L-A and M1
viral RNAs as described in Materials and Methods. (D) Programmed 1
ribosomal frameshifting phenotypes of selected mutants. JD1111 cells
harboring wild-type, C26U, C28U, C29A, or G30C 5S rRNA alleles were
transformed with either p-1 frameshift indicator or p0 control
plasmids, and efficiencies of L-A virus-promoted programmed 1
ribosomal frameshifting were monitored as previously described
(17). Assays were performed in triplicate, and bars denote
percent error.
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A number of factors can contribute to an observed killer phenotype.
These include loss or decreased copy numbers of the helper L-A virus
and/or the killer toxin-producing M1 satellite
virus, defects in the gene products that are responsible for processing the killer toxin precursor into the mature toxin, and defects in the
apparatus responsible for secretion of the toxin out of the cell. It
has been shown that two types of flaws in the translational apparatus,
general 60S ribosomal subunit biogenesis defects (44) and
those that promote increased or decreased programmed 1 ribosomal frameshift efficiencies on the L-A viral mRNA (reviewed in reference 14), can lead to total or partial loss of L-A and/or
M1. In light of the data suggesting a role for 5S
rRNA in enhancing translational fidelity, the effects of the 5S rRNA
mutants on the ability of cells to maintain these viral dsRNA genomes
was examined (Fig. 4C). RNA hybridization analyses of a selected subset
of the alleles revealed that loss of the killer phenotype could result
from either (i) complete loss of both L-A and M1
(e.g., C28U; sample 4 in Fig. 4C), (ii) loss of
M1 with or without attendant decreases in L-A
copy number (e.g., compare U55A [no. 15]) with U53A [no. 13], or
(3) the production of defective-interfering M1
deletion mutants (e.g., G99A [no. 31] and G101C [no. 34]). The
Kw phenotype was found to be due to decreases in
M1 copy numbers either with or without
accompanying decreases in L-A concentrations (e.g., compare U31A [no.
7]) with U54C [no. 14]). In no cases were effects on the killer
phenotype found associated with wild-type M1
levels, suggesting that all the translational defects of the 5S rRNA
mutants affected virus propagation rather than processing and export of
the toxin.
The inability to maintain both L-A and M1 is
unusual: to date, mutants from only three other complementation groups,
MAK3, MAK10, and PET18, have been
shown to confer this phenotype (reviewed in reference 61).
A preliminary characterization of the programmed 1 ribosomal
frameshifting phenotypes of four examples of this class of 5S rRNA
mutants revealed that they all promoted strong decreases in
frameshifting efficiencies (Fig. 4D). We suggest that the reason for
L-A loss in these mutants is due to decreased availability of the
Gag-Pol dimer, excluding encapsidation of the L-A dsRNA into nascent
viral particles.
| |
DISCUSSION |
A powerful system for rRNA structure and function studies of a
model eukaryotic organism.
The results presented here demonstrate
the strength of the rdn1 strains as a tool to address
issues related to rRNA structure and function in a model eukaryotic
system. We have introduced modifications to the original strain and
plasmids that make such analyses more practical, specifically
stabilizing the clones by separating the RNA polymerase I and III
transcribed operons onto two plasmids and making LEU2
available as a selectable marker. Although this report focuses on 5S
rRNA for both historical and scientific reasons, the results presented
here provide proof of the principle that this system is amenable for
mutagenesis-based structure and function studies of all of the yeast
rRNAs. It should also be possible to use this system for large-scale
substitution studies, e.g., replacement of either entire yeast rRNA
genes or regions thereof with homologous sequences from other organisms.
Genotype, phenotype, structure, and function: clustering of
sequences responsible for mutant phenotypes at the critical
contacts between 5S rRNA and the major functional regions of the
ribosome.
Overall, just 4 of the 246 alleles were lethal, and only
5 more conferred either cold or heat sensitivity (Table 1). These findings support suggestions from other investigators that the rRNAs
are resilient molecules that can retain their functions despite
mutations at universally conserved bases within critical regions, e.g.,
the peptidyltransferase center or the sarcin-ricin loop (31, 32,
47, 58). The presence of multiple endogenous genetic markers in
this strain enabled us to qualitatively address 5S rRNA function by
examining the effects of the 5S rRNA mutants on other phenotypes. At a
general level, nucleotides at 64 of the 121 positions of yeast 5S rRNA
correspond to the eukaryotic consensus 5S rRNA sequence, and mutations
introduced into these evolutionarily conserved positions were
approximately twice as likely to promote mutant phenotypes than those
at the nonconserved positions. There are notable exceptions to this
trend: mutations of some highly conserved bases did not elicit
phenotypes (e.g., U33, G49, G85, G87, etc.), while some mutations of
some of the nonconserved bases promoted strong mutant phenotypes (e.g.,
nucleotide C19).
Charting the various phenotypes of the 5S rRNA mutants onto a consensus
map of the eukaryotic 5S rRNA and projecting these onto a topological
map of the H. marismortui 5S rRNA crystal structure (2) reveal that the phenotypes tended to cluster into two
minor and three major discrete regions (Fig.
5). The minor regions were (i) bases 69 to 71 and (ii) helix I. The contribution of bases 69 to 71 was
minor, only affecting the maintenance of the killer virus. The helix I
alleles tended either to be lethal or to only affect the killer
phenotype. The importance of this region of the gene for correct
posttranscriptional 3' end processing of 5S rRNA may provide an
explanation for the lethal phenotypes produced by the U114C and C116A
alleles. With regard to nucleotide 116, whereas the C116A allele
exhibited a strong dominant lethal phenotype, cells harboring either
the C116U allele or the C116A plus G5U double mutant appeared to
be defect free. Though a likely conclusion from these data suggests
that, while the identity of the base at position 116 is important, the
effects of changing it can be overcome by ensuring base pairing with
the nucleotide at position 5. Overexpression of the C116G allele from a
high-copy-number plasmid in an RDN1 wild-type background
also produced no discernible defect (29). Thus, the answer
to this quandary awaits more detailed biochemical and molecular
analyses. Mutations in the highly conserved loop A region (and adjacent
sequences, notably the conserved G67-U111 and C68-G110 base pairs) also
had no significant effects. Thus, it appears either that specific
nucleotides in this region do not play a major role in the function of
5S rRNA in the context of translational fidelity or that, if this arm
of the molecule has a specific function, our analysis was not
sufficient to elucidate it.
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|
FIG. 5.
Mapping the different phenotypes of the 5S rRNA mutants
onto the eukaryotic 5S rRNA consensus sequence and H.
marismortui 5S rRNA crystal structure reveals phenotypic
clustering. (Left) Eukaryotic 5S rRNA consensus sequence. R, purine; Y,
pyramidine; *, any base. Red bases are conserved in the yeast 5S rRNA
sequence. Classes of phenotypes are colored as indicated. Killer,
impact on the ability of cells harboring mutant 5S rRNA alleles to
maintain the killer phenotype as shown in Fig. 4; growth and viability,
impact of the mutants on cell viability (lethality at 13, 25, or
37°C) as shown in Fig. 2; accuracy, nonsense-suppression phenotypes
as shown in Fig. 3; allele-specific phenotypes, regions in which
individual mutant alleles promote specific phenotypic defects. (Right)
Crystal structure of H. marismortui 5S rRNA
(2). Red, bases from this study corresponding to
suppressors of the ade2-1 allele; arrows, analogous
regions of the molecule as depicted by the two different topological
representations.
|
|
In contrast, mutations in three defined regions of the molecule had
very significant impacts on ribosome function. These were (i) the loop
Bloop C arm, (ii) the loop E-helix IV interface, and (iii) the
bottom of helix IV and the highly conserved G91 base in loop D. These
regions are topologically located at the head, middle, and tail of the
molecule, respectively (Fig. 5). With regard to the atomic scale
structures of H. marismortui and T. thermophilus
ribosomes, 5S rRNA appears to form a chain running from the top of the
central protuberance of the large subunit, down through the ASF and
ending at the GTPase-associated center (Fig.
6) (2, 66). Mutations in the
loop Bloop C arm tended to produce a broad range of defects. While
the most severe of them appear to map along a helical face of the
molecule from nucleotide 19, crossing over to positions 59 to 51 and
back again to nucleotides 28 to 35, those in the bases on the other
face of this helix tended to have fewer severe consequences
(Fig. 5 and 6B). The yeast loop Bloop C 5S rRNA mutants also
coincide with regions from E. coli 5S rRNA that were
previously shown to be affected by the binding of the small subunit
(56). This region of the molecule interacts with several
ribosomal proteins (in T. thermophilus, these are L5 [yeast
L11] and L18 [yeast L5]) and helps to form a critical contact
between the large and small ribosomal subunits (66). This
region of 5S rRNA also interacts indirectly with the peptidyl-tRNA (66), and we have demonstrated that mutant alleles
encoding the yeast ribosomal protein L5 have 5S rRNA-dependent
peptidyl-tRNA binding defects, promoting increased rates of programmed
ribosomal frameshifting, which in turn inhibit propagation of both the
M1 killer and Ty1 viruses
(34). The existence of this phenotypic cluster suggests
that loop Bloop C region of 5S rRNA may be involved both in
communicating with the small subunit and in maintaining translational
reading frame at the P site.
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|
FIG. 6.
Structure and function mapping of the yeast 5S rRNA
mutants onto the H. marismortui
large-subunit rRNA. (A) Ribbon diagram of the H.
marismortui large-subunit rRNAs. Blue, 5S rRNA; aqua, 23S rRNA.
CP, central protuberance; GTPase, GTPase-associated center. (B) 5S rRNA
as a bridge to mediate communication between the decoding center and
the GTPase-associated center. Dashed box and arrow, region of the large
subunit enhanced. Red nucleotides map mutations in 5S rRNA which
suppress the ade2-1 allele of yeast (this
study) and those in 23S rRNA which also decrease translational accuracy
in E. coli. Nucleotides in which substitutions
lead to the hyperaccurate phenotype are blue. Shown are 5S rRNA (blue);
the ASF (green); helices 89 (yellow), 91 (magenta), and 92 (magenta);
and the sarcin-ricin loop (SRL) (green) of the 23S rRNA of H.
marismortui (2). (C) 5S rRNA as a bridge to
mediate communication between the peptidyltransferase center and the
elongation factor binding center. Dotted box and arrow, region of the
large subunit enhanced. Nucleotides increasing their reactivity upon
mutation at E. coli 23S rRNA residue 960 are in red, and
residues promoting decreased reactivity are in blue (54).
The latter nucleotides coincide with the residues protected by the P
site-bound tRNA and may influence helix 89 (indigo) and 5S rRNA.
Mutations in 5S rRNA at positions 87 and 91 or 5S rRNA (red) also
affect translational accuracy. Helices 39 (black) and 80 (green) of the
large-subunit rRNA are located in immediate proximity to the
peptidyltransferase center. We suggest that an allosteric signal
transmission pathway through 5S rRNA could serve to coordinate the
peptidyltransferase reaction with subsequent EF-2 binding and GTP
hydrolysis.
|
|
The loop E-helix IV interface is the site of the second important
cluster of 5S rRNA alleles: these map to a point of contact between 5S rRNA and helix 38, also known as the ASF (Fig. 6B). The
+A104 allele is of particular interest because the associated region of
5S rRNA contains an A minor motif that makes the most significant contact with 23 S rRNA through an "A patch"
(36). We speculate that the addition of an extra
nucleotide into this region interfered with formation of this critical
structural determinant to an extent sufficient to prevent viability.
The C98G and G99A alleles are also of special interest in that they
were previously shown to promote a significant conformational change in
the 5S rRNA molecule (59), and their overexpression in a
wild-type background promoted increases in both 1 and +1 programmed
ribosomal frameshifting (17). The lethality of the C98G
mutation is suggestive of ribosomes with catastrophic translational
fidelity defects, while the temperature sensitivity-conferring G99A
allele likely has a similar but less severe impact. The existence of
this phenotypic cluster suggests that this region of 5S rRNA is
involved in helping the ribosome monitor the status of the aa-tRNA in
the A site.
The third cluster of mutations, those at the bottom of helix IV, tended
to be allele (compare U86A to U86C) and phenotype (e.g., C93 and C94)
specific. The highly conserved G91 was the only base in loop D with any
phenotypic effects. The yeast G91 is located at the same structural
region of the molecule as the E. coli U89, which has been
shown to make multiple contacts with the large subunit rRNA (reviewed
in reference 35). These findings suggest that specific
base contacts are important for function in this region of the 5S rRNA
molecule. Structurally, the helix IV-loop D region is where 5S
interacts with helices 89 and 39 of the large-subunit rRNA: mutants in
this region lie at crucial contact points between 5S rRNA, the
GTPase-associated center, and the peptidyltransferase center,
respectively (Fig. 6B and C). Thus, this phenotypic cluster links 5S
rRNA to the two remaining functional centers of the ribosome.
5S rRNA as a physical transducer of information within the
ribosome.
We propose that a major function of 5S rRNA could be to
enhance translational fidelity by acting as a physical transducer of
information between all of the different functional centers of the
ribosome. For example, though the decoding of the genetic information
encoded in the mRNA is considered to be the function of the small
ribosomal subunit, the genetic and biochemical evidence leads us to
hypothesize that 5S rRNA may help to enhance translational fidelity by
coordinating the transfer of aa-tRNA from eukaryotic elongation factor
1A to the ribosomal A site by linking this small-subunit functional center with the large-subunit GTPase center. In support of
this, random-mutagenesis approaches have demonstrated that several
components of the 23S rRNA are involved in maintaining translational
fidelity (42). One of these, the loop of helix 92, interacts with the A site-bound aa-tRNA (26). This loop also interacts with helix 89, which in turn contacts 5S rRNA (Fig. 6B).
Thus, 5S rRNA could communicate with helix 92 through helix 89. There
is also a link from the tip of 5S rRNA through helix 89 to the
sarcin-ricin loop via helices 92 and 91. Both error-prone and
hyperaccurate mutants are known to occur at the sarcin-ricin loop
(33, 43), and thus it is possible that upon the binding of
cognate aa-tRNA an allosteric signal could be transduced from the small
subunit to the large subunit to 5S rRNA through the intersubunit
contact through L5 (yeast L11). From there, the signal would be
transduced through 5S rRNA to the GTPase-associated center, activating
the GTPase activity of eEF-1A. By our model, the translational fidelity
phenotypes associated with the mutant alleles at position 91 in
particular are a consequence of defects in their abilities to
efficiently transmit information across the gap between the tip of 5S
rRNA and helix 89, thus inhibiting communication between the small
subunit and the GTPase-associated center.
The grouping of mutants in the loop E-helix IV region of 5S rRNA
suggests the presence of another critical contact between the aa-tRNA
and the GTPase-associated center. We propose that the reason that these
mutant 5S rRNAs affect translational fidelity is that they
inefficiently convey information along this transmission line. The
existence of this phenotypic cluster suggests that 5S rRNA may also be
involved in monitoring the transfer of aa-tRNA from eEF-1A to the
ribosomal A site. Thus, accommodation of the CCA end of the aa-tRNA
could be monitored via transmission of a signal through this contact to
the other functional centers of the ribosome. It is important to note
that we have drawn the 5S rRNA loop E as a base-paired region because
the high-resolution crystal structures show it as such (2, 36,
66). However, most depictions of 5S rRNA prior to elucidation of
the crystal structures show this region as a loop. As has been noted
elsewhere, many different crystal forms of ribosomes can be obtained,
each of which diffract X rays with various degrees of resolution,
presumably because of differences in the extent of order or disorder
among them (51, 64). Thus, the reason that loop E is
closed in these crystal structures may simply be that this conformation
contributes to the overall order of the crystals, i.e., those that have
been used in the analyses because they diffract X rays with the highest resolution. Viewed in this light, it is possible that the loop E region
may indeed alternate between open and closed conformations, contributing to the allosteric signaling potential of 5S rRNA.
Another putative signal transmission chain could coordinate the
activities of the peptidyltransferase and elongation factor binding
centers (Fig. 6C). The existence of this chain and the involvement of
5S rRNA in this pathway were first postulated in cross-linking
experiments (19, 20, 55) and were further supported by
site-directed mutagenesis of helix 39, which is in contact with the D
loop of the 5S rRNA (54). These mutants specifically alter
the chemical reactivity of nucleotides that have been implicated in
binding of the CCA end of the peptidyl-tRNA at the P site. Helix 39 interacts with helix 80, which base pairs with the P site-bound
peptidyl-tRNA; thus the interaction of 5S rRNA with helix 39 means that
5S rRNA may influence the interactions between the ribosome and
peptidyl-tRNAs and vice versa. The third critical cluster of 5S rRNA
mutants, those at the tip of helix IV of 5S rRNA, and G91 lie in
close proximity to helix 39 (Fig. 6C). As noted above, this
phenotypic cluster is also a site of interaction between 5S rRNA and
the functional region formed by helices 89, 92, and 91 and the
sarcin-ricin loop. Thus, this cluster points to a functional connection
through 5S rRNA between the peptidyltransferase center and the major
sites of interaction between the elongation factors and the ribosome.
This suggests that 5S rRNA is also involved in the communication
between the peptidyltransferase center and the elongation factor
binding center. An allosteric signal transmission pathway through 5S
rRNA could thus serve to coordinate the peptidyltransferase reaction
with subsequent eEF-2 binding and GTP hydrolysis.
In conclusion, we propose that all of the different functional centers
of the ribosome are able to coordinate their activities by transmission
of allosteric signals through 5S rRNA. In this manner, 5S rRNA could
serve to ensure the fidelity of each step in the translation program.
The 5S rRNA mutants described in this study will enable us to perform
meaningful biochemical tests of this model. Additionally, in light of
this model it is interesting that the assembly of the 5S rRNA into the
ribosome is one of the last steps in ribosome biogenesis. One can
speculate that the translational apparatus has evolved so as to ensure
that ribosomes are only functionally activated at a late stage in the
biogenesis program by addition of 5S rRNA to ensure protein synthesis
in the appropriate cellular compartments.
This work was supported by grants to J.D.D. from the National
Institutes of Health (R01 GM58859 and R01 GM62143).
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Abstract
Introduction
Present address: Department of Microbiology, University of
Washington Regional Primate Research Center, Seattle, WA 98195.
