Our results suggest that the mutant IF1 strain is perturbed at the level of ribosomal subunit association, and the suppressor mutations partially com-pensate for this defect by disruptin
Trang 1by alterations in the 23S rRNA maturation region
Jaroslav M Belotserkovsky, Georgina I Isak and Leif A Isaksson
Department of Genetics, Microbiology and Toxicology, Stockholm University, Sweden
Introduction
Bacterial protein synthesis, as directed by the action of
the ribosome, can be broadly divided into four main
phases – initiation, elongation, termination, and
recy-cling Initiation is the rate-limiting step [1] This phase
is mediated by initiation factor 1 (IF1), initiation
fac-tor 2, and initiation facfac-tor 3 IF1 is the smallest of the
initiation factors [2] Its structure has been determined
by NMR spectroscopy, revealing that IF1 is a member
of the oligomer-binding (OB-fold) family of proteins,
with structural similarities to cold shock proteins [3]
In addition, IF1 has been shown to complement
lesions in several cold shock response proteins in
Bacil-lus subtilis[4] The interaction of IF1 with the bacterial
ribosome has been investigated by chemical probing
[5], mutagenesis studies [6], and crystallography [7,8]
These data indicate that IF1 makes contacts with the
functionally important bases G530, A1492 and A1493
in 16S rRNA In addition to its direct involvement in
translation initiation [1], IF1 has been shown to be an
RNA chaperone [9], as well as playing a role in tran-scriptional antitermination in Escherichia coli [10] More recently, it has been reported that IF1 acts as a sensor of cis-elements in mRNA [11] and, together with initiation factor 3, determines the rates of ribo-somal subunit joining by inducing conformational changes in the 30S subunit [12]
Each of the seven rRNA operons in E coli is tran-scribed as a single primary transcript The order of gene products relative to the start of transcription is 16S, 23S, and 5S, with some tRNA species between 16S and 23S as well as at the end of the transcript As transcription proceeds, the rRNA forms secondary structures that are substrates for the binding of ribo-somal proteins and maturation factors [13] Among these structures are double-stranded stems that are composed of terminal flanking sequences of 16S and 23S rRNAs These ‘processing’ stems are substrates for RNase III, and other ribonucleases that trim the stems
Keywords
Escherichia coli; RNase III; rRNA mutation;
rRNA processing; translation
Correspondence
L A Isaksson, Department of Genetics,
Microbiology and Toxicology, Stockholm
University, S-10691 Stockholm, Sweden
Fax: +46 8 164315
Tel: +46 8 164197
E-mail: Leif.Isaksson@gmt.su.se
(Received 24 January 2011, revised 1 March
2011, accepted 14 March 2011)
doi:10.1111/j.1742-4658.2011.08099.x
Genetic selection has been used to isolate second-site suppressors of a defective cold-sensitive initiation factor I (IF1) R69L mutant of Escherichia coli The suppressor mutants specifically map to a single rRNA operon on a plasmid in a strain with all chromosomal rRNA operons deleted Here, we describe a set of suppressor mutations that are located in the processing stem
of precursor 23S rRNA These mutations interfere with processing of the 23S rRNA termini A lesion of RNase III also suppresses the cold sensitivity Our results suggest that the mutant IF1 strain is perturbed at the level of ribosomal subunit association, and the suppressor mutations partially com-pensate for this defect by disrupting rRNA maturation These results support the notion that IF1 is an RNA chaperone and that translation initiation is coupled to ribosomal maturation
Abbreviations
Amp, ampicillin; Cm, chloramphenicol; IF1, initiation factor 1; Kan, kanamycin; Tet, tetracycline; TIR, translation initiation region.
Trang 2to eventually produce mature rRNA termini [14,15].
The processing of 23S rRNA is strictly dependent on
the action of RNase III A deletion in the gene
encod-ing this enzyme results in immature 23S rRNA with
extended termini, whereas 16S rRNA is matured to
completion [16]
There is ample evidence that the maturation of the
two ribosomal subunits is interdependent, and that
subunit maturation events are functionally linked to
translation initiation [13] Here, we have isolated
muta-tions in the 23S rRNA processing stem that suppress a
cold-sensitive mutant of IF1 This serves as additional
evidence that ribosome maturation and translation
initiation are intimately linked
Results
Mutations in the processing stem of 23S rRNA
The existence of D7 E coli strains, in which all seven
rRNA operons are deleted, has facilitated studies with
rRNA that exists as genetically pure populations of
cellular ribosomes This is possible because a plasmid
with any one of these genes can compensate for the
deleted rRNA genes We have used such a strain in
order to select for mutations in rRNA that suppress
the cold-sensitive phenotype of a mutant IF1 (R69L)
This mutation in IF1 leads to significant growth
inhibi-tion at low temperatures [17] A D7 strain containing
IF1R69L was screened for spontaneous revertants that
were cold-resistant at the nonpermissive temperature
of 23C We specifically sought revertants that carried
second-site suppressor mutations on the
plasmid-encoded rRNA operon Here we report a set of
sup-pressor mutations that are located in the 23S rRNA
processing stem (Fig 1) This structural region is
sub-ject to cleavage by RNase III and other ribonucleases
during 23S rRNA maturation [16] In particular, the
positions of mutated residues overlap with known
RNase III cleavage sites [18] The same base was
found to be mutated in the mutant plasmids pD1 (G
to T) and pD6 (G to A) as a result of independent
selections, indicating that such suppressors are
com-mon in this structure (Fig 1) When plasmids with the
mutated rrnB gene were introduced into the IF1
mutant strain JB69, thus providing the sole source of
rRNA, noticeable growth enhancement was observed
on solid medium as well on as rich liquid medium
upon downshift to the nonpermissive temperature of
23C (Figs 2A and 3) It should be noted that the
R69L mutation is not lethal in the downshift
condi-tion, but merely deleterious This explains why JB69
continued to grow, albeit slowly, following the
down-shift (Fig 3) The obvious question was whether these mutations have any effect on 23S stem processing Pri-mer extension analyses were used to check the termini
of 23S rRNA from total cellular extracts in these mutants Additional bands corresponding to accumula-tion of precursors of 23S rRNA in mutant plasmids were detected (Fig 4A) The expected 5¢ mature and )7 termini, as well as additional bands corresponding
to approximate )41 (e1) and )46 (e2) termini, were identified in strains carrying the mutant plasmids pD1 and pD6, which carry mutations on the 5¢-side of the processing stem, gave rise to rRNA with the majority
of termini in the mature form, whereas pD3, with a mutation on the 3¢-side, gave rise rRNA with the majority of the termini in the)7 form In addition, for
Fig 1 Secondary structure of 23S rRNA processing stem, show-ing sites of suppressor mutations Cleavage sites of RNase III on naked RNA (solid arrows) and on the ribosome (dashed arrows) are indicated The sequence of mature termini of 23S rRNA is in bold Sites of mutations are encircled Mutants are designated as fol-lows: pD1 and pD6 carry mutations in position G8, and are G to T and G to A substitutions, respectively pD3 has a C to A substitu-tion at posisubstitu-tion C + 2 Figure reproduced from [18].
Trang 3pD1, the e1 ()41) terminus was more prominent Thus,
there were apparent differences between the mutant
plasmids in extended termini, depending on the
mutated position in the processing stem Taken
together, these results suggest that the processing stem
mutations block nucleolytic processing, most likely by
RNase III or other RNases; however, the blockage is
incomplete, as shown by the existence of mature 23S termini in all mutants As these mutations were iso-lated as suppressors of a cold-sensitive IF1 mutant, we considered the remote possibility that IF1 has some involvement in the processing of this structure We reasoned that, if IF1 has such a role, we would detect differences in the relative amounts of bands corre-sponding to mature 23S and other extended termini when total cellular RNA was extracted at the nonper-missive temperature of 23 C and when it was extracted at 37C Processing of mutant plasmids was compared with that of the wild type in both JB69 and SQZ10, using total RNA extracted from cells grown at
23C and 37 C No significant differences were found, indicating that processing stem mutations affect the processing of 23S rRNA irrespective of incubation temperature or strain background (not shown)
Lack of processing suppresses the IF1 cold sensitivity phenotype
Next, we investigated whether the suppression pheno-type results from the nature of the 23S processing stem mutations themselves, or whether a general lack of 23S processing stem maturation would result in the same phenotype To this end, we generated a large deletion
in the ORF of rnc, a gene encoding RNase III, in the cold-sensitive JB69 and CVR69L strains The deletion was designed such that only the first 15 and last 38 amino acids were left intact in the ORF The reason was to avoid potential disruption or polar effects on the downstream and overlapping ORF that encodes the essential Era GTPase [19] We found that the RNase III lesion in JB69 resulted in the same apparent phenotype as the 23S processing stem mutations when
Fig 2 Phenotype of cold-sensitive IF1 mutants with various suppressors (A) A plate incubated at 23 C for 72 h with the D7-derived strains, where: IF1 is pKK3535 ⁄ JB69; IF1Drnc is pKK3535 ⁄ JB69Drnc; IF1 + pD1, pD3 and pD6 are suppressor plasmids pD1, pD3 and pD6
in JB69, respectively (B) A plate incubated at 20 C for 72 h with the MG1655-derived strains, where: IF1 is CVR69L; IF1Drnc is CVR69LDrnc.
Fig 3 Growth properties of suppressor, wild-type and IF1 strains
in rich liquid medium Cultures were grown to D590 nm0.2 at 37 C,
after which they were shifted to 23 C (shown as dotted line).
Growth trajectories of strains are labeled as follows: ¤,
pKK3535 ⁄ SQZ10; j, pD3 ⁄ JB69; m, pKK3535 ⁄ JB69;•, pKK3535 ⁄
JB69Drnc Suppressor plasmids pD1 and pD6 in JB69 are omitted
for clarity, as they have identical trajectories to pD3 ⁄ JB69 Straight
lines were fitted to data generated from at least three independent
experiments.
Trang 4the strain was grown on solid medium (Fig 2A)
How-ever, in rich liquid medium, the suppression effect was
less apparent for this strain (Fig 3) It can be seen
that, in these culture conditions, the rnc strain had a
different growth trajectory from the processing stem
mutants (pD3) This suggests that the RNase
III-dependent suppressor may act in a different manner
from the processing stem mutations As a deletion in
rnc acts as a suppressor of JB69, a D7-derived IF1
mutant strain, it was of interest to determine whether
this deletion would also give rise to a similar
suppres-sion phenotype in the MG1655 (wild type)-derived IF1 mutant strain CVR69L Indeed, we observed weak but obvious growth enhancement of CVR69L carrying a lesion in RNase III as compared with the CVR69L mutation alone at 20C (Fig 2B) This indicates that the suppression phenotype (on solid medium) is allele-specific, depending on the presence or absence of
RNa-se III Figure 4B shows primer extension analysis of the 5¢-terminus of 23S rRNA in strain JB69Drnc There were various extended termini, with the major extension products corresponding to an approximate
Fig 4 Primer extension analysis of 5¢-termini of 23S rRNA from mutant strains (A) Extension products from total cellular RNA (B) Compari-son of processing stem mutant plasmid extension products and the RNase III deletion strain (Drnc) (C) Extension products from sucrose gradient fractions, where 50 and 70 are fractions corresponding to the 50S and 70S subunit peaks Plasmids and strains are as follows: pKK3535 is the wild-type rrnB+ plasmid; pD1, pD3 and pD6 are processing stem mutant plasmids; SQZ10 is the wild-type strain (IF1wt); JB69 is the IF1 mutant strain (IF1R69L); JB69Drnc is the IF1 mutant with a deleted RNase III gene Extension products are labeled as fol-lows: M is mature 23S rRNA (55 bases long); )3 and )7 are immature 23S rRNA extension products; e1 and e2 are additional immature extension products observed in processing stem mutant strains (41 and 46 bases, respectively); e3 is the RNase III lesion-specific extension product (24 bases) Sizes of marker lane products are given in (A) The tables below (A) and (C) show quantification of extension products for each corresponding panel calculated by IMAGE analysis software.
Trang 5)24 (e3) terminus as well as the )46 terminus, as is the
case with the processing stem mutants Interestingly,
there was a total absence of a fully mature 23S
termi-nus; instead, a slightly truncated product was
observed To rule out any possible involvement of Era
in cold sensitivity suppression of mutant IF1, we
cloned the era ORF (which is immediately downstream
of rnc) under control of an isopropyl
thio-b-d-galacto-side-inducible promoter No cold sensitivity
suppres-sion was observed either with or without isopropyl
thio-b-d-galactoside induction when the era plasmid
was introduced into CVR69L (not shown) In fact,
overexpression of Era was deleterious at both 20C
and 37C, consistent with another report [20] The
corresponding experiment could not be performed in
JB69, owing to the presence of multiple plasmids in
this strain Taken together, our results suggest the
RNase III deletion results in suppression of cold
sensi-tivity JB69, probably as a result of incomplete
matura-tion of 23S rRNA, as is also the case with the
processing stem mutants However, other effects
asso-ciated with this lesion could also account for the
suppressor phenotype
The IF1 mutant has an altered sucrose gradient
ribosomal profile when shifted to 23C
Having shown that processing of rRNA is involved in
the suppression phenotype, we next examined how
such processing defects would affect sucrose gradient
ribosomal profiles of the mutant strains As the
sup-pressor effect is manifested in the cold, we employed a
temperature downshift from 37C to 23 C during
cul-turing before examining the ribosome profiles During
the course of these experiments, we noted that, upon
downshift to the nonpermissive temperature of 23C,
there were clear differences between the sucrose
gradi-ent ribosomal profiles of the IF1 mutant and wild-type
strains In particular, in the case of JB69, there
appeared to be a slight decrease in the proportion of
free ribosomal subunits and a concurrent relative
increase in the 70S ribosomes as compared with
SQZ10 (Fig 5), suggesting that JB69 is perturbed at
the level of subunit association in the cold It is known
that the Mg2+ concentration influences ribosome
sub-unit association As the differences are rather subtle,
the sucrose gradient experiments were carried out at
several Mg2+ concentrations Following a downshift
to 23C, we lysed the cells in a 6 mm Mg2+ buffer,
and applied them to sucrose gradients with varying
Mg2+ concentrations from 6 mm to 20 mm We also
performed experiments in which cells were lysed in
10 mm Mg2+buffer and applied to the same gradients,
with similar results (not shown) First, both the sucrose gradient profiles and the accompanying quanti-fication suggest that JB69 exhibits a decreased ratio of free 30S and 50S subunits relative to 70S particles as compared with SQZ10 Thus, throughout the Mg2+ titration range, there was an increased proportion of 70S particles relative to the free subunits This was evi-dent when looking at the amount of 30S subunits that were incorporated into the 70S particles as a percent-age of the total amount of 30S (value a in Fig 5) In SQZ10, this value ranged from 33% to 58% through-out the Mg2+titration range, whereas in JB69, it ran-ged from 50% to 60% at the same Mg2+ concentrations When the traces and the quantification
of peak areas in JB69 and SQZ10 were examined, it appeared that there was a stoichoimetric imbalance of 30S and 50S subunits, whereby the 30S subunits were
in excess in JB69 This was noticeable at lower Mg2+ concentrations, when only 33% of the subunits (30S) were in the 70S particles in SQZ10, as compared with 50% in JB69 However, there was little difference in the stoichiometric amounts of 30S and 50S subunits between these two strains in the 20 mm Mg2+titration – a condition where most of the 30S and 50S subunits were in the 70S particles (approximately 60%) in both strains In addition, the apparent ratio of 30S to 50S subunits in SQZ10 varied with increasing Mg2+ con-centration Thus, the observed difference may reflect the limitations in quantifying the peak areas of traces when most of the free subunits are in the 70S trace, as was the case with JB69 throughout the Mg2+titration, and with SQZ10 at high Mg2+ conentrations Taken together, the data suggest that JB69 has an increased amount of 70S particles relative to the free subunits in the cold, and that this effect is probably not attribut-able to stoichiometric imbalances of the 30S and 50S subunits The same analysis revealed that there was a general decrease in the amount of 50S subunits in the sucrose gradient in the case of the suppressor plasmid pD3 In particular, there was a consistent increase in the 30S⁄ 50S ratio of approximately 15% when pD3 was the sole source of rRNA in either SQZ10 or JB69 This suggests that there was a stoichiometric imbalance
of 30S and 50S particles, whereby 30S was in excess This effect was strain background-independent, and occurred throughout the Mg2+ titration range (com-pare traces and 30S⁄ 50S ratios of pD3 to pKK3535 in each strain background) In addition, whenever pD3 was the sole source of rRNA, there was a slight decrease in the degree of subunit association in both SQZ10 and JB69 (compare value a in the correspond-ing traces) Finally, when traces of the rnc lesion strain (JB69Drnc) were examined, it was apparent that there
Trang 6was a decrease in the total amount of ribosomal
parti-cles (30S, 50S, and 70S) as compared with all other
traces, even though the same amount of material was
applied to the gradients This decrease was consistent
and occurred throughout the Mg2+titration This
sug-gests that the total pool of ribosomes in this strain is decreased, most likely as a result of improper matura-tion of rRNA However, this effect could also be growth rate-related, as JB69Drnc has a long lag phase
in the downshift condition (Fig 3) Interestingly, the
Fig 5 Sucrose gradient profiles of ribosomes at different Mg2+ concentrations Each column is designated with the respective strain, where: pKK3535 is the wild-type rrnB+ plasmid; pD3 is a processing stem mutant plasmid; SQZ10 is the wild-type strain (IF1wt); JB69 is the IF1 mutant strain (IF1R69L); JB69Drnc is the IF1 mutant with a deleted RNase III gene Rows are designated with the corresponding
Mg2+concentration in the gradient Identities of the peaks are as indicated in the left gradient, second row a is the molar proportion of 30S subunits that are in 70S ribosomes over total 30S subunits [70S ⁄ (30S + 70S)], as a means of quantifying the extent of subunit association b
is the molar ratio of total 30S to 50S subunits (30S ⁄ 50S) Quantification is based on peak areas, whereby the 30S ⁄ 50S ⁄ 70S molar ratio is adjusted to 1 : 1.96 : 2.96 of the peak areas Each experiment was carried out three times The a and b values are given as means of these experiments, where the standard deviation does not exceed 10% of the value The figure is generated by the use of FYTIK software from raw data with a representative gradient profile.
Trang 7apparent extent of subunit association was greater in
the RNase III deletion strain than in JB69 throughout
the Mg2+ titration range Taken together, the results
suggest that the effect of the processing stem
suppres-sor mutations is to lower the available pool of mature
50S subunits as a result of incomplete 23S rRNA
mat-uration As a consequence, there is a decrease in the
extent of subunit association, as suggested in Fig 6 In
the case of the Drnc strain, this effect might have
resulted from decreases in the cellular pool of both
the 30S and 50S subunits, because of disruption of the
primary rRNA transcript maturation
As the analysis was carried out with D7 strains,
we were concerned that the observed increased
sub-unit association in the case of JB69 was an artefact
resulting from the altered genetic background
Indeed, it appeared that the parental strain SQZ10
was somewhat perturbed at the level of subunit
asso-ciation in the conditions used here To settle this, we
examined sucrose gradient profiles of the original
CVR69L IF1 mutant and its parental wild-type
strain, MG1655, when subjected to the same
down-shift to 23C (Fig 7) Here, a similar profile was
observed as in the case of JB69 as compared with
SQZ10 There was an increase in the relative amount
of 70S particles as compared with free subunits, indi-cating that the observed aberrant profile is not strain-specific, but is a function of the mutant IF1 allele
Fig 6 A model of possible mechanism of suppression of IF1R69 mutant by 23S processing stem mutations (A) Effect of the mutant IF1R69L on subunit association in the cold The forward reaction of subunit association⁄ dissociation is favored (bold forward arrow) (B) Out-come when processing stem mutations interfere with 23S rRNA processing This results in a decreased cellular pool of properly matured 50S subunits, favoring the reverse reaction of subunit association ⁄ dissociation (bold reverse arrow) III indicates sites of processing by
RNa-se III X indicates RNaRNa-se III sites blocked becauRNa-se of processing stem mutations.
Fig 7 Sucrose gradient profile of ribosomes from MG1655 (IF1wt) and CVR69L (IF1R69L) For this experiment, cells were lysed and analyzed on gradients with 10 m M Mg2+ Profiles are representative
of three independent experiments.
Trang 8Immature 23S rRNA termini are present in both
the 50S and the 70S fractions
We then investigated whether the observed extended
termini present in the 23S processing stem mutants
were incorporated into the 50S subunits and 70S
translating ribosomes To this end, we purified rRNA
from sucrose gradient fractions and checked the state
of processing of 23S rRNA termini by primer
exten-sion It can be seen in Fig 4C that when the
suppres-sor mutant plasmid pD3 was present in either SQZ10
or JB69, a significant proportion of 23S termini from
both the 50S and 70S peaks were in the immature
form, where the )7 species predominates The e2
extension species was also prevalent, and other minor
extension products were observed In the presence of
the suppressor plasmid, as little as 13% of the normal
23S 5¢-terminus was in the fully mature form In
con-trast, when the wild-type plasmid pKK3535 was the
sole source of rRNA in either SQZ10 or JB69, as
much as 59% of the termini were in the fully mature
form, with the )7 and, to a lesser extent, the )3
spe-cies accounting for the rest This was the case when
rRNA was purified from cells grown at 37C, as well
from those grown at 23C This indicates that
imma-ture extended 23S rRNA termini were incorporated
into the 50S subunits and functional 70S ribosomes,
irrespective of strain background or incubation
tem-perature It is worth noting that, in the case of the
wild-type pKK3535 plasmid in either SQZ10 or JB69,
there was a slight but consistent difference between the
extension products from the 50S and 70S fractions,
respectively In particular, there was an increase in the
relative amount of the fully mature terminus, with a
concurrent decrease of the )7 species in the 70S
frac-tion as compared with the 50S fracfrac-tion This suggests
that final maturation of 23S rRNA occurs on
translat-ing ribosomes, in agreement with other reports [13]
This effect was less apparent in the case of the
sup-pressor plasmid pD3, suggesting that extended 23S
rRNA termini are not fully matured on the translating
ribosome
Other defects in 50S subunit maturation do not
rescue cold sensitivity of mutant IF1
As we had established that processing defects in 23S
rRNA act as suppressors of a cold-sensitive IF1 mutant,
we reasoned that other similar defects in 50S maturation
as a whole may have the same effect To check this
possibility, we moved, by P1 transduction, deletions in
genes that have been shown to be involved in 50S
maturation into the cold-sensitive IF1 mutant strains
Specifically, we focused on the genes deaD, dbpA, and srmB, encoding 23S rRNA helicases DeaD, DbpA, and SrmB respectively [21–23] Deletions in these genes (one
at a time) were introduced, by P1 transduction from KEIO collection donor strains, into CVR69L as well as JB69, and checked for cold sensitivity suppression [24]
No such suppression was observed when the con-structed strains were grown at the nonpermissive tem-perature of 23C This indicates that the suppressive effect of processing stem mutants was not a function of general defects in 50S subunit maturation, but was specific to processing of 23S rRNA termini
On the basis of our results, we suggest that the dif-ference observed in sucrose gradient ribosome profiles between the IF1 mutant and wild-type strains is attrib-utable to a relative increase in the proportion of 70S ribosomes, and a concurrent decrease in the propor-tion of the free ribosomal subunits (Fig 6A) Taken together, these results indicate that at least one of the manifestations of the growth defect in the IF1 mutant
at nonpermissive temperatures is at the level of ribo-somal subunit joining One class of suppressor muta-tions that specifically alter 23S rRNA processing partially restores the growth defect by affecting the 30S to 50S stoichiometry
Discussion
Although IF1 has been the focus of studies for a few decades, there is still a considerable amount of interest
in this small initiation factor, largely because it is essen-tial for growth and has been found in all organisms investigated With the knowledge gained from struc-tural and mutagenic studies [6,7], as well as the more recent data indicating that IF1 is an RNA chaperone [9,10], all of which demonstrate that IF1 interacts with RNA, we set out to find functional interactions that IF1 may undergo with rRNA We employed a simple genetic approach to isolate second-site suppressor mutations that map to rRNA and that suppress a cold-sensitive IF1 mutant strain Such suppressor mutations should reveal the interactions between IF1 and rRNA that have not been evident from crystallographic or other studies Contrary to our initial expectations, the first set of suppressor mutations were found to be located in the 23S rRNA processing stem, and not in the structural part of the mature rRNA Our data sug-gest that the mechanism of suppression of cold sensitiv-ity in these double mutants is indirect, resulting from rRNA maturation defects According to structural data, the mature form of the processing stem of 23S rRNA (helix 1) is not proximal to the subunit interface
of 50S, suggesting that direct contacts between IF1 and
Trang 9helix 1 on the ribosome are unlikely The notion of
indirect suppression was further supported by the
observation that a lesion in RNase III, the enzyme
responsible for initial cleavage of the processing stem,
also suppresses the IF1 defect On the other hand, the
effect of the RNase III lesion was different from that
of processing stem mutations with respect to the
sup-pression effect in rich liquid medium As RNase III has
multiple RNA targets in the cell, it is possible that, in
this case, the suppressor effect is, in fact, not directly
related to the processing of rRNA However, such
effects cannot explain the mode of suppression of the
23S rRNA processing stem mutants
On the basis of the sucrose gradient data, we suggest
that the mutant IF1, when shifted to the nonpermissive
temperature, leads to an altered rate of subunit
associa-tion⁄ dissociation, at the expense of some functional
conformational change, presumably in the 30S subunit
Several lines of evidence support this First, it is known
from structural studies that IF1 induces conformational
changes in the 30S subunit [7,25,26] Second, the
partic-ular R69L alteration in IF1 results in a general increase
in expression of reporter genes [27], while also leading
to increased RNA chaperoning activity as compared
with wild-type IF1 [9] Moreover, recent data have
shown that the R69L IF1 mutation leads to increased
expression of reporter genes that is translation initiation
region (TIR)-dependent, and that the mutation shares
this effect with the antibiotic kasugamycin [11] Third,
it has recently been demonstrated that IF1 plays a role
in subunit joining, and has the ability to discriminate
between certain mRNAs on the basis of their TIRs [12]
In addition, IF1 influences ribosomal subunit
associa-tion–dissociation rates [28], and this function is
espe-cially necessary in the cold [29] Finally, it is known
that IF1 has a role in the cold shock response [29–32]
Taking these findings together, we suggest that the
R69L mutant of IF1 allows premature subunit joining
by either failing to discriminate between certain TIR
elements in mRNA, or inducing a conformation in the
30S subunit under nonpermissive cold shock conditions
such that the rate of association–dissociation with the
50S subunit is affected Analogously, the mutant IF1
may be defective in recognition of TIR elements in
mRNAs that are specifically translated by cold shock
nontranslatable ribosomes, as proposed in a model by
Jones et al [33] In this model, cold shock conditions
induce a conformation in the ribosomes that becomes
blocked in translation initiation, whereby only specific
mRNAs with an appropriate TIR can bypass this block
and be translated These workers have also described a
transient increase in the 70S ribosomes after a shift to a
low temperature
On the basis of our results, we suggest that the identi-fied 23S processing stem suppressors act by interfering with ribosome subunit joining by limiting the pool of available mature 50S subunits (Fig 6B) Moreover, as a similarly large fraction of 50S and 70S ribosomes of one
of the processing stem mutants (pD3) is composed of immature rRNA, it seems unlikely that there exists an active system that prevents immature 50S subunits from entering the translating ribosome pool Therefore, it is also unlikely that such immature subunits are preferen-tially degraded Instead, assembly of ribosomal proteins onto immature rRNA could be delayed in these mutants, thus accounting for an apparent decrease in the amount of ribosome subunits Defects that affect rRNA maturation or subunit association should lead to
a similar suppressor phenotype for the R69L mutant IF1 On the other hand, we found that general defects in 50S subunit maturation, when deletions in known 23S helicases were introduced into the IF1 mutant, did not rescue the cold sensitivity In addition, one would expect there to be many more potential targets for suppressor mutations in the 23S structural gene that may interfere with some step in 23S maturation, 50S assembly, and subsequent subunit joining, besides those that affect the 23S processing stem After an extensive selection, we were not able to find any such suppressor mutations in the structural part of 23S rRNA We have, however, isolated other suppressor mutations that map to the 16S rRNA structural gene These were found in helices 18,
20, 32, 34 and 41 in 16S rRNA Preliminary data indi-cate that these mutations interfere with 16S rRNA processing and subunit association (to be published else-where) In conclusion, we show a functional interaction between IF1 and the processing stem of 23S rRNA Our results suggest that ribosomal maturation and transla-tion are closely linked processes
Experimental procedures
Bacterial strains and plasmids
The strains and plasmids used in this study are listed in Table 1 All strains were grown in LB medium supplemented with ampicillin (Amp) 200 lgÆmL-1, kanamycin (Kan) 50 lgÆmL)1, tetracycline (Tet) 20 lgÆmL)1, or chloramphenicol (Cm) 35 lgÆmL)1, when necessary
Construction of strains
SQZ10 is an E coli strain with all seven chromosomally encoded rRNA operons deleted (D7 strain) This strain car-ries a Kan resistance plasmid encoding the rrnC operon, as well as a counterselectable sacB marker [34] E coli strain
Trang 10CAG18478 was used to transfer the Tet resistance marker
into the IF1 mutant strain CVR69L, with an arginine to
leucine substitution at position 69, by P1 transduction [17]
This strain was then used as a donor for subsequent
trans-fer of the mutant IF1 allele into SQZ10 to generate JB69
Plasmid pKK3535 [35], containing the rrnB of E coli and
an Amp resistance marker, was used to replace a resident
plasmid, pCsacB-KmR PCR-based site-directed
mutagene-sis was used to introduce mutations into pKK3535
A deletion was introduced into the gene rnc, encoding the
RNase III, with pKO3::Drnc [36] As a result, only 15
N-ter-minal and 38 C-terN-ter-minal amino acids remained in the ORF
The deletion fragment was constructed according to [37],
with the following primers: rnc5¢O NotI, GTCGGATC
CGCGGATCAGGTGGGGATGTATTA; rnc5¢I comp,
GGCAGTGGATGATGGGGTTCATGCGATACC; rnc3¢O
SalI, TGCGTCGACATTTGCCGCAATAGTGTCAACA;
and rnc3¢I comp, TGAACCCCATCATCCACTGCCAG
GTCAGCG The deletion was constructed in CVR69L and
JB69 to generate CVR69LDrnc and JB69Drnc, respectively
(Table 1) A pTrc99A vector was used for cloning and
over-expression of era, encoding the GTPase Era The primers
used were as follows: era_F_NcoI, CGACCATGGCGAAC
AGGCGTTGAAAAAAC; and era_R_SalI, CGAGTCGA
CAGCCTTCCATCGGAGTTACT The resulting vector
was termed pTrc99a::era Protein overexpression was assayed by SDS⁄ PAGE
Selection of second-site rRNA suppressors of cold-sensitive IF1
A direct selection procedure was used to isolate mutations in rRNA that suppress the cold-sensitive phenotype of JB69 Briefly, the cold-sensitive R69L mutant [17] of IF1 provided
a tool for isolation of second-site suppressors by selection at the nonpermissive temperature For this purpose, the mutant allele was transferred by P1 transduction into the D7 strain SQZ10 (Table 1) The IF1 mutant strain JB69 obtained exhibited pronounced cold sensitivity at 23C Second, the resident pCsacB-KmR plasmid was replaced by a high copy number plasmid, pKK3535 Stationary-phase cultures of JB69 carrying pKK3535 were plated directly on Amp plates
at 23C Colonies were pooled, and the extracted plasmid DNA was used to transform JB69 by selecting on plates at
23C containing Amp and 5% sucrose to displace the resi-dent Kan plasmid Candidate mutants were purified, plasmid DNA extracted, used to transform JB69 at 37C, and then streaked at 23C to confirm that the suppressor mutations were plasmid-borne Successful candidates that suppressed the cold-sensitive phenotype of JB69 were chosen, and plas-mid DNA was sequenced after propagation in DH5a Inde-pendent selections were performed 10 times To confirm the cold sensitivity suppression effect, mutants were recon-structed by site-directed mutagenesis
Preparation of total RNA
The strains were grown at 37C in LB to D590 nm0.7 or at
37C to D590 nm0.2, and the cultures were then shifted to the nonpermissive temperature of 23C and grown to
D590 nm0.7 Total RNA was isolated from 5-mL cultures using the RNeasy Mini kit (Qiagen, Hilden, Germany)
Primer extension
Primer extension analysis was used to analyze the 5¢-end of 23S rRNA, with the primer extension system avian myoblastosis virus reverse transcriptase (Promega, Madi-son, WI, USA) Probe MRA141 (CCTTCATCGCCTCT-GACTGCC) was labeled with [32P]ATP[cP] and used as a template for the 5¢-terminus of 23S rRNA The products of the primer extension reaction were separated on 6% or 8% acrylamide 8M urea sequencing gels The gels were dried and visualized by phosphor imaging
Ribosome preparation and sucrose gradient
A 500-mL culture was grown in 2· LB to log phase (D590 nm0.5–0.7) at 37C, or grown at 37C to
Table 1 Bacterial strains and plasmids used in this study.
Pertinent feature(s)
Reference
or source Plasmids
pKK3535 rrnB, Amp, pBR322-derived [35]
pD1 pKK3535 but suppressor
of infA R69L
This work
pD3 pKK3535 but suppressor
of infA R69L
This work pD6 pKK3535 but suppressor
of infA R69L
This work
Pharmacia Biotech pTrc99a::era pTrc99a with cloned era This work
pKO3::Drnc pKO3 with rnc deletion
fragment
This work Strains
SQZ10 DrrnA, DrrnB, DrrnC, DrrnD,
DrrnE, DrrnG, DrrnH, pCsacB-KmR, ptRNA67-SpcR
S Quan and
C Squires