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Journal of Bacteriology, September 1998, p. 4804-4813, Vol. 180, No. 18
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Transcription Analysis of Two Disparate rRNA
Operons in the Halophilic Archaeon Haloarcula
marismortui
Patrick P.
Dennis,*
Sonia
Ziesche, and
Shanthini
Mylvaganam
Department of Biochemistry and Molecular
Biology, University of British Columbia, Vancouver, British
Columbia V6T 1Z3, Canada
Received 12 January 1998/Accepted 22 April 1998
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ABSTRACT |
The genome of the halophilic archaeon Haloarcula
marismortui contains two rRNA operons designated rrnA
and rrnB. Genomic clones of the two operons and their
flanking regions have been sequenced, and primary transcripts and
processing intermediates derived from each operon have been
characterized. The 16S, 23S, and 5S genes from the two operons were
found to differ at 74 of 1,472 positions, 39 of 2,922 positions, and 2 of 122 positions, respectively. This degree of sequence divergence for
multicopy (paralogous) rRNA genes was 10- to 50-fold or more higher
than anticipated. The two operons exhibit other profound differences
that include (i) the presence in rrnA and the absence in
rrnB of tRNAAla and tRNACys genes
in the intergenic and distal regions, respectively, (ii) divergent 5'
flanking sequences, and (iii) distinct pathways for processing and
maturation of 16S rRNA. Processing and maturation of 16S and 23S rRNA
from rrnA operon transcripts and of 23S rRNA from
rrnB operon transcripts follow the canonical halophilic
pathway, whereas maturation of 16S rRNA from rrnB operon
transcripts follows an unusual and different pathway that is apparently
devoid of any 5' processing intermediate.
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INTRODUCTION |
During ribosome biogenesis, mature
rRNAs are endonucleolytically excised from a larger precursor
transcript, modified, folded into a complex three-dimensional
structure, trimmed, and assembled along with a complete complement of
ribosomal proteins into mature subunits. With few exceptions, the major
small- and large-subunit rRNA genes are linked within the genome of an
organism and cotranscribed in order to maintain control and
coordination of these complex processes. Because of the high metabolic
demand for protein synthesis, rRNA operons are often multicopy genes,
with all copies being identical or nearly identical in both coding and
transcribed flanking sequences (1, 13, 24). Any sequence
heterogeneity arising from mutations within one of the operons is
either eliminated or homogenized to other operons by recombination and
gene conversion type processes (11, 19). As a consequence of
these processes, rRNA coding and flanking sequences are highly uniform
within a species and immune to replacement during infrequent lateral
transfer of genetic material across the species boundary.
Although rRNA sequence uniformity is the rule, there are at least two
cases where heterogeneity within multicopy (paralogous) rrn
genes is substantial. In the eucaryotic parasite Plasmodium berghei, two types of paralogous small-subunit rRNA genes are present (18). These differ by substitution or
deletion-insertion at about 5% of the nucleotide positions. The two
types of genes appear to be preferentially expressed, respectively, in
the blood and insect stages in the life cycle of the parasite.
The second example is the halophilic archaeon Haloarcula
marismortui. This organism contains two defined rrn
operons in its genome (23). Alignment of the two paralogous
16S sequences has revealed substitutions at 74 of the 1,472 nucleotide
(nt) positions (i.e., 5% sequence divergence) (24). All of
the substitutions occurred at phylogenetically variable positions, and
none affected nucleotide positions that are known to be important in
ribosome function. The pattern of nucleotide substitution within the
16S alignment differed in at least one important respect from that expected of independently evolving orthologous 16S rRNA genes: the
substitutions were not distributed throughout the 16S gene alignment
but rather were concentrated within limited regions of the gene. For
example, two-thirds of the nucleotide sequence differences (51 of 74)
were located within a 300-nt region (between positions 520 and 820) of
the 16S sequences, whereas, for example, only two differences were
observed in the adjacent region between positions 220 and 520. In
diverging orthologous genes, substitutions occur frequently at many
positions that are more or less evenly spread between positions 220 and
820 of the 16S sequence (24, 32). Under standard laboratory
conditions of growth, both of the H. marismortui rrn operons
appear to be expressed (24). Despite the differences, both
of these sequences reside within the halophilic archaeal cluster in
small-subunit rRNA phylogenies.
If the situation of sequence heterogeneity within paralogous 16S genes
is more common than presently imagined, it could complicate rRNA-based
phylogenetic analysis and cause problems with the interpretation of
biodiversity as detected by most PCR-based methods (27, 32). Moreover, this situation raises fundamental questions about the composition of genomes and uniformity within the protein synthesis apparatus. How much sequence variability can be accommodated within rRNA without compromising efficiency and accuracy? What roles do the
conserved rRNA flanking sequences play in the biogenesis of ribosomes?
To appreciate better this complex situation and to understand more
thoroughly the potential impact, we have completely sequenced the
rrnA and rrnB operons from the genome of H. marismortui and analyzed the in vivo intermediates that accumulate
during the processing and maturation of the rrnA and
rrnB operon transcripts. The features distinguishing the two
operons that have been revealed include (i) sequence heterogeneity
within 16S, 23S, and 5S genes; (ii) the presence or absence of spacer
and distal tRNA genes; (iii) divergent 5' flanking sequences; and (iv)
distinct pathways for processing and maturation of 16S rRNAs.
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MATERIALS AND METHODS |
H. marismortui was grown at 37 to 42°C in rich
medium (26). The two rrn operons designated
rrnA and rrnB were previously cloned as an 8.0-kb
HindIII-ClaI fragment and as a 10-kbp
HindIII fragment cloned into the vector pBR322,
respectively. For some DNA sequence determinations, various fragments
were subcloned into pGEM3 or pGEM7 vectors. Sequences were generated
with the universal T7 or SP6 primers or with sequence-specific internal
primers with the T7 Sequenase system. In some cases, deletions were
generated within the inserts of pGEM vectors by using exonuclease III
(20). The 7 deaza-2'-deoxyguanosine-5'-triphosphate analog
of dGTP was used to help resolve sequence compressions resulting from
secondary structure and the high GC content of the template.
Total cellular RNA was isolated by the boiling-sodium dodecyl sulfate
lysis method, and the recovered RNA was used for both S1 nuclease
protection and primer extension analysis. These methods have been
described in detail (7). For S1 protection experiments, 1 to
5 µg of total RNA was hybridized to either 5'- or 3'-end-labeled DNA
probes (about 0.05 pmol). The DNA fragments were obtained by
restriction enzyme digestion of complete or partial clones of the
rrnA or rrnB operons followed by fragment
purification in acrylamide gels. Probe DNA fragments were 5' end
labeled with polynucleotide kinase and [
-32P]ATP or 3'
end labeled with the Klenow fragment of DNA polymerase I and the
appropriate [
-32P]deoxynucleoside triphosphate. The
hybridization temperature (between 52 and 58°C) was empirically
determined for each fragment such that rehybridization of the probe DNA
was minimized without interfering with the formation of RNA-DNA
hybrids. The molecular length standards employed were the fragments of
pBR322 generated by digestion with MspI and 3' end labeled
with the Klenow fragment of DNA polymerase and
[-32P]dCTP. In some cases, size ladders were also
generated by using end-labeled DNA fragments in the A and A+G Maxam
Gilbert sequencing reactions. Estimates of the length of protection
products are accurate to within a few nucleotides and in some instances
have been confirmed by primer extension analysis. The protection
products often exhibit heterogeneous ends. This heterogeneity could
result from either heterogeneity in the RNA ends that enter into the formation of RNA-DNA hybrids or from under- or overdigestion at the
ends of the hybrids by the S1 nuclease.
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RESULTS |
Operon structures.
Southern hybridization indicated that the
genome of H. marismortui contained two defined rRNA operons.
These were cloned as an 8-kbp HindIII-ClaI
fragment and as a 10-kbp HindIII-HindIII fragment and were designated rrnA and rrnB,
respectively (23). The nucleotide sequences of the
rrnA and rrnB operons and their 5' and 3'
flanking sequences were determined (accession numbers AF034619 and
AF034620). The rrnA operon contains 16S, 23S, and 5S genes,
as well as a tRNAAla gene in the intergenic spacer and a
tRNACys gene in the 3' distal position (Fig.
1). The rrnB operon contains 16S, 23S, and 5S genes but lacks both the intergenic and the distal tRNA genes. The absence of the distal tRNACys gene from the
3 kbp of unsequenced region distal to the rrnB operon was
confirmed by Southern hybridization with a tRNACys-specific
oligonucleotide. A subclone from the rrnA operon containing the tRNACys gene exhibited strong hybridization to the
oligonucleotide probe, whereas the entire rrnB-containing
10-kbp clone exhibited no hybridization signal (data not shown).
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FIG. 1.
Structure of rrnA and rrnB operons
of H. marismortui. The chromosomal structures of the
rrnA and rrnB operons are indicated. The 16S,
23S, 5S, tRNAAla, and tRNACys genes are
represented by solid boxes, the processing inverted repeats are
indicated by hatched boxes, and the other flanking and spacer sequences
are indicated by open boxes. Flanking and spacer regions longer than 50 nt and greater than 90% identical in sequences between rrnA
and rrnB are indicated by brackets. All gene sequences are
greater than 95% identical (see Table 1). The complete nucleotide
sequences of rrnA and rrnB have been deposited in
GenBank (rrnA, AF034619; rrnB, AF034620).
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Gene sequences.
Analysis and comparison indicated that the
16S, 23S, and 5S genes from the rrnA and rrnB
operons contain an abnormally high number of nucleotide sequence
differences. This was first noted for the 1,472-nt 16S genes where
substitutions were observed at 74 positions (24). Similarly,
for the 2,922-nt 23S genes substitutions were observed at 39 positions,
and for the 122-nt 5S genes substitutions were observed at 2 positions
(Table 1). As in the rrnA and
rrnB 16S genes, differences in the 23S and 5S sequences
occur at positions that are phylogenetically variable, and many of the
differences are compensatory, affecting both components of a
Watson-Crick base pair. None of the substitutions affect positions
known to be important in 50S subunit function.
A previous study described the 23S and 5S gene sequence from
H. marismortui (
2). The sequence was apparently obtained
by
a chromosome walk starting from a fragment containing an
rrnA-like
5S gene. For simplicity, this sequence has been
designated
rrnC.
In reality,
rrnC may be a
composite of the
rrnA and
rrnB operon
sequences
that was generated either in vivo through recombination
or in vitro
through the cloning of overlapping fragments from
the two operons.
Support for this supposition is based on the
following. First, the 5'
portion of the
rrnC 23S gene is more
similar to the
rrnB 23S sequence, the central portion is a composite
of
rrnA and
rrnB, and the 3' portion is very similar
to
rrnA.
Second, the
rrnA and
rrnC
23S-5S intergenic spacers, 5S genes,
and 5S distal sequences are
identical. Third, there were 7 nt
substitutions unique to the
rrnC 23S sequence, whereas there were
15 nt substitutions
unique to the
rrnB 23S sequence and 22 substitutions
unique
to the
rrnA 23S sequence. The
rrnC 23S gene is
only 2,917
nt in length and contains single-nucleotide deletions at
positions
680, 1280, 1641, 2417, and 2357 relative to the
rrnA and -
B 23S
sequences. Some of these features
suggest that
rrnC may be a composite
operon sequence rather
than an independently evolving entity.
Inverted repeats surrounding the 16S and 23S genes.
Further
analysis of the primary sequences indicated that in both operons, the
16S and 23S genes were surrounded by long inverted repeats (Fig. 1).
These repeats are believed to form helical structures in the primary
rRNA operon transcripts and to contain the recognition motifs for
excision of pre-16S and pre-23S (3, 6, 15). In most archaea,
this motif consists of two three-base loops on opposite strands of the
helix and separated by four nucleotide base pairs. The motif is
believed to be recognized and cleaved between the second and third
bases of the two respective loops by a recently characterized
bulge-helix-bulge (BHB) endonuclease (15, 21, 30). The
concerted cleavages liberate the pre-16S or pre-23S rRNA from the
primary transcript. The BHB motif was present within both the 16S and
the 23S repeats of rrnA and the 23S repeat of
rrnB (Fig. 2A). It was
noticeably absent from the 16S repeat of rrnB; this implies
that a distinct pathway is used to excise and process 16S rRNA from
rrnB operon transcripts.
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FIG. 2.
Important sequence motifs located in the flanking and
spacer regions of rrnA and rrnB. (A) Small
portions of the helical structures of the processing inverted repeats
surrounding the 16S and 23S genes in rrnA and
rrnB are indicated. The BHB motif is noticeably absent from
the rrnB 16S helix. The sites of cleavage detected within
the bulges on the 5' side (left) and 3' side (right) of each helix are
indicated by scissors, and the position of the nucleotide 3' to the
scissile phosphate is indicated. Where negative, numbers correspond to
5' flanking sequences, with the first nucleotide of 16S rRNA being 1 for the rrnA and rrnB regions, respectively. i,
16S-23S intergenic spacer positions with the first nucleotide after 16S
being iA1 or iB1; 23Sd, 23S-5S intergenic spacer with the first
nucleotide after 23S being 23Sd1. The 23S-5S intergenic spacer is
identical in rrnA and rrnB. (B) The point of
sequence convergence at the junction between the 5' flanking sequences
and the beginning of the 16S genes in the rrnA and
rrnB operons is indicated. The dots indicate identical
nucleotides. (C) A 5' flanking sequence element conserved between
rrnA, rrnB, and the rrn operons of
other genera of halophilic archaea are aligned. The numbers in
parentheses are the first nucleotide positions of the sequences shown.
Dots indicate conserved nucleotides in pairwise comparisons. The 3-nt
bulge located in the 5' portion of the 16S processing helix in
rrnA and the single rrn operon of H. cutirubrum is boxed. A conserved sequence proposed to play a role
in folding of a conserved pseudoknot within 16S rRNA is indicated
(9). (D) 16S-23S intergenic sequence around the
tRNAAla in rrnA and the corresponding region of
rrnB. Endonucleolytic cleavages occur at or near the
indicated positions at the 3' end of the tRNAAla and
tRNAAla-like sequences. Regions of secondary structure
within tRNAAla are indicated by brackets; the potential for
similar secondary structures in rrnB is also indicated. Dots
represent identical nucleotides. The 3' transcript end detected in the
anticodon loop of the tRNAAla structure (position iA139) is
indicated; the anticodon sequence is overlined. The position of a
corresponding 3' end in rrnB (position iB137) is also
indicated. Finally, a 5' end resulting from cleavage by RNase P at the
5' end of tRNAAla is indicated (position iA105). No
corresponding end was observed in rrnB (position iB121). (E)
The sequence at the point of convergence within the 16S-23S intergenic
space between rrnA and rrnB is shown. The UUAA
sequence (overlined or underlined) immediately 3' to the point of
perfect convergence represents the archaeal TATA box for the putative
intergenic promoters in rrnA and rrnB. The dots
represent identical nucleotides. (F) The sequence at the point of
divergence in the 5S 3' flanking region is shown. 5Sd represents the 5S
3' flanking sequence with the first nucleotide beyond 5S being 5Sd1.
The dots represent identical nucleotides.
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Transcription initiation and precursor processing in the 5'
flanking regions of rrnA and rrnB.
The 5'
flanking regions of the rrnA and rrnB operons
contain multiple elements exhibiting a high degree of sequence
similarity interspersed within otherwise unrelated sequences. All but
one of these conserved elements were identified as putative promoters based on two criteria: sequence similarity to each other and to other
well-characterized halophile rrn promoters (5, 6) and (in all but one instance) the presence of a 5' transcript end
mapping to a position at or near the anticipated dinucleotide initiation sites. The rrnA operon contains four
promoter-like elements, whereas the rrnB operon contains
three such elements (Fig. 3A). Each
element possesses a conserved archaeal TATA box centered 22 to 24 nt
upstream of the initiation site, which was usually G within a conserved
TG dinucleotide. Under standard laboratory culture conditions, both
operons are actively transcribed.
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FIG. 3.
Alignment of promoter-like sequences from the 5'
flanking and intergenic spacer regions of rrnA and
rrnB. The nucleotide sequences of the 5' flanking
promoter-like motifs from rrnA and rrnB (A) and
the intergenic promoter-like motifs from rrnA and
rrnB (B) are indicated. The sequences are aligned at the
archaeal TATA box element and at the dinucleotide transcription
initiation site (dot; usually G). The promoter designations and the
nucleotide positions of the putative transcription start sites are
indicated on the left.
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Transcripts derived from the 5' flanking regions of the
rrnA
and
rrnB operons were detected by S1 nuclease protection
assays
by using 5'-end-labeled DNA probes specific for each operon
(Fig.
4). With the
rrnA
operon-specific probe, 5' transcript ends were
located at or near
position 1 of the 16S rRNA gene and at positions
A95,
A176,
A286,
A364, and
A446 in the 5' flanking regions
(Fig.
4A). The
rrnA DNA probe hybridizes mature 16S rRNA derived
from both
rrnA and
rrnB but only hybridizes precursor
transcripts
from
rrnA. This is because the point of sequence
divergence between
the two operons is at the junction between the 5'
flanking region
and the start of the 16S gene (see Fig.
2B). The 5' end
of the
product mapping to position
A95 was presumably derived by
protection
with trailer products generated by endonucleolytic cleavage
by
the BHB enzyme within the 3-nt bulge on the 5' side of the 16S
rRNA
processing stem. The structure of the putative cleavage site
is
depicted in Fig.
2A. The products with 5' ends at positions
A446,
A364,
A286, and
A176 correspond to transcripts initiated
at the
P1A, P2A, P3A, and P4A promoters, respectively.
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FIG. 4.
Analysis of 5' transcript ends located in the 5'
flanking regions of rrnA and rrnB. Two respective
AflIII restriction fragments specific for rrnA
and rrnB were 5' end labeled on the () strand at position
98 within the 16S sequence and used as probes against total cellular
RNA in S1 nuclease protection assays. (A) The protection products were
separated on denaturing polyacrylamide gels. The sizes (in nucleotides)
of protection products and molecular length markers (MLM) are indicated
beside the autoradiograms. (B) A diagram of rrnA and
rrnB is shown. Each protection product is represented by an
arrow with the length in nucleotides given on the right and the
position of the 5' transcript end given above the leftward pointing
arrowheads. The positions of promoter-like sequence motifs and
precursor processing sites are indicated. The 16S gene is a solid box,
the 5' portion of the processing repeat is a hatched box, and the
remaining 5' flanking region is unfilled. The dashed arrow in
rrnB represents full-length protection of the probe by
transcripts that were presumed to have been initiated at the upstream
P1B promoter. At the bottom of panel B, the results of nuclease
protection assays with 3'-end-labeled rrnB probes are
illustrated (autoradiograms not shown). As above, the dashed arrows
represent full-length protection of the probe, and the solid arrows
represent partial-length protection of the probe. The length of each
product is indicated on the left end and the position of the 3'
transcript end is indicated on the right end of each arrow (see text
for details).
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With the
rrnB operon probe, 5' transcript ends were located
at or near position 1 of 16S rRNA and positions
B157 and
B259
(Fig.
4B). Also apparent was a product corresponding to full-length
protection of the DNA probe. The transcripts that protected the
entire
probe were presumably derived from the putative upstream
P1B promoter
(position
B458), whereas the two shorter products
were derived by
protection with transcripts initiated at the P2B
and P3B promoters,
respectively (Fig.
3A). No other abundant 5'-end
protection products
were observed, suggesting that there was no
endonucleolytic processing
within the 5' flanking region of the
rrnB transcript. As
mentioned above, the
rrnB 16S processing helix
noticeably
lacks the recognition motif used by the BHB processing
endonuclease to
excise a precursor 16S rRNA from the primary transcript
(Fig.
2A). To
confirm the absence of endonucleolytic processing
in the 5' flanking
region of
rrnB transcripts, the 424-nt
AflIII
fragment was 3' end labeled on the minus strand at position
B327
and
used as a probe in the nuclease protection assay. Only transcripts
derived from
rrnB protect this probe. Two protection
products
were observed. The first corresponds to full-length protection
of the probe by
rrnB transcripts initiated at the upstream
P1B
promoter. The second product was about 327 nt long and resulted
from partial protection of the probe by an RNA intermediate with
a 3'
end at or near position 1 of 16S rRNA (data not shown but
see Fig.
4B).
This result seems to indicate that the 5' region
of
rrnB
operon transcripts are subject to a single endonucleolytic
cleavage
that separates and releases the 5' end of 16S rRNA from
the intact 5'
leader. This result was confirmed by using a 245-nt
MspI
fragment from
rrnB, 3' end labeled at position
B240, as
a
probe (Fig.
4B).
Another somewhat longer and more highly conserved motif is also present
in the 5' flanking regions of both the
rrnA and the
rrnB operons and at a similar position in the
rrn
operons of other
genera of halophilic archaea. Between
rrnA
and
rrnB, the conserved
motif is 52 nt long and 92%
identical in sequence (Fig.
2C). When
compared to the motifs in other
halophilic archaea (e.g.,
Halobacterium cutirubrum),
similarity to the
rrnA motif extends a further 11
nt in the
5' direction and a further 16 nt in the 3' direction
(
6).
The 3' end of the
rrnA and
H. cutirubrum motifs
correspond
to the site of the BHB endonuclease cleavage in the 5'
portion
of the 16S processing helix. The 5' end of the motifs has been
proposed to play a role in folding of a universally conserved
pseudoknot within 16S rRNA (
9). Other sequences within this
motif may be involved in other as yet uncharacterized aspects
of 16S
rRNA processing, folding, or maturation. Outside of this
motif and the
multiple promoter elements, no other sequence similarity
was observed
between the 5' flanking regions of the
rrnA and
rrnB operons.
Intergenic spacer regions in rrnA and rrnB.
The 16S-23S intergenic spacer regions of rrnA and
rrnB are 383 and 405 nt, respectively. The two spacers are
identical in sequence over the 3'-distal 183 nt but show only
fragmentary similarity over the remaining 5' proximal regions (Fig. 1).
The rrnA operon contains a tRNAAla gene within
the 5' portion of the spacer, whereas the rrnB operon appears to contain only a partial remnant of a tRNAAla-like
gene at this position (see below).
Transcripts and processing intermediates derived from the 16S-23S
intergenic spaces of the two operons were detected by S1
nuclease
protection assays by using both 5'- and 3'-end-labeled
DNA probes
specific for each operon (Fig.
5). In
regions of perfect
or near-perfect sequence identity, transcripts from
both operons
hybridize to either end-labeled probe. These regions of
coincident
hybridization include the 3' end of the 16S rRNA (for
3'-end-labeled
probes) or the terminal 183 nt of the spacer and the 5'
end of
23S rRNA (for 5'-end-labeled probes). With the 3'-end-labeled
DNA probe from
rrnA, transcript 3' ends were located at or
near
positions iA1, iA70, iA139, iA177, iA350, and position 1 of 23S
rRNA. Also apparent was a product of full-length probe corresponding
to
protection by unprocessed transcripts from the
rrnA operon.
With the same
rrnA DNA probe labeled at the 5' end,
transcript
5' ends were located at or near positions iA70, iA105,
iA177,
iA193 to iA205, iA230, and iA350 and position 1 of 23S rRNA.
Again,
full-length protection of the probe by an unprocessed
rrnA operon
transcript was also observed (Fig.
5A and B).
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FIG. 5.
Analysis of 5' and 3' transcript ends located in the
16S-23S intergenic spaces of rrnA and rrnB. Two
respective AvaI restriction fragments specific for
rrnA and rrnB were 3' end labeled on the ()
strand at position 1365 within the 16S gene or 5' end labeled on the
() strand at position 157 within the 23S gene and used as probes
against total cellular RNA in S1 nuclease protection assays. The
protection products were separated on denaturing polyacrylamide gels.
The size in nucleotides of protection products and molecular length
markers (MLM) are indicated beside each autoradiogram. (A)
Autoradiograms with the rrnA probe either 3' labeled (left)
or 5' labeled (right). The lanes marked A and A+G are Maxam Gilbert
sequence reactions on the 3'-labeled probe. (B) Diagram illustrating
the rrnA 383-nt 16S-23S intergenic space and a summary of
the protection products obtained with the 3'-labeled probe (3'
transcript end sites are represented by rightward pointing arrows) and
the 5'-labeled probe (5' transcript end sites are represented by
leftward pointing arrows). The estimated length of each product is
indicated at the start of the arrow, and the positions of the
respective 3' or 5' transcript ends within the intergenic spaces are
indicated above the arrowhead. Opposing arrowheads represent sites of
endonuclease cleavage. The gray arrow represents a product with a 3'
end at position iA105 that was not observed. The overlapping arrowheads
at positions iA193 to iA205 represent multiple protection products. The
dashed double-headed arrow represents full-length protection of both
the 3'- and 5'-labeled probes with unprocessed transcripts from
rrnA. The 16S, tRNAAla, and 23S genes are
represented as solid boxes, the inverted processing repeats are
indicated as hatched boxes, and other intergenic sequences are shown as
open boxes. M16, maturation site at the 3' end of 16S rRNA; BHB, BHB
cleavage site; 5'TP, 5' tRNA processing site (presumably RNase P); PPS,
uncharacterized precursor processing site; 3'TP, 3' tRNA processing
site (apparently by an endonuclease; see the text); M23, 5'-end
maturation of 23S rRNA (apparently an endonuclease; see the text). (C)
Autoradiograms with the rrnB probe either 3' labeled (left)
or 5' labeled (right). (D) Diagram of the rrnB 405-nt
16S-23S intergenic space. Other details are as described for panel B.
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When both a leader 3' transcript end and a trailer 5' transcript end
map to a position at or near the same nucleotide position,
we have
inferred that they are generated by a single endonucleolytic
cleavage
event within the primary transcript. By this criterion,
putative
endonucleolytic cleavages occur at or near positions
iA70, iA177, and
iA350 and somewhat surprisingly at position 1
of 23S rRNA. Positions
iA70 and iA350 correspond to BHB endonuclease
recognition motifs that
occur within the 3' portion of the 16S
and the 5' portion of the 23S
processing stems, respectively (Fig.
2A). Detection of an endonuclease
cleavage at or very near position
1 of 23S rRNA infers that this
cleavage could precede at least
in some instances cleavage by the BHB
endonuclease in the 23S
rRNA processing stem (at position iA350). The
final cleavage at
or near position iA177 occurs at the 3' end of the
tRNA sequence.
The 5' end of the trailer released by this cleavage has
been mapped
by primer extension and appears to correspond to the first
base
following the encoded tRNA sequence (i.e., position iA177; data
not shown, but see Fig.
2D). If this primer extension result is
reliably detecting a 5' transcript end and is not the result of
a tRNA
structure-induced stop at the top of the acceptor stem,
it implies that
tRNA 3'-end generation is endonucleolytic.
The last 3' transcript end was mapped to position iA139 within the
anticodon loop of the tRNA sequence. Since no corresponding
5' end was
mapped to this position, the 3' end could have been
generated by either
exonuclease digestion from a site located
3' to position iA139 or by
endonuclease cleavage at this site
to produce a 3' trailer that has
escaped detection by the 5'-end-labeled
probe used here. The presence
of a 3' end within the tRNA anticodon
loop suggests that excision and
maturation of the tRNA
Ala are substoichiometric. Similar
cleavages have been observed within
the tRNA sequences in the
transcripts from the single-copy
rrn operon of
H. cutirubrum (
4).
There were three protection products with 5' ends located at positions
iA105, iA193 to iA206, and iA230 that were not paired
with
corresponding 3' protection products. The first, at iA105,
corresponds
to the mature 5' end of tRNA
Ala. The 5' end of all tRNAs is
believed to be generated by RNase
P endonucleolytic cleavage (
17,
28). The simplest explanation
for the absence of a product with a
3' end at this position is
that cleavage by the BHB endonuclease at
position iA76 preceded
cleavage by RNase P at the 5' end of the tRNA
gene; as a consequence,
only the shorter product with a 3' end at
position iA76 would
be visible. The second product representing a
heterogeneous collection
with 5' ends between iA193 and iA206
represents the area of sequence
convergence between the
rrnA
and
rrnB spacers. These protection
products resulted from
rrnB operon transcripts hybridizing to
the
rrnA
operon probe and from imprecise trimming by S1 nuclease
(Fig.
2E). The
third product, with a 5' end near position iA230,
corresponds to a
putative internal promoter (Fig.
3B) presumably
used to compensate for
premature transcription termination within
the operon and augment the
production of 23S and 5S rRNA. Similar
internal promoters have been
identified in the
rrn operons of
other halophilic archaea
(
22).
Similar experiments were carried out with 3'- and 5'-end-labeled probes
from
rrnB (Fig.
5C and D). Again, these probes hybridize
predominantly
rrnB operon transcripts but also some
rrnA transcripts
in regions of perfect or near-perfect
sequence identity. With
the 3'-end-labeled probe, transcript 3' ends
were located at or
near positions iB1, iB42, iB137, iB198, and iB372
and position
1 of 23S rRNA. With the same 5'-end-labeled probe,
transcript
5' ends were located at or near positions iB53, iB198, iB214
to
iB227, iB252, and iB372 and position 1 of 23S rRNA. Again,
full-length
protection of the 5' and 3' labeled probes by the primary
rrnB transcript was observed.
By the criteria stated above, there appear to be three endonucleolytic
cleavages in the intergenic spacer of
rrnB. The first
of
these, at position iB198, occurs immediately following the
tRNA remnant
in the
rrnB spacer (Fig.
2D). Alignment of this region
with
the tRNA
Ala from the
rrnA spacer reveals a
substantial degree of sequence
and structural similarity. It seems
likely that a single activity
may be responsible for both of these
cleavages. The second endonucleolytic
cleavage was at position iB372
and corresponded to the BHB endonuclease
cleavage in the 5' portion of
the 23S processing stem. The third
was at position 1 of 23S rRNA and
again indicates that BHB excision
of pre-23S rRNA does not always
precede the endonucleolytic cleavage
near or at the 5' end of 23S rRNA.
Because these last two cleavages
occur in a region of sequence identity
between
rrnA and
rrnB,
the trailer fragments
protecting the 5'-end-labeled probe were
from both
rrnA and
rrnB. In contrast, the 5' leader fragments
protecting the
3'-end-labeled probe were only derived from
rrnB.
There were two additional 5' protection products with ends located at
positions iB214 to iB227 and position iB252 that are
easily explained.
The first heterogeneous group maps to the area
where the
rrnA and
rrnB spacer sequences converge (Fig.
2E). The
protection products result from
rrnA operon
transcripts hybridizing
to the
rrnB operon probe and
imprecise trimming by S1 nuclease.
The second product with a 5' end
near position iB252 corresponds
to a putative internal promoter (Fig.
3B). As in the
rrnA operon,
this promoter is apparently
utilized to augment production of
23S and 5S rRNA. The 3' transcript
end detected at position iB137
occurs within the tRNA remnant and may
be related to the 3' end
detected at position iA139 within the
tRNA
Ala sequence in
rrnA (Fig.
2D). There is no
obvious explanation for
the 3' transcript end detected at position iB42
or the 5' transcript
end detected at position iB53.
23S distal region in rrnA and rrnB.
The 23S
3' flanking region of rrnA contains a 5S gene and a
tRNACys gene, whereas rrnB contains only a 5S
gene (Fig. 1). The 23S-5S intergenic spaces in the two operons are 139 nt long, identical in sequence, and designated 23Sd1 to 23Sd139 in a
5'-to-3' direction (Fig. 1). The 5S genes
are 122 nt long and differ in sequence at positions 73 and 106. The 5S
distal regions are identical in sequence for the first 61 nt except for
a single difference at position 5Sd2; beyond position 5Sd61, the two
operons show no significant sequence similarity (Fig. 2F). The
5S-tRNACys intergenic space in rrnA is 407 nt
long.
View larger version (20K):
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|
FIG. 6.
Analysis of 3' transcript ends located in 23S-5S
intergenic and flanking regions of rrnA and rrnB.
Transcripts from both rrnA and rrnB were analyzed
by using an AvaI fragment 3' end labeled on the () strand
at position 2859 within the 23S gene as a probe in S1 nuclease
protection assays. (A) Autoradiogram illustrating protection products
with the sizes in nucleotides of protection products (left) and of
molecular length markers (MLM; right) indicated. (B) Diagram of the
23S-5S intergenic and 5S 3' flanking regions. The 23S, 5S, and
tRNACys genes are represented by solid boxes, the 23S
inverted repeat is indicated by a hatched box, and other intergenic and
flanking regions are indicated by open boxes. The dashed region,
including the tRNACys gene, is unique to the
rrnA operon. Other designations are as described in the
legend to Fig. 5.
|
|
Processing of primary rRNA transcripts in the region distal to the 23S
genes was examined by using a 3'-end-labeled 369-nt
AvaI
fragment that overlapped the last 65 nt of the 23S gene,
the 23S-5S
intergenic space, the 5S gene, and the first 43 nt
of the 5S distal
regions (Fig.
6). Transcripts from both operons
hybridized efficiently
to this probe in spite of the three nucleotide
substitutions that exist
within and immediately following the
5S sequence. With this probe, 3'
transcript ends were located
at or near positions 23Sd1, 23Sd62,
23Sd132 to 23Sd137, and 5Sd6.
There was no apparent protection of the
entire probe, and the
products resulting from cleavages around the 5S
sequence were
very faint. This indicates that primary transcripts
containing
the 5S sequence are rapidly and efficiently processed and
therefore
difficult to detect by nuclease protection assays. The
shortest
product with an end at 23Sd1 corresponds to the mature 3' end
of 23S rRNA, and the somewhat longer product with an end near
position
23Sd62 corresponds to the BHB endonuclease processing
site in the 3'
side of the 23S processing stem (Fig.
2A). The
other two very faint 3'
ends were located about 4 to 8 nt upstream
of the 5S sequence
(positions 23Sd132 to 23Sd137) and about 6
nt downstream of the 5S
sequence (5Sd6). These locations suggest
that the 5S sequence may be
excised as a precursor from the primary
transcript and subsequently
trimmed by a few nucleotides at the
5' and 3' ends. In
Escherichia coli, pre-5S is excised by RNase
E and
subsequently trimmed a few nucleotides at the 5' and 3'
ends
(
16). An RNase E-like endonuclease activity has been
recently
identified with
H. marismortui and may be
responsible for these
pre-5S cleavages (
14). Although
precursor transcripts containing
the tRNA
Cys gene have been
detected by nuclease protection assays, the results
were neither
definitive nor informative because of their low abundancy
and rapid
processing.
| |
DISCUSSION |
Two rrn operons were initially identified on 20- and
10-kb fragments from the genome of H. marismortui and
subsequently cloned as 8.0-kb ClaI-HindIII
(rrnA) and 10-kb HindIII (rrnB)
fragments (23), respectively. Characterization of these
clones indicates that they are surprisingly disparate: (i) the
respective 16S, 23S, and 5S genes contain a substantial number of
nucleotide sequence differences (Table 1); (ii) except for a short
region of sequence conservation (Fig. 2C) and general promoter
arrangement, the 5' flanking regions exhibit little sequence
similarity; (iii) processing of 16S rRNA from the rrnA
operon transcript follows the canonical archaeal pathway, whereas the
processing of 16S rRNA from the rrnB operon occurs by direct
endonucleolytic cleavage at the 5' maturation site; and (iv) the
rrnA operon contains tRNAAla and
tRNACys genes in the 16S-23S intergenic and 5S distal
regions, respectively, whereas the rrnB operon is apparently
devoid of functional tRNA genes. Although only qualitative, the results
of the S1 nuclease protection experiments reported here and in our
earlier work (24) indicate that both operons contribute more
or less equally to the production of ribosomes.
Implications for rRNA-based phylogeny and biodiversity.
Based
upon sequence similarity alone, it would normally be concluded that the
two 16S genes were orthologs derived from distinct species of
halophilic archaea. Procaryotic divergence of 16S rRNA proceeds at a
rate of about 1% per 50 × 106 years, suggesting that
the two 16S sequences (with 5% divergence) were derived from a common
ancestor about 250 × 106 years ago. This represents
the same degree of 16S sequence divergence that separates E. coli and Serratia marcescens (8, 25).
However, there is ample reason to believe that these sequences are not true orthologs. First, both were present in genomic DNA prepared from a
culture derived from an isolated single colony. Second, the nucleotide
sequence differences are not distributed throughout the 16S gene but
rather are concentrated within limited regions; 51 of the 74 substitutions occur between nucleotide positions 520 and 820 of the 16S
gene. A much more even distribution of substitutions across the entire
sequence was observed for independently evolving orthologous 16S genes
from several genera of halophilic archaea (24). Third,
although the two 16S sequences are dissimilar, other regions of the
rrnA and rrnB operons, including the 3' portion of the 16S-23S intergenic spacer, the 23S-5S spacer, and the first 62 nt of the 5S 3' flanking sequence, are virtually identical (Fig. 1).
When orthologous halophilic rrn operons are compared, sequence divergence in spacer and flanking regions is more rapid and
extensive than is divergence within structural rRNA genes.
From the above considerations, we conclude that the
rrnA and
rrnB operons are both present within the genome of
H. marismortui.
However, the evolutionary history of the operons
cannot be ascertained
with any degree of certainty. They may be strict
paralogs, i.e.,
generated by a gene duplication event and never
separated by a
speciation event. If this were the case, it suggests
that the
rrnB operon has lost both the tRNA
Ala
and tRNA
Cys genes and developed an alternative pathway for
processing 16S
rRNA, since these features appear to be ancestral within
halophilic
rrn operons. Alternatively, the two operons may
be ancient orthologs
brought back together by lateral transfer to
create a chimeric
genome. If so, it suggests that recombination or gene
conversion-type
processes have partially rehomogenized certain parts of
the two
operons, whereas other parts of the operons have retained their
ancestral features. Whatever the correct scenario, it is clear
that the
different rRNA sequences do not necessarily mean distinct
species.
Moreover, the potential complications for interpreting
phylogeny or
biodiversity assessments based upon partial 16S rRNA
sequences are
evident (
25,
27,
32).
Structural considerations.
Essentially all of the ribosomal
protein genes from H. marismortui have been cloned and
sequenced (29); each gene appears to be unique and have a
single copy, and each protein appears to be capable of assembling into
particles with either the rrnA or rrnB operon
RNAs (24). This implies that the sequence or structural
requirements for the RNA-protein interactions are maintained in spite
of numerous nucleotide substitutions within or near certain protein
binding sites. The 50S ribosomal subunits from H. marismortui have been crystallized for X-ray diffraction analysis
and three-dimensional structural characterization (12). If
these crystals contain both rrnA and rrnB 23S
sequences, it implies that the nucleotide differences have little or no
effect on the overall structure of the 50S subunit. The majority of
substitutions in both 16S and 23S rRNAs occur in duplex regions and are
compensatory. Such compensatory mutations would have little or no
effect on the higher-order structure of 23S rRNA.
Maintenance of two disparate rrn operons.
Are
there selective forces that contribute to the maintenance of two
disparate rrn operons in H. marismortui and, if
so, what might they be? In P. berghei, where there are also
two types of 18S rRNA designated A and C, it has been suggested that
differential expression may play a role in the types of proteins
synthesized during the different developmental stages of the complex
life cycle (18, 31). The switch from A to C expression
occurs through the control of rRNA processing. In gametocytes,
precursor transcripts from C-type genes are not processed, and
ribosomes containing A-type RNA predominate. In the zygote and the
early ookinete, processing of C-type RNA is accelerated and A-type
ribosomes are targeted for selective degradation. In H. marismortui, both rrnA and rrnB RNAs are
present in approximately equal amounts in ribosomes during cultivation
under standard laboratory conditions (24). Moreover, it is
interesting that, as in P. berghei, the pathways for
processing 16S rRNA from the rrnA and rrnB
transcripts are fundamentally different. We have previously suggested
that salinity fluctuations in hypersaline environments pose unique
challenges to resident halophilic organisms (4, 8). The
presence of two distinct ribosome populations may allow H. marismortui to maintain essential protein synthesis during periods
of environmental stress (4). Other characterized halophile
species contain either a single rrn operon or two identical
rrn operons (6, 15).
Processing of precursor transcripts from rrnA and
rrnB.
The products observed in S1 nuclease protection assays
indicate that there are at least four sites of endonuclease cleavage in
the 383-nt 16S-23S spacer of transcripts derived from the
rrnA operon. The first was at the BHB motif in the 3'
portion of the 16S processing helix. It is unclear whether maturation
at the 3' end of 16S is an endo- or exonucleolytic event. If it is
endonucleolytic, it is a late event and occurs only after endonuclease
cleavage either at the BHB motif in the 16S processing stem or at some other downstream site.
The second detectable endonuclease cleavage occurs at the 3' end of the
tRNA. In
E. coli, an endonuclease cleaves several
nucleotides 3' to the mature tRNA sequences, and 3'-end maturation
is
subsequently carried out by a regiment of exonucleases (
10).
We have mapped the site of endonuclease cleavage to position iA177
by
primer extension. If this result is correct (see Results),
it suggests
that 3' exonuclease trimming is not involved in
H. marismortui. In
E. coli, the 3' CCA end of the tRNA is
encoded
in the tRNA gene, whereas in archaea the addition of CCA is
posttranscriptional
and presumably added by a tRNA
nucleotidyltransferase (see Fig.
2D). The 5' end of tRNA is known to be
generated by RNase P endonuclease
(
17,
28); the 3' end of
the leader released by RNase P cleavage
was not detected by an S1
protection assay, whereas the 5' end
of the trailer was detected. This
implies that RNase P cleavage
is a late event and occurs only after the
BHB endonucleolytic
cleavage in the 3' portion of the 16S processing
stem. The detection
of a 3' end site at position iA139 in the middle of
the tRNA suggests
the presence of an alternate processing pathway that
leads to
tRNA degradation. It is unclear whether this product was
generated
by an exo- or endonuclease.
The third and fourth endonuclease cleavages occurred at the BHB motif
in the 5' portion of the 23S processing helix and near
or at the 5' end
of 23S rRNA, respectively. The fact that the
leader product of the
maturation cleavage was observed indicates
that 23S 5'-end maturation
can on occasion occur before any of
the other cleavages in the
intergenic space, including excision
of pre-23S from the primary
transcript.
Only three endonucleolytic processing events were observed in the
405-nt 16S-23S spacer of transcripts derived from the
rrnB operon. These correspond to three of the cleavages within the
rrnA spacer: at the 3' end of the tRNA-like sequence, at the
BHB
motif in the 5' portion of the 23S processing helix, and at the
5'
23S maturation site. Again, it is clear that 23S 5'-end maturation
can
occur on occasion before any of the other cleavages in the
intergenic
space.
As illustrated in Fig.
2D, the
rrnB intergenic spacer
appears to contain a tRNA-like sequence capable of forming a
well-defined
acceptor and T
C stem. This sequence is almost certainly
not a
functional tRNA for several reasons: (i) it lacks the conserved
GG dinucleotide in the DHU loop and the UUC trinucleotide in the
T
C
loop, and (ii) the DHU and anticodon stems and loops are poorly
conserved and organized. Based upon the presence of GU base pairs
at
position 3 in the acceptor stem, this sequence appears to be
the
remnant of a tRNA
Ala gene. Nonetheless, formation of a
coaxial stack between the acceptor-like
and the T
C-like stems is
apparently sufficient for endonucleolytic
cleavage at the 3' end of the
structure at position iB198. Somewhat
surprisingly, no products with
either 5' or 3' ends located near
the 5' end of the tRNA-like structure
were detected, although
the structure should be a suitable substrate
for RNase P. It is
possible that RNase P cleavage, if it occurs at all,
occurs late,
after both upstream and downstream cleavages have taken
place.
Because of this, the product would not be visible in the S1
nuclease
protection assay that was employed. The product with a 3' end
at iB137 within the
rrnB tRNA-like sequence was detected.
Comparison
with the
rrnA 3' end at position iA139 revealed
that both ends
are located immediately 5' to the tetranucleotide CAAG;
in
rrnA,
this forms part of the anticodon loop in
tRNA
Ala (Fig.
2D).
Processing and maturation of 16S rRNA from the
rrnA operon
transcript follow the canonical halophilic archaeal pathway that
utilizes the BHB endonuclease to excise pre-16S rRNA. This is
followed
by maturation at the 5' and 3' ends. The
rrnB operon
transcript lacks the BHB motif in the 16S processing stem. No
precursor
5' ends were detected in the 5' portion of the 16S processing
stem.
Instead, the
rrnB primary transcript appears to be cleaved
directly by a maturation endonuclease at the 5' end of 16S rRNA.
In the
intergenic region, a 3' transcript end at position iB42
and a 5'
transcript end at position iB53 were located in the 3'
portion of the
stem. The significance of these ends and their
relationship to each
other is unknown.
| |
ACKNOWLEDGMENTS |
This work was supported by a grant from the Medical Research
Council of Canada.
We thank Deidre de Jong-Wong for technical assistance.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Department of
Biochemistry and Molecular Biology, University of British Columbia,
2146 Health Sciences Mall, Vancouver, BC V6T 123, Canada. Phone: (604) 822-5975. Fax: (604) 822-5227. E-mail:
pdp1{at}unixg.ubc.ca.
| |
REFERENCES |
| 1.
|
Branlant, C. A.,
M. A. Krol,
A. Machott,
J. Pouyet,
J.-P. Ebel,
K. Edwards, and H. Kossel.
1981.
E. coli 23S rRNA heterogeneity.
Nucleic Acids Res.
9:4303[Abstract/Free Full Text].
|
| 2.
|
Brombach, M.,
T. Specht,
V. A. Erdmann, and N. Ulbrich.
1989.
Complete nucleotide sequence of a 23S ribosomal RNA gene from Halobacterium marismortui.
Nucleic Acids Res.
17:3293[Free Full Text].
|
| 3.
|
Chant, J., and P. P. Dennis.
1986.
Archaebacteria: transcription and processing of ribosomal RNA sequences in Halobacterium cutirubrum.
EMBO J.
5:1091-1097[Medline].
|
| 4.
| Dennis, P. P. Expression of ribosomal RNA
operons in halophilic archaea, In A. Oren (ed.),
Microbiology and biogeochemistry of hypersaline environments, in press.
CRC Press, Inc., Boca Raton, Fla.
|
| 5.
|
Dennis, P. P.
1985.
Multiple promoters for the transcription of the ribosomal RNA gene cluster in Halobacterium cutirubrum.
J. Mol. Biol.
186:456-461.
|
| 6.
|
Dennis, P. P.
1991.
The ribosomal RNA operons of halophilic archaebacteria, p. 251-257.
In
F. Rodriguez-Valera (ed.), General and applied aspects of halophilic microorganisms. Plenum Press, New York, N.Y.
|
| 7.
|
Dennis, P. P., and J. Chow.
1994.
Analysis of RNA transcripts in archaebacteria, p. 163-178.
In
F. T. Robb, A. R. Place, K. R. Sowers, H. J. Schreier, S. Das Sarma, and E. M. Fleischmann (ed.), Protocols for archaebacterial research. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 8.
|
Dennis, P. P., and L. C. Shimmin.
1997.
Evolutionary divergence and salinity-mediated selection in halophilic archaea.
Microbiol. Mol. Biol. Rev.
61:90-104[Abstract].
|
| 9.
|
Dennis, P. P.,
A. G. Russell, and M. Moniz de Sá.
1997.
Formation of the 5' end pseudoknot in small subunit ribosomal RNA: involvement of U3-like sequences.
RNA
3:337-343[Abstract].
|
| 10.
|
Deutscher, M.
1993.
Ribonuclease multiplicity diversity and complexity.
J. Biol. Chem.
268:13011-13014[Free Full Text].
|
| 11.
|
Dover, G. A.
1982.
Molecular drive: a cohesive mode of species evolution.
Nature
299:111-117[Medline].
|
| 12.
|
Evers, U.,
F. Franceschi,
N. Boddeker, and A. Yenath.
1994.
Crystallography of halophilic ribosomes: the isolation of an internal ribonucleoprotein complex.
Biophys. Chem.
50:3-16[Medline].
|
| 13.
|
Fleishmann, R. D.,
M. D. Adams,
O. White,
R. A. Clayton,
E. F. Kirkness, et al.
1995.
Whole-genome random sequencing and assembly of Haemophilus influenzae.
Science
269:496-512[Abstract/Free Full Text].
|
| 14.
|
Franzetti, B.,
B. Sohlberg,
G. Zaccai, and A. von Gabain.
1997.
Biochemical and serological evidence for an RNase E-like activity in halophilic Archaea.
J. Bacteriol.
179:1180-1185[Abstract/Free Full Text].
|
| 15.
|
Garrett, R. A.,
J. Dalgaard,
N. Larson,
J. Kjems, and A. Mankin.
1991.
Archaeal rRNA operons.
Trends Biochem. Sci.
16:22-26[Medline].
|
| 16.
|
Ghora, B. K., and D. Apirion.
1978.
Structural analysis and in vitro processing of p5 rRNA of a 9S RNA molecule isolated from an rne mutant of Escherichia coli.
Cell
15:1055-1066[Medline].
|
| 17.
|
Gopalan, V.,
S. J. Talbot, and S. Altman.
1995.
RNA-protein interactions in RNase P, p. 103-127.
In
K. Nagai, and I. Mattaj (ed.), RNA protein interactions. Oxford University Press, Oxford, England.
|
| 18.
|
Gunderson, J. H.,
M. L. Sogin,
G. Wollett,
M. Hollingdale,
V. F. de la Cruz,
A. P. Waters, and T. F. McCutchan.
1987.
Structurally distinct, stage-specific ribosomes occur in Plasmodium.
Science
238:933-937[Abstract/Free Full Text].
|
| 19.
|
Hancock, J. M., and G. A. Dover.
1990.
Compensatory slippage in the evolution of ribosomal RNA genes.
Nucleic Acids Res.
18:5949-5954[Abstract/Free Full Text].
|
| 20.
|
Henikoff, S.
1987.
Exonuclease III generated deletions for DNA sequence analysis. Promega Notes, no. 8, p. 1-3.
Promega Corp., Madison, Wis.
|
| 21.
|
Kelman-Leyer, K.,
D. W. Armbruster, and C. J. Daniels.
1997.
Characterization of the Haloferax volcanii tRNA intron endonuclease gene reveals a relationship between the archaeal and eucaryal tRNA intron processing system.
Cell
89:839-847[Medline].
|
| 22.
|
Mankin, A. S.,
E. A. Skripkin, and V. K. Kagramanova.
1987.
A putative internal promoter in the 16S/23S intergenic spacer of the rRNA operon of archaebacteria and eubacteria.
FEBS Lett.
219:269-273[Medline].
|
| 23.
|
Mevarech, M.,
S. Hirsch-Twizer,
S. Goldman,
E. Jakobson,
H. Eisenberg, and P. P. Dennis.
1989.
Isolation and characterization of the rRNA gene clusters of Halobacterium marismortui.
J. Bacteriol.
171:3479-3485[Abstract/Free Full Text].
|
| 24.
|
Mylvaganam, S., and P. P. Dennis.
1992.
Sequence heterogeneity between the two genes encoding 16S rRNA from the halophilic archaebacterium Haloarcula marismortui.
Genetics
130:399-410[Abstract].
|
| 25.
|
Ochman, H., and A. Wilson.
1987.
Evolution in bacteria: evidence for a universal rate in cellular genomes.
J. Mol. Evol.
26:74-86[Medline].
|
| 26.
|
Oren, A.,
P. P. Lau, and G. E. Fox.
1988.
The taxonomic status of Halobacterium marismortui from the Dead Sea: a comparison with Halobacterium vallesmortis.
Syst. Appl. Microbiol.
10:251-258[Medline].
|
| 27.
|
Pace, N. R.
1997.
A molecular view of microbial diversity and the biosphere.
Science
276:734-740[Abstract/Free Full Text].
|
| 28.
|
Pace, N. R., and J. W. Brown.
1995.
Evolutionary perspective on the structure and function of ribonuclease P, a ribozyme.
J. Bacteriol.
177:1919-1928[Free Full Text].
|
| 29.
|
Scholzen, T., and E. Arndt.
1992.
The alpha operon equivalent genome region in the extreme halophilic archaebacterium Haloarcula (Halobacterium) marismortui.
J. Biol. Chem.
267:12123-12130[Abstract/Free Full Text].
|
| 30.
|
Thompson, L. D., and C. J. Daniels.
1990.
Recognition of exon intron boundaries by the Halobacterium volcanii tRNA intron endonuclease.
J. Biol. Chem.
265:18104-18111[Abstract/Free Full Text].
|
| 31.
|
Waters, A. P.,
C. Syin, and T. F. McCutchan.
1989.
Developmental regulation of state-specific ribosome populations in Plasmodium.
Nature
342:438-440[Medline].
|
| 32.
|
Woese, C. R.
1987.
Bacterial evolution.
Microbiol. Rev.
51:221-271[Free Full Text].
|
Journal of Bacteriology, September 1998, p. 4804-4813, Vol. 180, No. 18
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
