Genomic Analysis of the Genes Encoding Ribosomal
Proteins in Eight Eubacterial Species andSaccharomyces cerevisiae

Katsutoshi Fujita

1

fujita@biol.kobe-u.ac.jp

Tomoya Baba2

baba@staff.or.jp

Katsumi Isono

1

isono@biol.kobe-u.ac.jp

1

Graduate School of Science and Technology, Kobe University
Rokkodai, Kobe 657-8501, Japan
2 STAFF, Kamiyokoba, Ippaizuka, Tsukuba 305-0854, Japan
Abstract

The complete genomic nucleotide sequence data of more than 10 unicellular organisms havebecome available. During the past years, we have been focusing our attention to the analysis of thestructure and function of the ribosome and its protein components. By making use of the genomic
sequence data, our work can now be extended to comparative analysis of the ribosomal components at the genomic level. Such analysis will contribute to our understanding of the structurefunction relationship of the ribosome that is vital to the expression of genetic information. Bearing
these in mind, the ribosomal protein genes of organisms whose genomic sequence data are available were analyzed, which included Aquifex aeolicus; Archaeoglobus fulgidus; Borrelia burgdorferi;
Bacillus subtilis; Escherichia coli; Haemophilus in uenzae; Helicobacter pylori; Methanococcus jannaschii; Mycoplasma genitalium; Mycoplasma pneumoniae; Synechosystis sp., and Saccharomyces
cerevisiae. In addition, the amino acid sequence data of Bacillus stearothermophilus ribosomal proteins were used in the evolutionary evaluation. The results indicate that, in eubacteria including
two species of Mycoplasma, the operon structure of ribosomal protein genes is well conserved, while
their relative orientation and chromosomal location are diverged into several classes. The operon
structure in M. jannaschii on the other hand is quite di erent from the eubacterial one and wenoticed that its many genes show similarity to rat ribosomal protein genes. The degrees of sequenceconservation di er from one ribosomal protein gene to another, but several genes encoding proteinsthat are considered to be of structural importance are conserved throughout the bacterial species
including archaebacteria and further in S. cerevisiae.

1 Introduction
Recent remarkable progresses in the methodology and instrumentation for performing large scale
nucleotide sequencing have resulted in the completion of genomic nucleotide sequencing of more thanten organisms and the data for additional ten or more organisms are expected to become available in
the near future. Furthermore, genomic sequencing of organisms that are important in agriculture andmedicine is also in progress. The resultant data will drastically a ect the ways of performing researchin biological sciences. With the vast amount of sequence data available, we can select genes of ourinterest and characterize them in silico before performing actual in vitro or in vivo experiments. Bysuch cyber-biological analyses, we will be able to predict the features of the genes and their protein
products and to determine the way of approaching to their experimental characterization.
Escherichia coli and Haemophilus in uenzae are rather closely related, both belonging to the samebranch within the gamma subdivision of purple bacteria. While E. coli K-12 is benign, H. in uenzae
as well as some wild E. coli strains are pathogenic, causing diseases of varying degrees of harmfulness.
The di erences must lie within their individual genomic contents: virulence related factors could be
identi ed by subtracting from H. in uenzae those genes that have a homolog(s) in E. coli [13]. Such
3

analysis would be not only useful for therapeutic purposes but also for the identi cation of di erencesamong organisms.
There are many structural entities that are involved in the translation of genetic messages within
the cell. Of them, the ribosome is a pivotal apparatus which is composed of two subunits of unequal sizeand contains several RNA and more than 70 di erent protein molecules. Because of the high degree offunctional importance, interaction between ribosomal subunits and their individual components musthave been highly conserved during the course of evolution. A mutation in one of the components
will a ect its local conformation, changing its ability to be assembled with other components, andconsequently the function of the whole ribosome may be impaired. Since all ribosomal proteins (rproteins) of E. coli have been extensively characterized and their in vitro reconstitution into active
ribosomes has been made possible [21], it would be interesting to analyze to what extent we might
be able to correlate the evolutionary conservation and structural importance of individual r-proteins.
Furthermore, comparative studies of r-protein genes at the genomic level would clarify whether any
one or more r-proteins are, either partly or totally, dispensable or not, and if so, what would be aprerequisite for that to occur. As a rst step to such an approach, we carried out extensive similarity
search of the r-protein genes in the genomic sequence data of nine bacterial species and yeast as will
be described below.

2 Materials and Methods
The genomic nucleotide sequence data were retrieved either directly from the DDBJ/EMBL/GenBank
nucleic acid sequence databases or through the World Wide Web server at TIGR (http://www.tigr.org/).
The amino acid sequence data were retrieved from the SWISSPROT and PIR databases. To compareindividual ribosomal proteins, we used the FASTA program [24]. Since r-proteins have not been fully
characterized in organisms other than E. coli, their genomic nucleotide sequences were translated intoamino acid sequences in six reading frames and subjected to FASTA analysis using the r-protein aminoacid sequence data of E. coli and Bacillus stearothermophilus [30]. The FASTA scores obtained were
subsequently converted into 'degrees of conservation' by normalizing them with the corresponding'self-examination' data of E. coli and B. stearothermophilus r-proteins.

3 Results and Discussion
3.1

Ribosomal protein genes are conserved across kingdoms.

Putative r-proteins were identi ed in the amino acid sequence data translated from the genomic nucleotide sequences of individual organisms by comparing similarity to the r-protein sequences of E.
coli and B. stearothermophilus. We calculated the degrees of conservation of individual r-proteins as
described in Materials and Methods and used them for comparison. The genomic nucleotide sequencesof eubacteria, Bacillus subtilis [19], E. coli [2], H. in uenzae [5], Helicobacter pylori [28], Mycoplasma
genitalium [6], Mycoplasma pneumoniae [10], Synechosystis sp. [15], and Aquifex aeolicus [4] wereanalyzed. Of these, B. subtilis is classi ed as Gram-positive, E. coli, H. in uenzae and H. pylori as
Gram-negative, A. aeolicus as an extreme thermophile, and Synechosystis as cyanobacteria. In addition to these eubacteria, the data of a spirochete Borrelia burgdorferi [7] as well as two archaebacteria,
Methanococcus jannaschii [3] and Archaeoglobus fulgidus [16] and a unicellular fungus Saccharomyces
cerevisiae [8] were included in the analysis. We treated ORFs whose amino acid translation data
showed FASTA scores higher than 100 as putative r-protein genes. The results of analysis with E.
coli r-proteins are summarized in Table 1. It is readily evident that r-proteins are widely conserved
amongst eubacteria analyzed and to a much less extent in archaebacteria and yeast. Some proteins
known to be important for ribosomal structure and function such as S4, S5, S8, S10, S12, S13, L1,
L2, L5, and L11 are well conserved throughout the bacterial kingdom as expected. Moreover, some4

of the E. coli and/or B. stearothermophilus r-proteins show a considerable degree of similarity to thecytoplasmic r-proteins of S. cerevisiae as well.
Table 1: Comparison of r-proteins from various organisms (a)=====================================================================================================
Ec
Size
Hi
Bs
Hp
Ss
Bb
Aa
Mg
Mp
Af
Mj
Sc
Prot
(a.a.)
mit cyt-1 cyt-2----------------------------------------------------------------------------------------------------E-S1
557
+++
+
+
+
+
+
+
+
E-S2
241
+++
++
++
++
++
++
++
++
+
+
E-S3
233
+++
++
++
++
++
++
++
++
+
+
E-S4
206
+++
++
++
++
++
++
++
++
+
+
E-S5
166
+++
++
++
++
++
++
++
++
+
+
+
+
E-S6
135
+++
++
+
+
+
+
+
E-S7
178
+++
++
++
++
++
++
++
++
+
E-S8
130
+++
++
++
++
++
+
++
++
+
+
E-S9
128
+++
++
++
++
++
++
++
++
+
++
E-S10
103
+++
+++ +++
++
++
++
++
++
++
++
+
+
E-S11
129
+++
+++
++
++
++
++
++
++
++
+
+
+
E-S12
124
+++
++ ++++++ +++
+++
++
++
+
+
++
E-S13
118
+++
+++
++
++
++
++
++
++
+
+
+
+
+
E-S14
98
+++
++
+
++
+
+
+
+
+
E-S15
89
+++
+++
++
++
++
++
++
++
++
E-S16
82
+++
++
++
++
++
++
++
E-S17
84
+++
++
++
++
++
++
++
+
++
++
++
E-S18
74
+++
++
++
++
++
++
++
++
E-S19
92
+++
+++
++
+++
++
++
++
++
+
+
++
+
E-S20
87
+++
++
+
+
+
++
+
E-S21
71
+++
++
++
++
++
++
E-S22
45
----------------------------------------------------------------------------------------------------E-L1
234
+++
++
++
++
++
++
++
++
+
+
+
E-L2
273
+++
++
++
++
++
++
++
++
+
+
+
+
+
E-L3
209
+++
++
+
++
++
++
++
++
+
+
++
E-L4
201
+++
++
+
++
++
+
+
+
E-L5
178
+++
+++
++
+++ +++
+++
++
++
+
+
+
+
E-L6
176
+++
++
++
++
++
++
++
++
+
+
+
E-L9
148
+++
++
+
++
++
++
+
+
E-L10
165
+++
+
+
+
+
+
+
E-L11
142
+++
++
++
+++
++
++
++
++
++
++
++
E-L12
121
+++
+++ +++
++
++
+++
++
++
+
++
E-L13
142
+++
++
++
++
++
++
++
++
+
++
E-L14
120
+++
++
+++
++
++
++
++
++
+
+
+
+
E-L15
144
+++
++
++
++
++
++
++
++
+
++
E-L16
136
+++
++
++
+++
++
++
++
++
++
E-L17
127
+++
++
++
++
++
++
+
+
++
E-L18
117
+++
++
+
++
++
++
+
+
+
E-L19
115
+++
++
++
++
++
++
++
++
+
E-L20
118
+++
+++
++
++
++
++
++
++
E-L21
103
+++
++
++
++
++
++
+
+
E-L22
110
+++
++
++
++
++
++
++
++
+
E-L23
102
+++
+
+
++
++
++
+
+
+
E-L24
103
+++
++
+
++
++
+
+
+
+
+
E-L25
94
+++
+
E-L27
85
+++
++
++
++
++
++
++
++
++
E-L28
78
+++
+
++
E-L29
63
+++
++
++
++
++
++
+
++
E-L30
58
+++
++
++
+
E-L31
70
+++
++
++
++
+
++
++
++
E-L32
57
+++
+
++
++
E-L33
55
+++
++
++
++
++
++
++
++
++
+
E-L34
46
+++
+++
++
++ +++++ +++
+++
++
E-L35
65
+++
++
++
+
++
++
++
++
+
E-L36
38
+++
+++ ++++++ ++++++++ +++++=====================================================================================================(a)Symbols indicate: +, degree of conservation of less than 30%; ++, between 30 and 70%; +++, more than 70%.
Abbreviations of organism names are: Aa, Aquifex aeolicus; Af, Archaeoglobus fulgidus; Bb, Borrelia burgdorferi; Bs,
Bacillus subtilis; Ec, Escherichia coli; Hi, Haemophilus in uenzae; Hp, Helicobacter pylori; Mg, Mycoplasma genitalium; Mj, Methnococcus jannaschii; Mp, Mycoplasma pneumoniae; Sc, Saccharomyces cerevisiae; Ss, Synechosystis sp.
Abbreviations mit and cyt, respectively, indicate mitochondrial and cytoplasmic ribosomes.

5Protein S22 was identi ed as an r-protein in E. coli [29], but its counterpart could not be identi ed
in other organisms including the most closely related bacterium, H. in uenzae. S22 may be an E.
coli-speci c r-protein. H. in uenzae harbors a highly conserved L25 homologue. However, this was
not the case with other bacteria except for B. burgdorferi which showed the presence of a proteinof weak similarity to L25. In B. subtilis both S14 and L31 are encoded by two genes which are not
identical. Similarly, the gene for S15 is duplicated in H. in uenzae. More surprisingly, the extreme
thermophile, A. aeolicus, contains two sets of S7 and S12 genes and a gene which shows a weakerhomology to S1 in addition to the one listed in Table 1. Although A. aeolicus has been placed closest
to the branch point of eubacteria and archaebacteria [4], it appears to a typical eubacteria without
any doubt.
Of the highly conserved r-proteins, E. coli S3 protein has been shown to interact with 16 S rRNA
and was found to be located in the head of the small subunit near the site at which the polypeptide
release factor RF-2 binds. Together with S4 and S5, protein S12 participates in a region designated as
the recognition complexes [22, 23] (see below). S4 in turn is one of the primary rRNA-binding proteinsthat initiates assembly of the 30 S subunit and is known to protect several 16 S rRNA regions fromchemical modi cation during assembly. Proteins S8 and S11 interact with a highly conserved region
in the central domain of the 16 S rRNA termed the platform ring [23]. S19 interacts with proteins S7,
S9 and S14. S10 and S13 are later-assembling proteins and has been shown to rely relatively less onprotein-RNA interactions and more on protein-protein interaction.
L13 L21
S9 L27

IF-3
L35
L20

S16
L19

S12

S6
S18
L9
IF-3
L35
L20
L21
L27

L11
L1
L10
L7/L12

L10
L7/L12
S16
L19

L13
S9

L11 S6L1 S18
L11
L1
L10
L7/L12

L9

L13
S9

S16
L19
L21
L27

L33
L11
S6 L1
S18 L7/L12 S16
L19

L13
S9
L21
L27

IF-3
L35
L20

L11
L1
L10
L7/L12

L35
L20

L13
S9

L21
L27
S16 L21L19 L27

IF-3
L10
L35
L20 L21 L7/L12
L27 L32

S18 IF-3
L9 L35
S6
L20
S18
L9

L13
S9

L11
L1
L10
L7/L12

L13
S9
S6
S18
L9 L21
L27

L10
L7/L12
L32

IF-3
L35
L20

L13
S9

L13
S9

1,000

2,000

3,000

4,000

kb

Figure 1: The genomic distribution of transcription/translation-related protein genes in individual organisms.

Boxes representing the genes/ORFs homologous to those of E. coli are placed one after another in the vertical direction
on the w (above) and c strand (below), respectively, of the horizontal line indicating the chromosome of each organism
drawn to scale. In addition, those of M. jannaschii and A. fulgidus showing similarity to rat r-proteins are similarly

6placed in light grey. The left end of each chromosome is arbitrarily set at the position of the L3 gene of the S10 operon.
Some of the genes are marked with the name of their protein products for easier identi cation.

In E. coli, the three highly conserved proteins L1, L2 and L11 belong to the early-assembly proteinsof the large subunit [21]. L1 has been shown to take part in the formation of the ridge on the large
subunit [23]. Proteins L2 and L3 form part of the peptidyltransferase center and L5 binds to 5 S
rRNA. The highly conserved L14 proteins is one of the late-assembly proteins and does not binddirectly to 23 S r RNA [21].
S. cerevisiae has two sets of ribosomes, functioning in the cytoplasm and mitochondria, respectively.
Considering the putative prokaryotic origin of mitochondria during evolution, it is expected thatmitochondrial ribosomes contain bacterial r-protein homologues that are structurally well conserved.
The results obtained were largely as expected. However, some bacterial r-proteins such as S11 andS17 showed no discernible similarity to any of the mitochondrial r-proteins, but rather to cytoplasmic
ones.
3.2

Genomic distribution of the genes for r-proteins and related protein factors.

Since in E. coli the genes for r-proteins are often clustered with those for transcription and translationrelated proteins such as RNA polymerase subunits, initiation factors, elongation factors, and so on,
we surveyed not only r-protein genes but also the genes for other transcription/translation-related
proteins. In addition, we analyzed the genes encoding r-protein modifying enzymes. The distributionof these genes was compared in the genome of each organism. The relative positions and orientationof r-protein genes were aligned with each other by placing the gene for L3 protein of the S10 operon
at the left end as shown in Fig. 1.
The L13 and S9 genes are located near the S10 operon in E. coli, B. subtilis and Synechocystis,but they are not in the corresponding region in other organisms and its relative orientation are also
di erent. The genes encoding L21 and L27, IF3 and L35, as well as L20 are similarly located andclustered in the eubacterial species analyzed. From these data it can be generalized that, while
individual genes for r-proteins and related protein factors have been evolved by forming separate geneclusters which are well conserved amongst eubacteria, the locations and relative orientations of thesegene clusters are quite diverged in other bacteria. There are exceptions: the genes for L11, L1, L10
and L7/L12 proteins, for example, compose a single operon in most eubacteria, but in H. in uenzae
they are split into two operons and their relative orientation is also di erent. Although E. coli and
H. in uenzae are most closely related amongst the eubacterial species analyzed, there are several
di erences in the organization of genes involved in transcription and translation in the two species.
Unlike other bacteria H. in uenzae has an ORF (see bellow) inserted between the S17 and L14 genesof the S10 operon and the S12, S7, EF-G and EF-Tu gene cluster is located on the opposite strand
with respect to the S10 operon. H. in uenzae has already been compared at the genomic scale with
E. coli and M. genitalium. A comparison of the positions of the orthologous genes in the E. coli and
H. in uenzae chromosomes clearly showed lack of a long-range colinearity as reported by Tatusov etal. [27]. In comparison, lack of conservation of the gene localization in the genome of M. genitalium
is striking [17].
We anticipated that the distribution pattern of these protein genes would be conserved in closely
related species such as M. pneumoniae and M. genitalium, both belonging to Mycoplasmataceae.
However, their distribution was found to be rather di erent from each other: while the genes for IF-3,
L35 and L20 are located between the S10 operon and the cluster of L21 and L27 genes in M. genitalium,
another cluster containing the S6, S18 and L9 genes is located in place of the IF-3-L35-L20 clusterin M. pneumoniae. Thus, the positions of the IF-3-L35-L20 and S6-S18-L9 clusters are exchanged
between the two Mycoplasmataceae bacteria. Recently, comparative analysis of the two bacteria was
performed by Himmelreich et al. [11], suggesting that the two genomes could be subdivided into six
7segments. The order of orthologous genes was well conserved within individual segments but the orderof these segments was di erent. They explained that the di erent organization of the segments has
been brought in by translocation via homologous recombination. A similar feature has been reported
by Kunisawa [18] concerning E. coli and B. subtilis. There are many instances in which a contiguous
r-protein gene segment in one genome is split into two or more segments that are located at di erent
positions in the other. Unlike the two mycoplasma species mentioned above, E. coli and B. subtilis
are rather distantly related: a phylogenetic study of their 5 S rRNA sequences has given an estimateof their divergence time of ca. 1.2 billion years [12].
Despite various di erences mentioned above, there is one feature that is common to the eubacterial
genomes we have analyzed; namely, the genes for many r-proteins are clustered in the region near the
S10 operon. Since the expression of all ribosomal components must be regulated coordinately andthe amount of each gene product be equal to each other, it would be bene cial that many r-protein
genes are clustered with each other so that the regulation of their transcription would be rather easily
facilitated.
Since we were interested in characterizing the r-protein genes from an evolutionary point of view,
we then performed a similar analysis with the M. jannaschii and A. fulgidus r-protein genes. It wasevident that the degree of similarity of their r-proteins to those of eubacteria was not very high.
Therefore, the data for translation-related proteins of rat were also included in subsequent FASTA
analysis. The genes in these archaebacteria have been diverged in clusters as in the case of those of
eubacteria. The clusters are not identical, but some of the r-protein genes contained are similar toS12

E. coli

S10
S7

EF-G

EF-Tu

L23
L3 L4

S19
L2

L16S17 L24 S14
S3

L22

S10

L5
L29 L14L18 L30

L36 S11

L6 S5
S8

rpoA

L15

S13

S17

S19

L17

secY
S4

L17

L36
secY

H. influenzae
EF-Tu

L22

EF-G
S10
EF-G

L14

S19

m ap L36

S17

EF-Tu

L17

secY adk

B. subtilis

L3

S10
EF-G

rpoA

L22

L14

S19

S17

rpoA
if1

L36

EF-Tu

secY

L17

adk

L13 L31

rpoA

Synechocystis sp.

S6

S18

S10

L22

L14

infA

S19

S17

truA

L17

L36

EF-G

S9

secY adk m ap

rpoA

glnQ

M. genitalium
ssb

L9

S10

L22

L14

S19

S17

infA

hlyB

L17

L36

EF-G

secY adk m ap

rpoA

hisP

M. pneumoniae
ssb

L22

L3

L23

infA

L14

S19

cysA

S18 S11 L29E S16

EF-G

M. jannaschii
L4

S9

L2
S3

L14 S4E

S8

L13

L32

L18

L15
secY

S10
EF-Tu

L22

L29

S17 L24

L5

L6

L19

adk

S5 L7

Figure 2: Alignment of the S12-S10-rpoA-operons. The r-protein genes (marked by E. coli protein names) areindicated by boxes of various patterns for easier identi cation and other protein genes are indicated by white boxes. The
r-protein names that are boxed indicate those similar to rat r-proteins.

those in eubacteria, while many others are similar to those of rat which are often inserted between
eubacteria-type r-protein genes. Fig. 2 shows the results obtained with the r-protein genes of M.
jannaschii. It was also noticed that many of the eubacteria-type r-protein genes are either smaller or8larger in size than their eubacterial counterparts.
3.3

Comparison of the S10 and S12 operon structures.

The fundamental structure of the S10-spc-rpoA operons is well conserved amongst the eubacterial
species analyzed along with the relative gene order within the operons (Fig. 2). However, there arecases of insertions of other genes into the r-protein operons as well as translocations of some of thegenes within the operon. In H. in uenzae, for example, an ORF of hitherto unknown function has
been inserted between the S17 and L14 genes. In B. subtilis, three protein genes exist between secY
and the L36 gene, and the S4 gene is translocated between the S11 gene and rpoA encoding the-subunit of RNA polymerase. The size of each gene is nearly identical from one species to anotherin most cases. A notable exception is the case of the L23 gene of M. pneumoniae which contains an
extra stretch at the 3'-terminus which shows no similarity to any other known bacterial proteins. It is
likely that the extra stretch of L23 has been acquired after the diversion of M. pneumoniae, although
the precise branching point is not known. In Synechocystis, the genes for L13, S9 and L31 have beentranslocated at the end of the S10-spc-rpoA operons. That the L13 and S9 genes are clustered can beobserved in other eubacteria. The S10 gene of Synechocystis is not in the S10-spc-rpoA operons, but
it is located in the S12 operon. Since the S10 gene leads the S10-spc-rpoA operons in E. coli and B.
subtilis and is just downstream of the S12 operon, during the course of genomic evolution, the S10gene of Synechocystis have apparently moved to the 3'-end of the S12 operon. The order of the genes
for S12, S7 and EF-G is also conserved, but the genes following the operon di ers from one species toanother: in E. coli, H. in uenzae, Synechocystis, and B. subtilis the gene encoding EF-Tu is locatedimmediately downstream of the EF-G gene, while it is the S6-ssb-S18-L9 cluster that follows the S12
operon (Fig. 2). The S6-ssb-S18-L9 cluster is also conserved as such, though at a di erent locus,
except for Synechocystis sp. in which these protein genes are dispersed at various loci. Furthermore,in Gram-negative bacteria including Synechocystis, the adk gene encoding adenylate kinase exists at
the end of the S10 operon, but another copy exists at a di erent locus in Synechocystis. Therefore, thegenomic organization of the genes for ribosomal and related proteins in Synechocystis is more di erent
than expected from their nucleotide sequence divergence. Recently, the structure of the S10-spc-rpoA
operons in B. subtilis have been analyzed and the promoters identi ed [20]. While the three operons
in E. coli are transcribed from individual promoters, the B. subtilis counterparts are transcribed fromone and the same promoter. Thus they form a single operon. We have compared the sequence between
the S7 and EF-G genes and found that they are quite diverse (data not shown). Similarly, the size ofthe inter-cistronic regions vary a great deal from one organism to another.
In M. jannaschii, the genes in the S10 operon are not conserved, although the order of identi abler-protein genes are relatively well conserved as shown in Fig. 2. There are many genes the protein
products of which are di erent from eubacterial r-proteins: they are rather similar to eukaryotic
proteins. Thus, in M. jannaschii the r-protein gene clusters are composed of genes some of which aresimilar to eubacterial, while some others to eukaryotic r-protein genes. The corresponding region inanother archaebacterium, A. fulgidus, resembles that of M. jannaschii. However, the S10 homolog
itself is not included in the region in both archaebacteria as in the case of Synechocystis sp. The genes
encoding S12, S7, S10 proteins and the elongation factor 1a of a third archaebacterium, Sulfolobus
solfataricus, are located in regions comparable to those of the corresponding genes in E. coli [14].
Interestingly, the genes for L11, L1, L10 and L12 proteins in S. solfatarcius, S. acidocaldarius andHalobacterium cutirubrum are located in the same order as in the case of their E. coli counterparts
[25].
3.4

A high degree of conservation of the S12 gene.

S12 protein was found to be one of the most highly conserved r-proteins amongst the organisms
we have analyzed including S. cerevisiae. A yeast ORF termed YNR036c is likely to encode its
9mitochondrial homolog which showed a very high degree of similarity to E. coli S12 (57.4 % identity
in FASTA). In E. coli, S12 is essential for translational accuracy and is known to be a streptomycintargeted protein [1]. For these reasons, we compared S12 genes and their deduced protein products
from individual organisms in detail (Fig. 3). The amino acid residues that are known to be altered bystreptomycin resistance mutations in E. coli were found to be highly conserved even in S. cerevisiae andarchaebacteria. Therefore, we thought that these residues would also be important for the ribosomal
function in other organisms. In fact, in S. cerevisiae, the amino acid residues of the cytoplasmic
S12 homolog, RPS28 (indicated as Sc-c), were reported to be related to paromomycin resistance andtranslational accuracy [1]. However, there are di erences between Gram-positive and Gram-negative
bacteria. The S12 proteins
Ec
Hi
Ss
Bb
Hp
Bt
Bs
Mp
Mg
Sc-m
Af
Mj
Sc-c

Ec
Hi
Ss
Bb
Hp
Bt
Bs
Mp
Mg
Sc-m
Af
Mj
Sc-c

*************
1 MATVNQLVR-KPRARKVAKSNVPALE-------------ACPQKRGVCTRVYTTTPKKPNSALRKVCRVRL-TNGFEVTSYIGGE
1 ...I.....-...VK..V........-------------.............................I..-.............
1 .P.IQ..I.-SE.SKVQK.TKS....-------------Q...R......................A....-.S.....A..P.I
1 .P.I...I.-...KSQTEKTAS...Q-------------N...R..IC...M.V............A....-S......A..P.I
1 .P.I...I.-.E.KKV.K.TKS...V-------------E...R......................AK...-.SK...I..IP..
1 .P.I.....-.G.EK..F..KS...NKGYNSFKKEQTNVAS..........G.M...........YA....-...I...A..P.I
1 .P.I...IR-.G.VS..EN.KS...NKGYNSFKKEHTNVSS..........G.M...........YA....-...I...A..P.I
1 ...IA..I.-...KK.KV..KS...HYNLNLLNKKVTNVYS.L........G.M...........YAK...-......LT..P..
1 ...IA..I.-...QK.KV..KS...HYNLNLLNKKTTNVYS.L........G.M..R........YAK...-......LA..P..
29 ..L..IK.GSGPP.RKKI.TAPQLD-------------Q...RK..VL..MVLK.......Q..A.....-...NV.SA..P..
36
38

AKADP..-------------GA.MA..IVLEKIGIEARQ....I..AV..Q.IK..RQI.AFCP.D.EKYDP..-------------GA.MA..IVIEKVGLEA.Q....I..CV..Q.IK..RV..AFCP.N

41

SPFG-------------GSSHAK.IVLEKLGIES.Q....I..CV..Q.IK..KK..AFVPND

71 G--HNLQEHSVILIR--GGR-----VKDLPGVRYHTVRGALDC--SGVKDRKQARSKYGVKRPKA
71 .--........V...--...-----..................--A.......G...........
71 .--........V...--...-----..........I...T..A--T.......G.....TREKAKK
71 .--........V...--...-----..........I....K.T--L..NN..KG.....T.K...
71 .--.......IV.V.--...-----.......K..I......T--A..NK.TVS.....T.KA..TDKKATDNKKK
84 .--........V...--...-----....R.....II..G..T--A..AN.M.G.....A.K...AKK
84 .--........V...--...-----..N..R....I......T--A..EN.A.G.P...T.K...K
84 .--........T.L.--...-----..........I...T..T--V..EK.R.Q..A..A.KP..KS
84 .--........T.L.--...-----..........I...T..T--V..DK.R.Q..A..A.K..PKS
99 .--.DA....IVYVR--...-----CQ.....K..VI...G.L--...VN.ISS.....A.K.SKS
89 .AINFID..DEVIVEKI...MGRS-MG.I.....KV.KVNNTSLREL.RG..EK.LR
95 HAINFID..DEVI.EGI..PKGPRAKG.I...K.KVIMVGRNSLREL.RG.QEKIKR
91 .CLNFVD.NDEV.LA--.FGRKGKAKG.I....FKV.KVSGVSLLALWKEK.EKPRSFigure 3: Alignment of S12 proteins. The amino acid residues altered by streptomycin resistance mutations in E.

coli are boxed. An extra stretch found in Gram-positive bacteria is indicated by asterisks. In S. cerevisiae, mitochondrialand cytoplasmic homologues are indicated as Sc-m and Sc-c, respectively. Dots represent residues identical to those of
E. coli and hyphens indicate residues inserted for maximal alignment.

of the latter contain additional 13 amino acid residues in the N-terminal region which are well conserved in the four species belonging to Bacillaceae and Mycoplasmataceae. This feature is apparently
characteristic to Gram-positive bacteria. In spite of this rather long insertion, the E. coli S12 can becompletely substituted with the B. stearothermophilus S12 in poly(U)-dependent poly-Phe synthesis invitro [9]. This might suggest that the extra residues in this protein are not essential for the ribosomal
function. Interestingly, the stretch does not exist in archaebacteria and in either of the two homologues
in S. cerevisiae. It might indicate that the stretch emerged after the diversion of Gram-positive andGram-negative bacteria. Similarly, in several other highly conserved r-proteins, functionally importantamino acid residues were found to be conserved across kingdom (data not shown).

Concluding Remarks
In this study, we analyzed the genes encoding r-proteins as well as other transcription/translationrelated proteins of unicellular organisms whose genomic sequence has been completed. Many of the10

genes encoding these proteins are clustered (most likely forming operons) and their relative orientation is generally rather well conserved even in archaebacteria, although the genomic positions of
r-protein gene clusters in the two mycoplasmas analyzed, for example, were found not to be identical.
Furthermore, some of the r-protein genes are missing, while some others are duplicated, in some bacterial species. It remains to be investigated further to what extent the regulation of the expression ofthese r-protein gene clusters is conserved. Ribosomal proteins, especially those playing pivotal roles
in translation such as S12, are structurally highly conserved in all organisms we have analyzed in thiswork. Since the genetical and biochemical aspects of r-proteins and their genes have been extensively
characterized in E. coli, but not so much in other organisms, we hope that the analysis of r-proteins
and their genes reported here will be useful to obtain important clues as to how the genomic architecture of essential genes such as those encoding r-proteins was established and has been evolved sincethen and what will be their functional relevance.

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