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Evolution of Phenylalanyl-tRNA Synthetase by Domain
Losing
LIN
Jun1,2, HUANG Jing-Fei1,3*
( 1 Key Laboratory of Cellular
and Molecular Evolution, Kunming Institute of Zoology, the Chinese Academy of Sciences,
Kunming 650223, China; 2 Graduate School of the Chinese Academy of
Sciences, Beijing 100039, China; 3 Yunnan Key Laboratory of
Molecular Biology of Domestic Animals, Kunming 650223, China )
Abstract The
gene duplication, fusion and horizontal transfer are the frequent events during
evolution of many proteins, including the aminoacyl-tRNA synthetases (AARSs).
However, in this work, it was shown that the main event during evolution of
phenylalanyl-tRNA synthetase (PheRS) is a domain loss, and the
function/activity of PheRS is not affected by domain losing. Generally, the
size of genome and number of genes are increased during evolution from bacteria
to eukaryote, but the interesting thing is that the type and number of PheRS
domains in eukaryote are obviously less than those in bacteria. The evolution
of PheRS by domain losing seems to be related to the functional evolution of
some AARSs from the multiple specificities to the single specificity.
Key
words phenylalanyl-tRNA synthetase;
domain loss; molecular evolution
Phenylalanyl-tRNA synthetase (PheRS)
contains the most special structure in the twenty aminoacyl-tRNA synthetases
(AARSs). Usually, the PheRS is a heterotetramer enzyme (α2β2)
with multiple domains. Except the PheRS and glycyl-tRNA synthetase (GlyRS) in
some bacteria, all other AARSs are the monomer or homomer. The PheRS with
multiple domains holds complicated domain architecture. The bacterial PheRS β subunit contains a long α helix hairpin N-terminal domain (NTD)
and another catalytic core domain (CCD). The core structure of CCD is the α helices surrounded by the antiparallel β sheets. The β subunit in bacterial PheRS contains
several different domains including B1, B2, B3/B4, B5, non-catalytic core
domain (NCD) and anticodon binding domain (ABD)[1－3].
Most of these domains hold different structures, but some of them are similar,
such as CCD and NCD, or B1 and B5 domains. As to AARSs, it is very special to
contain two similar structural core domains. In the complex structure of
Thermus thermophilus PheRS with the cognate tRNA, one tRNAPhe molecule binds
across all four PheRS subunits, and the α2β2 architecture was absolutely needed for
the interactions of PheRS with tRNA[3]. However, some experiments confirm the
existence of single subunit PheRS. For example, the mitochondrial
phenylalanyl-tRNA synthetase (mtPheRS) possesses only one single subunit and is
active as a monomer (α)[4,5]. The N-terminal 314
amino acid residues in human mtPheRS appear to be homologous with the CCD in α subunit of prokaryotic PheRS, but about
100 amino acid residues in the C-terminus correspond to a region of the β subunit, which have been defined to
interact with the anticodon of tRNAPhe [6]. The interesting is that
aminoacylation capacity of mtPheRS is nearly equal to that of Escherichia coli
PheRS, and the misaminoacylation is not observed in the mt-tRNAPhe-mtPheRS
system[7]. The accuracy of mtPheRSs appears to be even better than that of the
cytoplasmic PheRS[8].
The evolutionary researches on AARSs show
that lots of complicated horizontal gene transfer events occurred during AARS
gene evolution, including transferring of PheRS genes in archaea to
spirochetes[9]. Although the evolutionary origins of the mtPheRS keep unknown,
it has been suggested that it is a result of the simplification of the
bacterial PheRS with α2β2 subunits, in which the specific
functional regions of bacterial PheRS are retained in mtPheRS[6]. In addition,
the monomeric PheRS from the yeast mitochondrion has been regarded as a chimera
of the α and β chains of the standard tetrameric
protein[10]. Because the PheRS subunit and domain architectures are very
special and complicated, the evolutionary study on the PheRS subunits and
domains may be helpful to reveal the evolution of AARS and of proteins with
multiple domains.
In
this work, the evolution of the sequence and structure of PheRS subunits and
domains of bacteria, archaea and eukaryote were studied, which suggests the
gene fusion and domain loss being the main events of PheRS evolution.
1 Materials
and Methods
The complex structure of Thermus
thermophilus PheRS with tRNAPhe from X-ray has been analyzed as a model with
its structural data from protein structure database (PDB)[11] (PDB code: 1EIY).
All sequences of PheRSs are from Swiss-Prot[12], including 44 α subunit sequences from eukaryote,
archaea, bacteria, and mitochondria respectively, and 50 β subunit sequences from eukaryote,
archaea, bacteria, and chloroplast, where the α
and β subunit sequences of 1EIY in
Swiss-Prot are named as SYFA-Theth and SYFB-Theth respectively. For the
sequence parts of known structures, the definition of domains in PheRS α and β subunits is from SCOP database[2]. For the sequence parts of unknown
structures, the domains are defined by sequence alignments using ClustalW[13],
and the specific sites denoted by Pfam database[14] are also considered. The
defined domains are checked by PSI-BLAST[15] searching against the
non-redundant (NR) database. The structure-based sequence alignments between
different domains with similar structures use the same methods as our previous
work[16,17]. Evolutionary trees are constructed by Phylip Program[18], where
the outgroup includes two CCD sequences of Lysyl-tRNA synthetases, and 1000
bootstrap test has also been done. The bootstrap confident values are marked at
each node of the tree. The distributions of PheRS subunits in various genomes
are obtained by PSI-BLAST searching against NCBI microbial genomes blast
databases at the National Center for Biotechnology Information (NCBI, USA). The
E values≤0.005 are used during the
databases searching.
2 Results
and Discussion
The analyses of the domains of PheRSs and
their distributions in various genomes show that the α subunit in bacteria, archaea or
eukaryote PheRSs contains a CCD with catalytic activity. The main difference
between PheRS α subunits in various genomes
is the N-terminus. For example, the N-terminus is a NTD domain in bacterial
PheRS, but an HTH domain in archaea and eukaryote PheRSs[9]. The β subunit of the bacterial PheRS contains
the several different domains, such as B1, B2, B3/B4, B5, NCD and ABD. The β subunits in archaea and eukaryote are
similar, but lack B2 and ABD compared with that in bacteria. The database
searching against the chloroplast genome[19] shows that the chloroplast PheRS β subunit is very similar to that in
bacteria except the absence of B2. The mtPheRS contains only one α subunit. The subunit and domains in
chloroplast have not been defined before this work yet. Because the core
domains of PheRS in both α and β subunits, the CCD and NCD, are
significant homologous, and the CCD has been suggested being closely related to
the PheRS functions[2,9], the evolutionary relationship of PheRSs in various
living organisms is analyzed further by using the both core domains in PheRS as
the model.
The result shows that the CCDs and NCDs
are clearly located in the different branches of evolutionary tree (Fig.1).
In the CCD subtree, all bacterial CCDs are clustered into a group, but the
archaea and eukaryote CCDs are in another branch. This similar relation can be
also observed in the NCD subtree. Thus, the archaea and eukaryote are closer in
evolution, which has also been confirmed from some rRNA molecular
evidences[20].
Fig.1 N-J tree of CCD and NCD (1000
bootstrap)
A, archaea; B, bacteria; E, eukaryote; M,
mitochondrial PheRS. The integers on each node indicate the bootstrap
confidence value. PORPU－NCD
is a PheRS β
subunit domain encoded in chloroplast of Porphyra purpurea. The OUTGROUP, SYK－DEIRA
and SYK－SULSO,
is the CCDs of Lysyl-tRNA synthetases. Both TREPA and BORBU are spriochaetes
belonging to bacteria, but their α
and β subunit genes are from archaea by
horizontal gene transferring[9].
The tree from both CCDs and NCDs shows
that the bacterial CCDs are firstly separated from others, which suggests that
the CCD in bacteria is an ancient domain. Since the CCD can be observed in all
AARSs in the class II, it can be thought of being very important in function.
Thus, in the evolutionary process from CCD to NCD, the function of activating
Phe and binding ATP has been lost in NCD.
The homologous domain of NCD is similar
to CCD in evolution (Fig.1), and all subunits in PheRS, two α and β
subunits, are necessary for keeping PheRS functions[3]. Thus the evolutionary
trend of CCDs and NCDs can reflect that of α
and β subunits in PheRS.
Most of PheRS contain both α and β
subunits, but the mtPheRS only contains one α
subunit, and only one β subunit can be observed in
the chloroplast PheRS. Although all PheRSs of bacteria, archaea and eukaryote
contain both α and β subunits, but the several domains in the
β subunit of bacterial PheRS
are not observed in archaea and eukaryote PheRSs (Fig.2). The CCD and
NCD, or B1 and B5 domains hold the similar structures, and have been defined as
homologous domains respectively. These domains are resulted from gene
duplications[2].
Fig.2 Evolution of PheRS by domain
losing
HTH, helix-turn-helix domain; CCD,
catalytic core domain; NCD, non-catalytic core domain; NTD, N-terminal domain;
ABD, anticodon binding domain; B1, B1 domain; B2, B2 domain; B3/B4, B3/B4
domain; B5, B5 domain.
Genome database searches show that the
bacteria PheRS genes encoding both α
and β subunits are in one operon.
But they are not adjacent in the archaea. As to spirochaetes PheRS, which have
been suggested being from archaea by the horizontal gene transfer, they are
also not adjacent in sequence[9]. In eukaryote, subunit genes are even located
in different chromosomes. For example, human PheRS α subunit gene located in chromosome 19,
but β gene in chromosome 10;
Schizosaccharomyces pombe PheRSα subunit gene in chromosome 3,
but β subunit gene in chromosome 1.
Thus, the bacterial PheRS genes in one operon encoding both α and β
subunits have been separated in sequence of archaea or been located in
different chromosomes of eukaryote during PheRS gene evolution (Fig.3).
These observations suggest that the gene regulation mechanism in archaea should
be more complicated than that in bacteria, and this mechanism in eukaryote be
the most complicated. Although the regulation mechanism of bacteria PheRS gene
may be the most simple, the subunit architectures or the types of domains in
bacteria are the most complicated in all PheRSs. The simple regulation
mechanism can be considered as the evidence to support the bacterial PheRS
being an ancient type. All the analyses above suggest that the genes encoding
the α and β subunits are adjacent in ancestor gene,
but they have been separated during PheRS gene evolution from bacteria to
eukaryote. On the other hand, in the evolutionary process, some gene fragments
encoding the NTD, ABD and B2 in bacterial PheRS have been lost (Fig.2, 3).
Fig.3 PheRS genes in genomes of
bacteria, archaea and eukaryote
In
bacteria, the genes encoding α
and β subunits are in one operon, but they
have been separated in sequence of archaea, and the genes are located in
different chromosomes of eukaryote.
Analyses on PheRS subunits and domains
show that the main evolutionary event in PheRS α
subunit is gene fusions at the N- or C-terminus of CCD (Fig.2). The
mtPheRS may be the simplification result of two subunits (α2β2)
in bacterial PheRS, but the specific functional regions of bacterial PheRS are
conserved[6,10]. In the β subunit, two domains in
bacterial PheRS, B2 and ABD, have been lost in archaea and eukaryote (Fig.2).
The chloroplast PheRS lacks B2 domain in the β
subunit, and no α subunit can be observed, but
the functions of chloroplast PheRS are not defined yet. So the evolutionary
relationships between chloroplast PheRS and other PheRSs are not discussed in
this work.
In molecular evolutionary process, the
gene duplications and fusions are one of the important pathways for protein
structural and functional evolution[21]. The early study shows that there are
lots of horizontal gene transfer events during AARS evolution, including the
PheRS genes in archaea being transferred to spirochaetes[9], which is also
supported by the evolutionary analyses on B1, B3/B4, B5, CCD and NCD in this
work.
Usually, the gene fragment loss, even a
single nucleotide in the gene loss can result in pseudogene, protein
deactivation or activity decreasing. But the results of this work show that the
main event during PheRS evolution is the gene fragments encoding the several of
PheRS domains being lost, and which do not affect the PheRS functions or
activities. Thus, the evolution of PheRS is very special comparing to that of
other proteins. The partial gene fragments encoding several PheRS domains have
lost in eukaryote, but the functions of eukaryotic PheRS can be conserved. One
of the probable reasons is that the number of enzymes or proteins in eukaryote
is increased in evolution, so that the functions of some enzymes have become
more special and the gene regulation also more accurate, thus the functions of
some domains in bacterial PheRS could have been replaced by other enzymes in
eukaryote. Despite the number of domains in eukaryotic PheRS comparing to those
in bacterial PheRS is decreased, the eukaryotic PheRS can activate Phe and bind
tRNAPhe. The PheRS gene duplication and fusion events should occur before
forming the entire PheRS with functions. Such homologous domains formed by gene
duplications and fusions as CCD and NCD or B1 and B5, can be observed in PheRSs
of all living organisms. However, in the evolutionary process of PheRS from
bacteria to eukaryote, the gene fragments of encoding B2 and ABD have been
lost.
Although the gene duplication, fusion and
horizontal transfer are the frequent events during evolution of many proteins,
the circular permutation[17] and gene fragment or domain loss are also the
important evolutionary events for some proteins. In general, the size of genome
and number of genes are increased during evolution from bacteria to eukaryote,
but the interesting is that the type and number of PheRS domains in eukaryote
are obviously less than those in bacteria. The evolution of PheRS by domain
losing seems to be related to the functional evolution of some AARSs from the
multiple specificities to the single specificity[22]. It is needed to study
further that the evolutionary direction of genomes or species is totally
opposite from that of the genes or proteins.
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____________________________
Received:
June 23, 2003 Accepted:
August 16, 2003
This
work was supported by the grants from Natural Science Foundation of China (No.
30170507, No. 30024004)
* Corresponding
author: Tel, 86-871-5195183; e-mail, [email protected]
Updated
at: 2003-12-16