Recognition of tRNAs with a long variable arm by aminoacyl-tRNA synthetases
In prokaryotic cells three tRNA species, tRNASer, tRNALeu and tRNATyr, possess a long variable arm of 11–20 nucleotides (type 2 tRNA) rather than usual 4 or 5 nucleotides (type 1 tRNA). In this review we have summarized the results of our research on the structural basis for recognition and discrimi...
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Інститут молекулярної біології і генетики НАН України
2013
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irk-123456789-1529972019-06-14T01:28:35Z Recognition of tRNAs with a long variable arm by aminoacyl-tRNA synthetases Tukalo, M.A. Yaremchuk, G.D. Kovalenko, O.P. Kriklivyi, I.A. Gudzera, O.I. Reviews In prokaryotic cells three tRNA species, tRNASer, tRNALeu and tRNATyr, possess a long variable arm of 11–20 nucleotides (type 2 tRNA) rather than usual 4 or 5 nucleotides (type 1 tRNA). In this review we have summarized the results of our research on the structural basis for recognition and discrimination of type 2 tRNAs by Thermus thermophilus seryl-, tyrosyl- and leucyl-tRNA synthetases (SerRS, TyrRS and LeuRS) obtained by X-ray crystallography and chemical probing tRNA in solution. Crystal structures are now known of all three aminoacyl-tRNA synthetases complexed with type 2 tRNAs and the different modes of tRNA recognition represented by these structures will be discussed. In particular, emphasis will be given to the results on recognition of characteristic shape of type 2 tRNAs by cognate synthetases. In tRNASer, tRNATyr and tRNALeu the orientation of the long variable arm with respect to the body of the tRNA is different and is controlled by different packing of the core. In the case of SerRS the N-terminal domain and in the case of TyrRS, the C-terminal domain, bind to the characteristic long variable arm of the cognate RNA, thus recognizing the unique shape of the tRNA. The core of T. thermophilus tRNALeu has several layers of unusual base-pairs, which are revealed by the crystal structure of tRNALeu complexed with T. thermophilus LeuRS and by probing a ligand-free tRNA by specific chemical reagents in solution. In the crystal structure of the LeuRS-tRNALeu complex the unique D-stem structure is recognized by the C-terminal domain of LeuRS and these data are in good agreement with those obtained in solution. LeuRS has canonical class I mode of tRNA recognition, approaching the tRNA acceptor stem from the D-stem and minor groove of the acceptor stem side. SerRS also has canonical class II mode of tRNA recognition and approaches tRNASer from opposite, variable stem and major groove of acceptor stem site. And finally, TyrRS in strong contrast to canonical class I system has class II mode of tRNA recognition. У клітинах евкаріотів тРНК трьох специфічностей – тРНКSer, тРНКLeu і тРНКTyr – мають довгу варіабельну гілку довжиною 11–20 нуклеотидів (2-га група тРНК) на відміну від чотирьох або п’яти нуклеотидів 1-ї групи тРНК. Підсумовано результати наших досліджень структурних основ упізнавання і дискримінації тРНК 2-ї групи серил-, тирозил- і лейцил-тРНК синтетазами з Thermus thermophilus (СерРС, ТирРС і ЛейРС), отриманих методами рентгенівської кристалографії і хімічної модифікації тРНК у розчині. На сьогодні кристалічна структура відома для всіх трьох комплексів аміноацил-тРНК синтетаз з відповідними тРНК 2-ї групи, різні типи впізнавання яких обговорюються в огляді. Зокрема, особливу увагу приділено результатам аналізу впізнавання гомологічними синтетазами характерних рис просторової структури тРНК 2-ї групи. У тРНКSer, тРНКLeu і тРНКTyr орієнтація довгої варіабельної гілки відносно основного тіла тРНК відрізняється і контролюється різною упаковкою корової частини молекули. У разі СерРС N-кінцевий, а в разі ТирРС – C-кінцевий домени зв’язуються з певними структурами довгих варіабельних гілок гомологічних РНК, упізнаючи таким чином унікальну структурну форму тРНК. Корова частина тРНКLeu має кілька шарів незвичайних пар основ, виявлених при вивченні кристалографічної структури комплексу тРНКLeu з ЛейРС із T. thermophilus та при дослідженні вільної тРНК у розчині методом хімічної модифікації з використанням специфічних реагентів. У кристалографічній структурі комплексу ЛейРС-тРНКLeu унікальна будова D-стебла впізнається С-кінцевим доменом ЛейРС і ці дані добре узгоджуються з результатами, отриманими в розчині. ЛейРС притаманний канонічний для синтетаз І структурного класу тип упізнавання тРНК – з боку D-стебла і малої борозенки акцепторного стебла. Для СерРС також характерний канонічний для синтетаз ІІ структурного класу тип упізнавання тРНК – з протилежного боку, тобто з боку варіабельного стебла і великої борозенки акцепторного стебла. І, нарешті, ТирРС на відміну від канонічного для ферментів І класу типу має тип упізнавання тРНК, властивий синтетазам ІІ класу. В клетках эукариотов тРНК трех специфичностей – тРНКSer, тРНКLeu и тРНКTyr – имеют длинную вариабельную ветку длиной 11–20 нуклеотидов (2-я группа тРНК) в отличие от четырех или пяти нуклеотидов 1-й группы тРНК. Суммированы результаты наших исследований структурных основ узнавания и дискриминации тРНК 2-й группы серил-, тирозил- и лейцил-тРНК синтетазами из Thermus thermophilus (СерРС, ТирРС и ЛейРС), полученные методами рентгеновской кристаллографии и химической модификации тРНК в растворе. На сегодня кристаллическая структура известна для всех трех комплексов аминоацил-тРНК синтетаз с соответствующими тРНК 2-й группы, разные типы узнавания которых обсуждаются в обзоре. В частности, особенное внимание уделено результатам анализа узнавания гомологичными синтетазами характерных черт пространственной структуры тРНК 2-й группы. У тРНКSer, тРНКLeu и тРНКTyr ориентация длинной вариабельной ветви относительно основного тела тРНК отличается и контролируется разной упаковкой коровой части молекулы. В случае СерРС N-концевой, а в случае ТирРС – C-концевой домены связываются с определенными структурами длинных вариабельных веток гомологичных РНК, узнавая тем самым уникальную структурную форму тРНК. Коровая часть тРНКLeu имеет несколько слоев необычных пар оснований, выявленных при изучении кристаллографической структуры комплекса тРНКLeu с ЛейРС из T. thermophilus и при исследовании свободной тРНК в растворе методом химической модификации с использованием специфических реагентов. В кристаллографической структуре комплекса ЛейРС–тРНКLeu уникальное строение D-стебля узнается С-концевым доменом ЛейРС и эти данные хорошо согласуются с результатами, полученными в растворе. ЛейРС свойствен канонический для синтетаз І структурного класса тип узнавания тРНК – со стороны D-стебля и малой бороздки акцепторного стебля. Для СерРС также характерный канонический для синтетаз ІІ структурного класса тип узнавания тРНК – с противоположной стороны, т. е. со стороны вариабельного стебля и большой бороздки акцепторного стебля. И, наконец, ТирРС в отличие от канонического для ферментов І класса типа имеет тип узнавания тРНК, присущий синтетазам ІІ класса. 2013 Article Recognition of tRNAs with a long variable arm by aminoacyl-tRNA synthetases / M.A. Tukalo, G.D. Yaremchuk, O.P. Kovalenko, I.A. Kriklivyi, O.I. Gudzera // Вiopolymers and Cell. — 2013. — Т. 29, №. 4. — С. 311-323. — Бібліогр.: 81 назв. — англ. 0233-7657 DOI: http://dx.doi.org/10.7124/bc.000825 http://dspace.nbuv.gov.ua/handle/123456789/152997 577.217 en Вiopolymers and Cell Інститут молекулярної біології і генетики НАН України |
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Reviews Reviews Tukalo, M.A. Yaremchuk, G.D. Kovalenko, O.P. Kriklivyi, I.A. Gudzera, O.I. Recognition of tRNAs with a long variable arm by aminoacyl-tRNA synthetases Вiopolymers and Cell |
| description |
In prokaryotic cells three tRNA species, tRNASer, tRNALeu and tRNATyr, possess a long variable arm of 11–20 nucleotides (type 2 tRNA) rather than usual 4 or 5 nucleotides (type 1 tRNA). In this review we have summarized the results of our research on the structural basis for recognition and discrimination of type 2 tRNAs by Thermus thermophilus seryl-, tyrosyl- and leucyl-tRNA synthetases (SerRS, TyrRS and LeuRS) obtained by X-ray crystallography and chemical probing tRNA in solution. Crystal structures are now known of all three aminoacyl-tRNA synthetases complexed with type 2 tRNAs and the different modes of tRNA recognition represented by these structures will be discussed. In particular, emphasis will be given to the results on recognition of characteristic shape of type 2 tRNAs by cognate synthetases. In tRNASer, tRNATyr and tRNALeu the orientation of the long variable arm with respect to the body of the tRNA is different and is controlled by different packing of the core. In the case of SerRS the N-terminal domain and in the case of TyrRS, the C-terminal domain, bind to the characteristic long variable arm of the cognate RNA, thus recognizing the unique shape of the tRNA. The core of T. thermophilus tRNALeu has several layers of unusual base-pairs, which are revealed by the crystal structure of tRNALeu complexed with T. thermophilus LeuRS and by probing a ligand-free tRNA by specific chemical reagents in solution. In the crystal structure of the LeuRS-tRNALeu complex the unique D-stem structure is recognized by the C-terminal domain of LeuRS and these data are in good agreement with those obtained in solution. LeuRS has canonical class I mode of tRNA recognition, approaching the tRNA acceptor stem from the D-stem and minor groove of the acceptor stem side. SerRS also has canonical class II mode of tRNA recognition and approaches tRNASer from opposite, variable stem and major groove of acceptor stem site. And finally, TyrRS in strong contrast to canonical class I system has class II mode of tRNA recognition. |
| format |
Article |
| author |
Tukalo, M.A. Yaremchuk, G.D. Kovalenko, O.P. Kriklivyi, I.A. Gudzera, O.I. |
| author_facet |
Tukalo, M.A. Yaremchuk, G.D. Kovalenko, O.P. Kriklivyi, I.A. Gudzera, O.I. |
| author_sort |
Tukalo, M.A. |
| title |
Recognition of tRNAs with a long variable arm by aminoacyl-tRNA synthetases |
| title_short |
Recognition of tRNAs with a long variable arm by aminoacyl-tRNA synthetases |
| title_full |
Recognition of tRNAs with a long variable arm by aminoacyl-tRNA synthetases |
| title_fullStr |
Recognition of tRNAs with a long variable arm by aminoacyl-tRNA synthetases |
| title_full_unstemmed |
Recognition of tRNAs with a long variable arm by aminoacyl-tRNA synthetases |
| title_sort |
recognition of trnas with a long variable arm by aminoacyl-trna synthetases |
| publisher |
Інститут молекулярної біології і генетики НАН України |
| publishDate |
2013 |
| topic_facet |
Reviews |
| url |
http://dspace.nbuv.gov.ua/handle/123456789/152997 |
| citation_txt |
Recognition of tRNAs with a long variable arm by aminoacyl-tRNA synthetases / M.A. Tukalo, G.D. Yaremchuk, O.P. Kovalenko, I.A. Kriklivyi, O.I. Gudzera // Вiopolymers and Cell. — 2013. — Т. 29, №. 4. — С. 311-323. — Бібліогр.: 81 назв. — англ. |
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Вiopolymers and Cell |
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UDC 577.217
Recognition of tRNAs with a long variable arm
by aminoacyl-tRNA synthetases
M. A.Tukalo, G. D. Yaremchuk, O. P. Kovalenko, I. A. Kriklivyi, O. I. Gudzera
Institute of Molecular Biology and Genetics, NAS of Ukraine and State Key Laboratory of Molecular and Cellular Biology
150, Akademika Zabolotnogo Str., Kyiv, Ukraine, 03680
mtukalo@imbg.org.ua
In prokaryotic cells three tRNA species, tRNASer, tRNALeu and tRNATyr, possess a long variable arm of 11–20 nuc-
leotides (type 2 tRNA) rather than usual 4 or 5 nucleotides (type 1 tRNA). In this review we have summarized the
results of our research on the structural basis for recognition and discrimination of type 2 tRNAs by Thermus
thermophilus seryl-, tyrosyl- and leucyl-tRNA synthetases (SerRS, TyrRS and LeuRS) obtained by X-ray crystal-
lography and chemical probing tRNA in solution. Crystal structures are now known of all three aminoacyl-tRNA
synthetases complexed with type 2 tRNAs and the different modes of tRNA recognition represented by these struc-
tures will be discussed. In particular, emphasis will be given to the results on recognition of characteristic shape
of type 2 tRNAs by cognate synthetases. In tRNASer, tRNATyr and tRNALeu the orientation of the long variable arm with
respect to the body of the tRNA is different and is controlled by different packing of the core. In the case of SerRS the
N-terminal domain and in the case of TyrRS, the C-terminal domain, bind to the characteristic long variable arm
of the cognate RNA, thus recognizing the unique shape of the tRNA. The core of T. thermophilus tRNALeu has several
layers of unusual base-pairs, which are revealed by the crystal structure of tRNALeu complexed with T. thermo-
philus LeuRS and by probing a ligand-free tRNA by specific chemical reagents in solution. In the crystal structure
of the LeuRS-tRNALeu complex the unique D-stem structure is recognized by the C-terminal domain of LeuRS and
these data are in good agreement with those obtained in solution. LeuRS has canonical class I mode of tRNA re-
cognition, approaching the tRNA acceptor stem from the D-stem and minor groove of the acceptor stem side.
SerRS also has canonical class II mode of tRNA recognition and approaches tRNASer from opposite, variable stem
and major groove of acceptor stem site. And finally, TyrRS in strong contrast to canonical class I system has class
II mode of tRNA recognition.
Keywords: type 2 tRNA, long variable arm, aminoacyl-tRNA synthetase, tRNA recognition, aminoacyl-tRNA syn-
thetase complexes.
Introduction. The fidelity of translation of the informa-
tion stored in nucleic acids into proteins is essential for
all living cells. The algorithm of translation of the gene-
tic code is established in the process of aminoacyl-
tRNA formation. Therefore, the fidelity of protein syn-
thesis depends to a large extent on a high specificity with
which aminoacyl-tRNA synthetases (aaRSs) charge their
cognate tRNAs with a correct amino acid. aaRSs cataly-
se the aminoacylation reaction in two steps, firstly, the
activation of amino acid using ATP to form the enzyme
bound aminoacyl-adenylate, and secondly, the transfer
of amino acid to the 2' or 3' hydroxyl of the 3' terminal
tRNA ribose. The ester linkage of the aminoacyl-tRNA
provides much of the energy required for peptide bond
formation on the ribosome. The accuracy of the amino-
acylation reaction is based on correct selection and recog-
nition by aaRSs their cognate tRNAs. The selection of
tRNA is a result of productive interaction between tRNA
and its corresponding aaRS mediated by the recogni-
tion-defined elements (identity determinants) and non-
productive interaction between this tRNA and 19 other
aaRS species mediated by the rejection-defined elements
(anti-identity determinants). Sum of identity and anti-
identity determinants forms the identity set of a given
tRNA system [1]. Given that tRNAs apparently have si-
milar secondary and tertiary structures, the question ari-
311
ISSN 0233–7657. Biopolymers and Cell. 2013. Vol. 29. N 4. P. 311–323 doi: 10.7124/bc.000825
Ó Institute of Molecular Biology and Genetics, NAS of Ukraine, 2013
312
ses about the structural basis for the specific recogni-
tion between aaRSs and tRNAs.
Evolution has resulted in two completely distinct
structural solutions of the aminoacylation problem. The
amino acid sequence analysis [2] and X-ray crystallo-
graphy [3] have shown that the aaRSs are partitioned in-
to two exclusive classes. The catalytic domain of class I
enzymes contains the well-known Rossmann fold as a
framework whereas that of class II enzymes is based
around a different anti-parallel fold. The catalytic do-
main of each class includes short sequence motifs, HIGH
and KMSK in class I and motifs 1, 2 and 3 in class II [2,
4]. A functional distinction between the two classes is
that class I synthetases charge the 2' hydroxyl and class
II synthetases (except for phenylalanyl-tRNA syntheta-
se) charge the 3' hydroxyl of the ribose of A76 [2, 5, 6].
Class II aaRSs are almost all functional dimers whereas
most class I enzymes are monomers except for tyrosyl-
tRNA synthetase and tryptophanyl-tRNA synthetase.
Now the crystal structures are available for all 20 cano-
nical aaRSs [4] as well as for the non-canonical pyrroly-
syl-tRNA synthetase and phosphoseryl-tRNA synthe-
tase [7, 8]. In both classes, the tRNA binding ability of
aaRS is augmented by RNA-binding modules which,
because of their greater structural variability, have pre-
sumably been added to the catalytic domain at a later
stage in evolution [9–11]. Classification of tRNAs ac-
cording to the length of their extra (variable) arm, leads
to dividing them into two classes: those with a short
variable arm of 4–5 nucleotide (type 1) and those with a
long variable arm, more than 10 nucleotides (type 2)
[12]. Prokaryotic tRNASer, tRNALeu and tRNATyr, are clas-
sified as the type 2 tRNAs. The long variable arm of
tRNASer and tRNALeu shows variation in both length and
sequence within the isoacceptor tRNAs. Thus, the long-
standing question was how the discrimination between
the type 2 tRNAs occurs and what is a role of the long
variable arm in the process. In this article we would like
to review what is known on the structural basis for re-
cognition and discrimination of the type 2 tRNAs by
Thermus thermophilus seryl-, leucyl- and tyrosyl-
tRNA synthetases.
The serine system. The serine system has a num-
ber of interesting features. Firstly, the bacteria Escheri-
chia coli and T. thermophilus possess five isoaccepting
tRNASers in order to cope with the six codons for serine
which correspond to distinct codon classes. In addition
tRNASec, the selenocysteine-specific tRNA species,
which has an opal stop anticodon, is also specifically
charged by seryl-tRNA synthetase (SerRS). As a result,
there is no consistency in the anticodon bases of tRNASers,
and their anticodons are apparently not involved in re-
cognition by SerRS. Another special feature is that pro-
karyotic tRNASer, as indicated above, has a long variab-
le arm and refers to type 2 tRNAs. In fact, the serine
aminoacylation system is unique in the sense that it is
the only system where class II aaRS recognizes type 2
tRNA. Therefore the main question has arisen in the
study of this system: what are the common features of
tRNASers that are recognized by synthetase? Clearly on-
ly a crystal structure of the complex can give a compre-
hensive picture of the recognition of SerRS by its cog-
nate tRNAs, but of course one expects it to be consis-
tent with the biochemical and mutagenesis results. The-
refore, we have studied the recognition of tRNASer by the
cognate synthetase using two approaches: X-ray crys-
tallography and chemical and enzymatic footprinting of
tRNA in solution. The extreme thermophilic bacterium
T. thermophilus has been chosen as a source for the iso-
lation of tRNA and SerRS to investigate their structures
and functions by biochemical and structural methods.
The proteins and nucleic acids from this organism are
very stable and crystallized easier than those from the
methophilic organisms. At the beginning the scheme of
simultaneous isolation of ribosome, tRNA, three elon-
gation factors, several aaRSs was developed, and purifi-
cation of SerRS from T. thermophilus HB27 was descri-
bed [13, 14]. To study the mechanism of amino acid ac-
tivation and specific recognition of cognate tRNA by T.
thermophilus SerRS (SerRSTT) we tried to crystallize
this enzyme alone and in a complex with substrates. The
crystals of SerRSTT, obtained using mixed solutions of
ammonium sulphate and 2-methyl-2,4-pentanediol, we-
re very stable and diffracted to at least 2 C [15]. The
enzyme from two strains of T. thermophilus HB8 and
HB27 has been cloned and sequenced (Tukalo et al. un-
published results). SerRSTTs from both strains have
421 residues per subunit, but differ in six positions, and
have an overall sequence identity with E. coli enzyme
of 37 %. The three-dimension structure of the HB27
SerRSTT has been determined and refined at 2.5 C
resolution [16]. The structures of T. thermophilus and
TUKALO M. A. ET AL.
E. coli SerRSs are very similar [3, 16] and made up a cen-
tral globular domain with a long coiled-coil extension
(Fig. 1, see inset). The globular, catalytical domain con-
sists of eight-stranded b-sheet, of which seven strands are
antiparallel, packed onto two a-helices. One of these a-
helices interacts with the corresponding one in the other
molecules of the dimer, forming the layer of four a-
helices. The antiparallel-fold is characteristic of class II
aaRSs and has been found in all synthetases from this
class [4]. N-terminal domain as coiled-coil extension of
the protein, a remarkable feature of SerRS, stretches
about 60 C in the solution and its role in tRNA recog-
nition will be discussed below.
The first step of the overall aminoacylation reaction
catalyzed by SerRS is activation of serine by Mg2+-
ATP leading to a stable enzyme-bound intermediate,
seryl-adenylate (Ser-AMP). The first information on
ATP-binding mode in the active site of class II synthe-
tase was obtained from the structure of SerRSTT in
complex with ATP-analogue [17, 18]. In spite of the me-
dium resolution of data, it was clear that ATP is bound
in a part of the active site pocket formed by the class II
conserved motifs 2 and 3. Later, a series of crystal struc-
tures at 2.3–3 C resolution of complexes involving Ser
RSTT, ATP, Mn2+ or Mg2+ and Ser-AMP (natural and
analogues) have been determined, which provide the
structural basis for explanation of the specificity and
mechanism of serine activation [19, 20]. In the presen-
ce of a divalent cation (Mg2+ or Mn2+) the ATP mole-
cule is found in an unusual U-shaped conformation in
which the b- and g-phosphates are bent back into an
arginine-rich pocket (comprising Arg271, Arg344 and
Arg386) towards the purine ring rather than extending
away from it. A similar conformation of ATP has been
observed in the yeast AspRS-tRNAAsp-ATP ternary
complex [21] and later in other class II synthetases [22].
This compact conformation of ATP is unique for class
II synthetases as in the active sites of class I enzymes
ATP adopts an extended conformation [23]. The super-
position of the enzyme-bound ATP and Ser-AMP struc-
tures provides strong support for an in-line displace-
ment mechanism of the serine activation [20]. The bent
conformation of ATP and the position of serine are con-
sistent with nucleophilic attack of the serine carboxyl
group on the a-phosphate leading to the release of in-
organic pyrophosphate.
Structure of SerRSTT-tRNASer complex. Since
the crystallization of the synthetase-tRNA complexes
might require relatively large quantities of pure tRNA
species we had to develop the method for preparative
isolation of tRNA. Separation of biologically active pu-
re and specific tRNAs is difficult due to the overall simi-
larity in tertiary structure of different RNA molecules
on the one hand and their heterogeneity on the other
hand. This heterogeneity is conditioned by both degene-
ration of the genetic code (6 anticodons for serine) and
the degree of maturation (post-transcriptional modifica-
tion of nucleotide bases and their transformation into
minor components). Therefore, we have developed a
method for tRNA isolation from T. thermophilus cells
which combines different techniques: chromatography
on benzoyl-DEAE-cellulose and HPLC on anion-ex-
change and reverse phase columns [24]. This methodo-
logical approach allowed us to obtain two highly purifi-
ed isoaccepting tRNASers, sufficient for the study of
their primary structures and for the crystallization trials
of tRNASer complexes with cognate SerRSTT. The nuc-
leotide sequence of two serine isoacceptor tRNAs from
T. thermophilus, containing different anticodons has be-
en studied by the ultramicrospectrophotometrical me-
thod and rapid gel sequencing procedure [25]. Compa-
rison of the sequences of tRNA1
Ser and tRNA2
Ser shows
that the acceptor stem and T-stem are identical in both
RNAs, and only three changes have been found in the
structures of D-stems. The most significant differences
were found in the anticodon stems and variable arms of
two tRNASers. These data are in good agreement with
the results of the study on the identity determinants in
E. coli tRNASer [26, 27]. The authors have shown that
the anticodon nucleotides are not involved in specific
recognition of tRNASer. However, the elements of ter-
tiary structure play a critical role. For example, it was
established that the length and orientation of the variab-
le arm of tRNASer are more important for aminoacy-
lation by SerRS than its sequence. In view of the fact
that SerRS selectively recognizes tRNASer on the basis
of its characteristic tertiary structure rather than the spe-
cific nucleotides, an important step in investigation of
the recognition mechanism for serine system was to de-
termine the characteristics of tRNASers spacial organi-
zation and topography of the complex with cognate
synthetase. The most direct and informative method for
313
RECOGNITION OF tRNAs WITH A LONG VARIABLE ARM BY AMINOACYL-tRNA SYNTHETASES
ISSN 0233-7657. Biopolymers and Cell. 2013. Vol. 29. N 4
Figures to article by M. A. Tukalo et al.
Le ucyl-tRNA
synthetase
Class I monomer with
editi ng activity
Se ryl-tR NA
synthe tase
C lass II dimer
Tyrosyl-tRN A synthe tase
Class I d imer
Catalytic domain
C-term inal
dom ain
N-terminal
domain
Editing
domain
Fig. 1. Structures of T. thermophilus aminoacyl-tRNA synthetases (SerRSTT, TyrRSTT and LeuRSTT), which recognize tRNAs with a long
variable arm
TyrRSTT-tRNA
Tyr
Se rRSTT-tRN A
Ser
LeuRSTT-tRNA
Leu
C-terminal dom ain
N-terminal dom ain
C-te rminal domain
Fig. 2. Complexes of aminoacyl-tRNA synthetases recognizing long variable arm tRNAs
achieving this goal is the X-ray analysis. However, it
should be noted that the crystallization conditions, on
the one hand, and the interaction with the enzyme, on
the other, may lead to changes in the spatial organi-
zation of tRNA. Therefore, extremely important is the
study of tRNASer in the free state and in the complex
with protein in solution under conditions close to phy-
siological.
In order to study the structure of tRNASer and the
molecular basis of its recognition by the synthetase, we
have obtained four crystal forms of the complex betwe-
en SerRS and its cognate tRNA from the T. thermophi-
lus [28, 29]. Of these, two (denoted by Form III and Form
IV) have tetragonal crystal forms and both diffract to
about 6 C resolution [28]. Form III crystals diffract to
about 3.5 C and contain two synthetase dimmers with
two tRNA molecules each. On the other hand, Form IV
crystals, which diffract to 2.8 C resolution, contain on-
ly one tRNA molecule bound to the synthetase dimer
[28]. Using Form IV crystals the structure of T. thermo-
philus SerRS complexed with tRNASer molecule was
solved at 2.9 C resolution [18]. A ribbon diagram of the
structure of SerRSTT-tRNASer complex is shown in Fig. 2
(see inset). The main conclusion can be summarized as
follows: (1) the tRNA binds across the two subunits of
the dimer; (2) the anticodon loop is not in contact with
the synthetase; (3) upon tRNA binding the coiled-coil
domain of the synthetase is stabilized in a particular ori-
entation and curves between the TYC loop and the long
variable arm of the tRNA; (4) the synthetase makes se-
veral backbone contacts but few base-specific interac-
tions; (5) the contacts with the tRNA long variable arm
backbone extend out to the sixth base pair, explaining the
need for a minimum length of the arm, but allowing lon-
ger arms (as, for instance, in tRNASec) to be accommo-
dated; and (6) the bases 20a and 20b inserted into the D
loop in the tRNASer both play novel roles in the core for-
mation of the tRNA. In particular, the base of G20b is
stacked against the first base pair of the long variable arm
and thus determines the orientation of the variable arm.
These crystallographic results show that both dis-
tinctive features of the serine system, the synthetase coi-
led-coil N-terminal domain and the tRNASer long vari-
able arm, play the major role in the mutual recognition
of these two macromolecules. Furthermore, this recog-
nition is based on the shape rather than on the specific
nucleotide sequence, which fits well with the biochemi-
cal data [26, 27]. These data are also in good agreement
with those obtained by us in the solution, where SerRS
TT protected from alkylation by ethylnitrosourea the
phosphates residues located in three regions of tRNASer:
at the variable arm (phosphates 46–47c, 47o, 47p), the T
stem-loop (P50, P53, P54) and the acceptor stem (P67-
P69) [30] (Fig. 3).
The structure of complex provides also the first de-
tailed description of the architecture of a type 2 tRNA.
314
TUKALO M. A. ET AL.
tRNASer tRNALeu tRNATyr
19
G
C
C
G
A
C
C
A
C
G
G
G
G
G
C
C
C
C
U
GA
GCG
G
CGCA
AU
U
Gm
G
U A G G
C
A
U
G
A
G
U
A
C
Y
U
U
C A
G
m1G
G
U
G
C
C
C
U
G
C
G
G
CGC
GCG
C
G
C
G
U
G
m1A
T Y
C
A
G
C A
G
A
1
15
20à
72
48
35
G
G
C
C
G
C
C
A
A
G
A
G
G
U
C
U
C
C
U
GA
CCCG
GGGA
GU
Gm
G
C
D G A A
C
A
C
G
A
G
U
G
C
Y
C
U
G G A
ms2i6A
A
U
A
G
G
G
G
G
G
C
U
C
C
C
U
C
C
CGC
GCG
C
G
C
G
U A
m1A
s2T Y
C
G
C
U
A
A
A
U
1 72
20b
15
19
48
35
19
G
G
C
C
A
C
C
A
G
C
A
G
G
C
G
U
C
C
s4U
GA
CCC
G
GGGA
GC
Gm
G
C
C A A G
A
C
G
G
U
U
G
C
C
A
C
U
G Y
A
A*
A
U
G
G
C
C
U
C
C
G
U
AGC
UCG
C
G
C
G
U
A
m1A
T Y
C
G
G
U A
U
1
15
20b
72
48
35
Fig. 3. Cleverleaf structures of T. thermophilus tRNASer, tRNALeu and tRNATyr with position of phosphates protected by cognate synthetase from
alkylation with ethylnitrosourea
The tertiary interactions in the core of the tRNASer dif-
fer from those observed previously for a type 1 tRNAPhe.
The core of the latter includes four parallel stacked pla-
nes; three of them consist of a base triplet. The equiva-
lent region in tRNASer (Fig. 4, A) is reorganized to ac-
commodate the insertion of 20a-20b from the D loop
and can be described by the notation D20a-[G15-C481,
A21-[U8-A14], G9-[A22-G13], and [G23-C12]. In or-
der to clarify the question, whether the resulting crystal
structure of the tRNASer corresponds to that under phy-
siological conditions, the elements of tertiary structure
of tRNASer from T. thermophilus were studied by the
methods of chemical modification in solution [31]. For
this, ethylnitrosourea has been used to modify the phos-
phate residues; guanosines and cytidines were modified
by dimethylsulphate, and adenosines by diethylpyro-
carbonate [32, 33]. Summarizing the results (which
will also be discussed below), we can conclude that the
same interactions, that define an architecture of the so-
lution structure of tRNASer, exist in the crystal form.
Thus, the crystal conformation of tRNASer in complex
with the synthetase is very close to that for the free
tRNA in solution.
In the original binary SerRSTT-tRNASer complex
crystal structure [18], the end of the acceptor stem of
tRNA was not ordered in the active site. However the
ternary complex of SerRSTT-tRNASer with a non-hyd-
rolysable seryl-adenylate analogue [34] shows a much
better ordering of the active site, and the interactions in-
side the acceptor stem mainly made by the motif 2 loop
of SerRS, are visible. Interestingly, in the absence of
tRNA, but in the presence of ATP or Ser-AMP [20, 34]
the motif 2 loop adapts a quite different conformation.
Upon tRNA binding a number of motif 2 residues pre-
viously found interacting with ATP or adenylate now
switch to participate in tRNA recognition. These results
combined with those, obtained previously [18], pro-
vide strong evidence that the functional binding of
tRNASer to SerRS occurs in at least two distinct steps:
firstly the initial recognition and docking which depend
largely on interaction of the N-terminal domain with a
long variable arm and secondly, the correct positioning
of the 3' end of the tRNA in the active site. The latter de-
pends critically on a conformation switch of the motif 2
loop after the adenylate formation.
The leucine system. A subfamily of class 1a aaRSs,
leucyl-, isoleucyl- and valyl-tRNA synthetases (LeuRS,
IleRS and ValRS, respectively) are particularly closely
related and probably evolved from a common ancestor.
The three enzymes are large monomers (» 100 kDa) and
contain an unusually large insertion often, called CP1
(connective polypeptide 1), [35] into the class 1 Ros-
smann-fold catalytic domain. Based on the structural
data of IleRS and on the mutagenesis experiments, a
putative hydrolytic editing active site was identified wi-
thin the CP1 domain [36–38]. LeuRS was the least stu-
315
RECOGNITION OF tRNAs WITH A LONG VARIABLE ARM BY AMINOACYL-tRNA SYNTHETASES
D-T
stem-loop
Acceptor
stem
Anticodon
Variable
arm
7
8
9
101112131415
20
a
20b
22
21
23 24 25 26
44
45
4647p
47q
4849
59
65
66
D-T
stem-loop
Acceptor
stem
Anticodon
Variable
arm
7
8
9
101112131415
20
a
47j
22
21
23 24 25 26
44
45
4647h
47i
4849
59
65
66
SertRNA T. thermophilus
LeutRNA T. thermophilus
A B
Fig. 4. Comparison of the core structure of T. thermophilus tRNASer (A) and tRNALeu (B)
died of this triad of editing enzymes. We have found
that T. thermophilus LeuRS is capable of editing homo-
cysteine, norvaline and norleucine, and have studied the
structure of editing active site of enzyme which binds
the distinct pre- and post-transfer editing substrates [39].
The second interesting feature of LeuRS is that this syn-
thetase recognizes tRNA with a long variable arm. How-
ever, unlike the other two such enzymes, SerRS and
TyrRS, the bacterial LeuRS surprisingly does not gene-
rally use the long variable arm of tRNALeu as an identity
element [40]. Neither does LeuRS use the anticodon
triplet [40]. Obviously, the complexity of LeuRS needs
a deep on complexes of the enzyme with various subst-
rates in combination with the mutagenesis and bioche-
mical results for the understanding of the molecular me-
chanisms of tRNA recognition and catalysis. For this,
the gene of T. thermophilus HB27 LeuRS has been clo-
ned and sequenced [41]. The open reading frame enco-
des a polypeptide chain of 878 amino acid residues in
length (molecular mass 101 kDa). Then the T. thermo-
philus LeuRS (LeuRSTT) was expressed in E. coli cells
by cloning the corresponding gene into pET29b vector
[41]. LeuRS from T. thermophilus was the first crystal-
lized LeuRS, for which the crystal structure was descri-
bed [ 42–44]. A high quality crystal form of the native
enzyme and its complexes with leucine and leucyl-ade-
nylate analogue diffracts to 1.9 C resolution and con-
tains one monomer in asymmetric unit [42]. The over-
all architecture of LeuRSTT is similar to that of IleRS,
except for the fact that the editing domain is inserted at
the different position in the primary structure [43] (Fig.
1, see inset). This feature is unique to prokaryotic LeuRS,
as well as the presence of novel additional flexibly in-
serted domain (designated leucyl-specific domain).
Comparison of the native enzyme and complexes with
leucine and a leucyl-adenylate analogue shows that the
binding of the adenosine moiety of leucyl-adenylate
causes significant conformational changes in the active
site required for amino acid activation and tight binding
of the adenylate. These changes propagated to more
distant regions of the enzyme, leading to a significantly
more ordered structure ready for the subsequent amino-
acylation and/or editing steps.
Structure of LeuRSTT-tRNALeu complex. To ob-
tain further insight into tRNA recognition by LeuRS
and into mechanisms of aminoacylation and editing, we
have determined the crystal structure of LeuRSTT com-
plexed with the tRNALeu transcript and leucine [45].
LeuRSTT-tRNALeu co-crystals have been obtained only
with the particular T7 transcript of T. thermophilus
tRNALeu(CAG), which normally has 87 nucleotides, but
in this work had a two base-pair deletion in the long va-
riable arm stem. Such a truncation of the long variable
arm does not affect the leucylation activity [40]. The
crystal structure shows (Fig. 2, see inset) that the tRNA
acceptor end enters the editing site in a fashion complete-
ly compatible with our previous structure of LeuRSTT
complexed with a post-transfer editing substrate analo-
gue, 2'-(L-norvalyl)amino-2'-deoxyadenosine (Nva2AA)
[39]. Furthermore, the structure at the improved resolu-
tion of 2.9 C, obtained by soaking the co-crystals with
Nva2AA, shows that this compound, bound in the editing
active site, displaces the 3' end of the tRNA. The anti-
codon stem of tRNALeu is packed against the helical do-
main characteristic of class Ia. Neither the long variable
arm, nor the anticodon loop, is in contact with the synthe-
tase. The C-terminal domain of the synthetase, hitherto
unseen in crystal structures of uncomplexed LeuRSTT,
for the first time could be traced as a compact alpha-
beta domain. To clarify the function of the C-terminal
domain in LeuRSTT, a truncation mutant LeuRSTTdC
with the C-terminal 60 residues deleted was expressed
and purified. The C-terminal truncation mutant shows
152-fold reduction (the kcat value) of the aminoacyla-
tion activity [46], indicating that the C-terminal domain
is strictly required for the tRNA charging activity of Leu
RSTT. This has also been demonstrated for the E. coli
and Pyroccocus horikoshii LeuRSs (LeuRSHP) [47,
48]. Analysis of the tRNALeu interaction with LeuRSTT
shows, that there are non-specific backbone contacts in
the regions of nucleotides 12–13, 22–26, and 42, invol-
ving residues in the regions 667–686 and 749–760. The
C-terminal domain makes more extensive contacts to
the base pair G19-C56 including some base specific in-
teractions. All these contacts agree reasonably well with
biochemical footprinting studies. The regions of the wild
type tRNALeu (the long variable arm has four base pairs)
protected by LeuRSTT against alkylation with ethylnit-
rosourea are located at the 5' side of D-stem (phosphates
P14 and P15), at the 3' side of the D-stem (phosphates
P24 and P25), at the 3' side of the anticodon stem (P38-
40) and at the variable arm (P47i). The anticodon loop
316
TUKALO M. A. ET AL.
ISSN 0233-7657. Biopolymers and Cell. 2013. Vol. 29. N 4
Figures to article by M. A. Tukalo et al.
tRNATyr
tRNASer
tRNALeu
Ac ceptor
ste m
Long variable
arm
50
o
D
C
B
A
tRNATyr(G YA)
tRNALeu(CAG )
tRNASer(GG A)
Fig. 6. Structural comparison of the long-variable arm tRNAs: T. thermophilus tRNASer (A), tRNATyr (B) and tRNALeu (C). The structural alignment
of tRNATyr , tRNASer and tRNALeu (D)
Ser RS
Class II synthetase
Class II mode l tRN A rec ognition
Tyr RS
Class I synthetase
Class II mode l tRN A rec ognition
GlnR S
Class I synthe tase
Class I model tRNA r ecognition
Fig. 5. A different modes of tRNA recognition by aminoacyl-tRNA synthetases. Adopted from [60]
and practically entire variable arm of tRNALeu are expo-
sed for chemical modification [49], consistent with their
not being in contact with the synthetase (Fig. 3). The
fact of the protection of P47i in solution which is not in
contact with the enzyme in the crystal may be explained
by using truncated form of tRNALeu for crystallization of
the complex. In the recently reported crystal structures
of E. coli LeuRS-tRNALeu complexes in the aminoacyla-
tion or edititing conformations, the long variable arm is
the same as in the wild type tRNALeu and contacts the C-
terminal domain via the variable arm phosphates P47i (in
the aminoacylation conformation) or P47f-P47i (in the
editing conformation) [50]. Regardless of the details of
the interaction with the long variable arm, LeuRSTT has
a canonical class I mode of tRNA recognition approa-
ching the tRNA acceptor stem from the D-stem and mi-
nor groove of the acceptor stem side. Ser RSTT also has
a canonical class II mode of tRNA recognition and ap-
proaches tRNASer from opposite, i. e. the variable stem
and major groove of the acceptor stem side.
For the first time, the study on the structure of the
LeuRS-tRNALeu complex has shown a unique spatial
structure of bacterial tRNALeu. There are two related as-
pects: firstly, the structure of the tRNA core and, second-
ly, the orientation of the long variable arm. The core
structure of T. thermophilus tRNALeu has several layers
of unusual base-pairs, which are revealed by both the
crystal structure and probing a ligand-free tRNALeu with
the specific chemical reagents in solution [45, 51] (Fig.
4, B). The orientation of the long variable arm in tRNALeu
is determined by the single unpaired base G48.1 which
stacks against the first base-pair of the variable arm
stem, G45:C48.2 (Fig. 6, C, see inset).
Unfortunately we failed to crystallize the LeuRSTT-
tRNALeu complex in aminoacylation conformation. But
this complex was obtained for archaea LeuRSHP [52].
Due to the high homology of key amino acid residues in
the catalytic domain of LeuRS, including LeuRSTT and
LeuRSPH, we proposed a model aminoacylation con-
formation of the 3'-end of tRNALeu, which is located in
the center of the catalytical domain of T. thermopilus
LeuRS [45]. A key observation from this modelling is
that the zinc-containing ZN-1 domain (residues 154–
189 in LeuRSTT) must be mobile during the amino-
acylation reaction. This model also shows a crucial role
of the conserved motif 418-RLRDWLISRQRYW-431
in positioning the 3' end of the tRNA, in particular, by
making specific main-chain hydrogen bonds to the dis-
criminator base A73. Basic residues Arg418, Arg420
and Arg426 are also probably important for tRNA bin-
ding. Interestingly that recently solved crystal structure
of E. coli LeuRS-tRNALeu complex in the aminoacyla-
tion conformation has confirmed the basic assumptions
for aminoacylation model of LeuRSTT [50]. Our study
on the ternary complex LeuRSTT with tRNALeu and
boron derivative AN2690, which is a selective inhibi-
tor to the editing active center of LeuRS, made it possib-
le to understand the initial binding conformation of
tRNA to the enzyme [53]. In the crystal it was obtained
stable adduct of AN2690-tRNALeu in the editing domain
of LeuRSTT, by chemical cross-linking the boron atom
of AN2690 with oxygen atoms of 2'- and 3'-OH end
groups of adenosine tRNA. In addition, we showed such
cross-linking in the solution. This indicates that the ori-
ginal, the most thermodynamically stable binding con-
formation of tRNALeu to LeuRSTT is the conformation
in which the 3'-end of tRNA interacts with the editing
active center of the enzyme. After the formation of ami-
noacyl-adenylate in the synthetic active site, 3'-end of
tRNA switches from the editing domain to the synthetic
one. A superposition of the tRNA conformations from
the various Pyroccocus and Thermus LeuRS-tRNALeu
complexes gives an impression of the trajectory of the 3'
end of the tRNA from aminoacylation to editing and exit
conformations. How does LeuRSTT recognize tRNALeu
and exclude noncognate tRNA? A biochemical insight
into this problem has mainly come from the attempts to
mutate tRNALeu in the E. coli system [40, 54–56]. The
generalisation of these results shows that A73, the con-
figuration of the D loop (notably the position of the G18-
G19 dinucleotide), the Levitt base pair A15-U48 and
the single unpaired nucleotide at 3' end of the long va-
riable arm are the crucial elements for effective leucyla-
tion. Examination of the crystal structures explained this
finding to some extent but failed to provide complete
understanding of the specificity of LeuRS for tRNALeu in
comparison to tRNATyr, which has the same discrimina-
tor nucleotide A73. We noted that for discrimination
between tRNALeu and tRNATyr the LeuRS uses the diffe-
rences in their tertiary structures, interacting simulta-
neously in several positions of a ribose-phosphate back-
bone of tRNA [45, 46].
317
RECOGNITION OF tRNAs WITH A LONG VARIABLE ARM BY AMINOACYL-tRNA SYNTHETASES
The tyrosine system. Tyrosyl-tRNA synthetase
(TyrRS) is a homodimeric class I aaRS, but is unusual-
ly a functional dimer, a feature only shared with trypto-
phanyl-tRNA synthetase [57]. This system is also uni-
que because of having two types of tRNATyr: with a long
variable loop for prokaryotes and eukaryotic organelles
and with a short variable loop for archaea and eukaryo-
tes. Besides, the acceptor stems for tRNATyr of proka-
ryotes, mitochondria and chloroplasts have the G1-C72
base pair found in most tRNAs while the first base pair
of tRNATyr of eukaryotic cytoplasm and archaea is C1-
G72 [12]. Eukaryote cytoplasmic and prokaryote TyrRS
cannot cross-aminoacylate their respective tRNAsTyr.
Although the crystallographic structure of the Bacillus
stearothermophilus TyrRS has been determined [58]
this structure comprises only the N-terminal 320 amino
acids of the molecule as the C-terminal 99 amino acids
are disordered in the crystal. A long time there were no
crystallographic data on the complex with ATP or with
tRNATyr. In order to fill up the gaps in structural infor-
mation, we have begun the work on the tyrosine system
from T. thermophilus. To obtain a sufficient amount of
TyrRS for crystallization, the gene encoding TyrRS
from the extreme thermophilic eubacterium T. thermo-
philus HB27 has been cloned and sequenced [59]. The
open reading frame encodes a polypeptide chain of 432
amino acid residues in length (molecular mass 48717
Da). Comparison of the amino acid sequence of the T.
thermophilus TyrRS (TyrRSTT) with that of TyrRS
from various organisms shows that the T. thermophilus
enzyme shares a branch in the phylogenetic tree of eu-
bacterial TyrRSs with the enzymes from Aquifex aeoli-
cus, Deinococcus radiodurans, Haemophilus influenzae
and Helicobacter pylori (40–57 % amino acid identity),
distinct from the branch containing E. coli, Chlamydia
trachomatis and Bacillus stearothermophilus, for
example (24–28 % amino acid identity). Non-bacterial
TyrRSs, which recognize type 1 tRNAs without a long
variable arm, are quite different and either lack (archaeal)
or have an alternative (eukaryotic) C-terminal domain.
We have determined a series of structures of TyrRSTT
complexed with various combinations of ATP and tyro-
sine, which causes several questions relating to the me-
chanism of tyrosine activation. Crystallization of Tyr
RSTT with tyrosinol (Fig. 1, see inset) allowed us to vi-
sualize for the first time the complete enzyme including
the C-terminal domain at 2.0 C resolution [60]. The fold
of C-terminal domain of TyrRSTT is similar to part of
the C-terminal domain of ribosomal protein S4 and its
role in tRNATyr recognition will be discussed below.
Structure of TyrRSTT-tRNATyr complex. We ha-
ve studied five different crystal forms of the complex
between TyrRS and native or transcript tRNATyr and de-
termined the structure of TyrRSTT-tRNATyr complex at
2.9 C resolution [60]. The structure of complex was no-
vel for several reasons since it (i) confirmed the cross-
subunit binding of the tRNA to TyrRS dimer, (ii) show-
ed that class I synthetase TyRS had a class II mode of
tRNA recognition, (iii) revealed the detailed interac-
tions of the TyrRS C-terminal domain with the tRNA
long variable arm and anticodon stem-loop, and (iiii)
demonstrated tertiary structural features in tRNATyr
which determine the orientation of the long variable arm
(Fig. 2, see inset).
The mode of binding tRNA to TyrRSTT is similar
to the earlier model of the TyrRS-tRNATyr complex pro-
posed by Bedouelle [61] on the basis of extensive muta-
tional studies and very similar to one proposed by us on
the basis of the study of phosphate protection upon
tRNATyr binding to the synthetase [62]. Despite having
an unambiguous class I catalytic domain, TyrRS in con-
trast to the canonical class I systems has a class II mode
of tRNA recognition [60]. This means that it interacts
with tRNATyr from the variable loop and acceptor stem
major groove side as, for instance, in the case of class II
AspRS [63] and SerRS [34] (Fig. 2, 5, see inset). This is
in strong contrast to the canonical class I systems such as
those of subclass Ib GlnRS [64], which approach cogna-
te tRNA from the acceptor stem minor groove side. Des-
pite the class II mode of tRNA recognition, TyrRS pre-
ferentially aminoacylates the 2' OH of A76 in accor-
dance with other class I systems [65]. The evolutional
scenario that led to those non-canonical features of TyrRS
is not obvious. There is a suggestion that dimerization of
synthetase and the class II mode of tRNA recognition
may be evolutionary linked in TyrRS [60]. This hypo-
thesis has been confirmed by the recent data on the
structures of the complex Methanococcus jannaschii
TyrRS with cognate tRNA [66] and the complex of hu-
man tryptophanyl-tRNA synthetase with tRNATrp [67],
where both homodimeric class I synthetases have the
class II mode of tRNA recognition.
318
TUKALO M. A. ET AL.
The C-terminal domain of TyrRSTT plays a critical
role in the recognition of tRNATyr, first by recognizing
the tRNA’s unique shape, and second by participating
in specific interaction with one of the anticodon bases.
These regions of contact agree very well with protec-
tion studies on the T. thermophilus system by chemical
modification and nuclease hydrolysis methods [62] (Fig.
3). The experiments showed that the tRNATyr interacts
with the cognate enzyme with the anticodon stem (on
the 5' side), the anticodon, the variable stem and loop (on
the 5' side) and the acceptor stem (on the 3' side). In the
complex, the anticodon triplet of tRNATyr (GYA) takes
up a novel conformation, in which G34 and A36 are
stacked on top of each other and Y35 bulges out in the
opposite direction. There is base-specific recognition
of Y35 by Asp423 and G34 by carboxyl group of
Asp259. It was shown also by biochemical methods,
that anticodon bases 34–35 are important recognition
elements by TyrRS [26, 68]. The acceptor stem of the
tRNATyr binds across the dimer interface onto the cata-
lytic domain of the opposing subunit. G1 is not specifi-
cally recognized by TyrRSTT; instead, C72 is recogni-
zed by one hydrogen bond with Glu154. Specific re-
cognition of the discriminator base A73 is made through
a hydrogen bound between the N6 position and the
main-chain carbonyl oxygen of Glu154 and a hydrogen
bond between the N3 position to Arg198. Interestingly,
the bacterial T. thermophilus TysRS and archaeal M.
jannaschii TyrRS (TyrSMJ) recognize the acceptor
stem of tRNA in a different manner [60, 66]. The ac-
ceptor stem of the M. jannaschii tRNATyr is the most im-
portant recognition element for TyrRSMJ, therefore
archeael enzyme strictly recognizes the C1-G72 base
pair, as it was shown by structural and biochemical me-
thods [66, 70].
Conformation comparison of the long variable
arm of the type 2 tRNAs. The structure of TyrRSTT-
tRNATyr complex completes the trilogy of structures of
long-variable-arm tRNAs with their cognate syntheta-
ses, allowing comparative studies of the unique confor-
mations of these tRNAs. The structures of three T. ther-
mophilus type 2 tRNAs, tRNASer, tRNATyr and tRNALeu
are compared in Fig. 6 (see inset). The crystal structures
of all three type 2 tRNAs show that the orientation of the
variable arm differs with the respect to the globular main
body of tRNA, depending on differences in the D and
variable arm regions [45]. As has been proposed, a key
determinant in the orientation of the long variable arm
of type 2 tRNA is the number of unpaired nucleotides at
the 3'-end of the long variable arm. In the case of tRNATyr,
this is of critical importance as a positive identity ele-
ment for recognition by TyrRS and as a negative ele-
ment which prevents the mischarging of tRNATyr by
LeuRS and SerRS [36, 40]. In tRNASer and tRNATyr the
D-loop has the same number of nucleotides and a simi-
lar conformation to nucleotide 20a, forming a planar
base-triple with the Levitt pair (G15-C48) and with the
base 20b inserted into the tRNA core. This makes the
backbone conformation of the two tRNAs, apart from
the variable loop, rather similar. However, the details of
the core packing are significantly different, resulting in
an » 50o change in orientation of the long variable arm
helix (Fig. 6, D, see inset), which clearly permits the
shape discrimination between these two type 2 tRNAs by
their respective synthetases [60]. In tRNASer, the signifi-
cant tilt of bases A21 and G9 allows deep penetration of
G20b into the core to stack against the first base pair of
the long variable arm (A45-U48-1). In contrast, in
tRNATyr, the first base pair of the long variable arm is
formed by reverse Hoogsteen base pair between A20b
and U48-2, against which the unpaired U48-1 stacks. In
bacterial tRNALeu there are highly conserved features
that distinguish it from the other two members of type 2
tRNAs (tRNASer and tRNATyr) as already discussed abo-
ve. Among them we should mention the less common
Levitt pair A15-U48; the a4-b3 configuration of the D
loop owing to the insertion of an additional base (nuc-
leotide 17) before the G18-G19 dinucleotide and to the
presence of base 20a but absence of 20b; and the occur-
rence of a single unpaired nucleotide (G48-1) at the base
of the long variable arm. As the result of this unique
configuration of tRNALeu core, G48-1 stacks against the
first base-pair of the variable arm and leads to its orien-
tation, different than that of tRNASer and tRNATyr (Fig. 6,
see inset). The different orientations of the variable arm
among the tRNAs thus depend on the identity of the in-
teracting D loop nucleotide, the number of other inser-
ted D loop nucleotides, and the number and identities
of the unpaired variable loop nucleotides that flank the
5' and 3' sides of the stem-loop motif.
Concluding remarks. Co-crystal structures of Ser
RS, LeuRS, and TyrRS that aminoacylate the type 2
319
RECOGNITION OF tRNAs WITH A LONG VARIABLE ARM BY AMINOACYL-tRNA SYNTHETASES
tRNAs together with the footprinting and biochemical
data show that the enzymes recognize the unique core
domain shape arising from the large stem-loop variable
region. The structural description of three bacterial
tRNAs with the long variable arms, tRNASer, tRNALeu
and tRNATyr, has provided an explanation of how the
systematic differences between them (correlate insertions
in the D loop and the base of the long variable arm) lead
to the unique core structure and long-variable-arm ori-
entation in each case [18, 30, 45, 51, 60, 62]. The re-
cognition by SerRS, LeuRS and TyRS of distinct glo-
bular shape in these type 2 tRNAs as a mechanism for
selectivity is related to so-called «indirect readout», be-
cause usually most or all of the interactions are made
with the sugar-phosphate backbone [45].
An example of indirect readout for the type 1
tRNA, is the recognition of the G15-G48 Levitt pair in
E. coli tRNACys in its natural context by CysRS [71].
Similarly, in the type 2 tRNALeu the substitution of A14,
which is not in direct contact with the enzyme in the
crystal structure of the complexes [45, 50], by any of the
other three nucleo- sides decreased the activity by
100-fold or more [40]. Since the mechanism of indirect
readout is important in the context of RNA-protein
interactions in general, it requires further studies using
different mutant forms of tRNAs and aaRSs by X-ray
methods together with footprinting analysis and kinetic
techniques.
Another important question, how the tRNA recogni-
tion mode for the type 2 tRNAs has evolved in associa-
tion with the evolution of synthetases? Eukaryotic cells
have only two of type 2 tRNAs (tRNASer and tRNALeu) as
the length of tRNATyr dramatically changed during evo-
lution. This has resulted in the fact that the recognition
style of eukaryotic tRNASer and tRNALeu varied in par-
ralel with some changes in the structure of these tRNAs
[72, 73]. There have also been changes in the structures
of relevant synthetases [74, 75]. Though the footprin-
ting studies have shown that in general the type of in-
teraction of eukaryotic tRNASer and tRNALeu with cognate
enzymes is similar to that for prokaryotic tRNAs [76–
78], the recognition elements of tRNAs are different
[72, 73, 79]. X-ray structures of the complexes of euka-
ryotic SerRS and LeuRS with cognate tRNAs should
shed light on the details of their recognition. The impor-
tance of information on tRNA selectivity by eukaryotic
SerRS and LeuRS that would emerge from such studies
is also related with a growing number of examples of
their participation in a variety of cellular functions and
pathological processes [80, 81].
Ì. À. Òó êà ëî, Ã. Ä. ßðåì ÷óê, Î. Ï. Êî âà ëåí êî, ². À. Êðèê ëè âèé,
Î. É. Ãóä çå ðà
Óï³çíà âàí íÿ òÐÍÊ, ÿê³ ìà þòü äîâ ãó âàð³àáåëü íó ã³ëêó,
àì³íî à öèë-òÐÍÊ ñèí òå òà çà ìè
Ðå çþ ìå
Ó êë³òè íàõ åâ êàð³îò³â òÐÍÊ òðüîõ ñïå öèô³÷íîñ òåé – òÐÍÊSer,
òÐÍÊLeu ³ òÐÍÊTyr – ìà þòü äîâ ãó âàð³àáåëü íó ã³ëêó äîâ æè íîþ
11–20 íóê ëå î òèä³â (2-ãà ãðó ïà òÐÍÊ) íà â³äì³íó â³ä ÷î òèðü îõ àáî
ï’ÿ òè íóê ëå î òèä³â 1-¿ ãðó ïè òÐÍÊ. ϳäñó ìî âà íî ðå çóëü òà òè íà -
øèõ äîñë³äæåíü ñòðóê òóð íèõ îñíîâ óï³çíà âàí íÿ ³ äèñ êðèì³íàö³¿
òÐÍÊ 2-¿ ãðó ïè ñå ðèë-, òè ðî çèë- ³ ëåé öèë-òÐÍÊ ñèí òå òà çà ìè ç
Thermus thermophilus (ÑåðÐÑ, ÒèðÐÑ ³ ËåéÐÑ), îò ðè ìà íèõ ìå òî -
äà ìè ðåí òãåí³âñüêî¿ êðèñ òà ëîã ðàô³¿ ³ õ³ì³÷íî¿ ìî äèô³êàö³¿ òÐÍÊ
ó ðîç ÷èí³. Íà ñüî ãîäí³ êðèñ òàë³÷íà ñòðóê òó ðà â³äîìà äëÿ âñ³õ òðüîõ
êîì ïëåêñ³â àì³íî à öèë-òÐÍÊ ñèí òå òàç ç â³äïîâ³äíè ìè òÐÍÊ 2-¿
ãðó ïè, ð³çí³ òèïè âï³çíà âàí íÿ ÿêèõ îá ãî âî ðþ þòü ñÿ â îãëÿä³. Çîê ðå -
ìà, îñîá ëè âó óâà ãó ïðèä³ëåíî ðå çóëü òà òàì àíàë³çó âï³çíà âàí íÿ ãî -
ìî ëîã³÷íè ìè ñèí òå òà çà ìè õà ðàê òåð íèõ ðèñ ïðî ñòî ðî âî¿ ñòðóê-
òóðè òÐÍÊ 2-¿ ãðó ïè. Ó òÐÍÊSer, òÐÍÊLeu ³ òÐÍÊTyr îð³ºíòàö³ÿ
äîâãî¿ âàð³àáåëü íî¿ ã³ëêè â³äíîñ íî îñíîâ íî ãî ò³ëà òÐÍÊ â³äð³çíÿ-
ºòüñÿ ³ êîí òðî ëþºòüñÿ ð³çíîþ óïà êîâ êîþ êî ðî âî¿ ÷àñ òè íè ìî ëå -
êó ëè. Ó ðàç³ ÑåðÐÑ N-ê³íöå âèé, à â ðàç³ ÒèðÐÑ – C-ê³íöå âèé äî ìå -
íè çâ’ÿ çó þòü ñÿ ç ïåâ íè ìè ñòðóê òó ðà ìè äîâ ãèõ âàð³àáåëü íèõ ã³ëîê
ãî ìî ëîã³÷íèõ ÐÍÊ, óï³çíà þ ÷è òà êèì ÷è íîì óí³êàëü íó ñòðóê òóð íó
ôîð ìó òÐÍÊ. Êî ðî âà ÷àñ òè íà òÐÍÊLeu ìຠê³ëüêà øàð³â íå çâè-
÷àé íèõ ïàð îñíîâ, âè ÿâ ëå íèõ ïðè âèâ ÷åíí³ êðèñ òà ëîã ðàô³÷íî¿
ñòðóê òóðè êîì ïëåêñó òÐÍÊLeu ç ËåéÐÑ ³ç T. thermophiles òà ïðè
äîñë³äæåíí³ â³ëüíî¿ òÐÍÊ ó ðîç ÷èí³ ìå òî äîì õ³ì³÷íî¿ ìî äèô³-
êàö³¿ ç âè êî ðèñ òàí íÿì ñïå öèô³÷íèõ ðå à ãåíò³â. Ó êðèñ òà ëîã ðà-
ô³÷í³é ñòðóê òóð³ êîì ïëåê ñó ËåéÐÑ-òÐÍÊLeu óí³êàëü íà áó äî âà
D-ñòåá ëà âï³çíàºòüñÿ Ñ-ê³íöå âèì äî ìå íîì ËåéÐÑ ³ ö³ äàí³ äîá ðå
óçãîä æó þòü ñÿ ç ðå çóëü òà òà ìè, îò ðè ìà íè ìè â ðîç ÷èí³. ËåéÐÑ
ïðè òà ìàí íèé êà íîí³÷íèé äëÿ ñèí òå òàç ² ñòðóê òóð íî ãî êëà ñó
òèï óï³çíà âàí íÿ òÐÍÊ – ç áîêó D-ñòåá ëà ³ ìà ëî¿ áî ðî çåí êè àê -
öåï òîð íî ãî ñòåá ëà. Äëÿ ÑåðÐÑ òà êîæ õà ðàê òåð íèé êà íîí³÷íèé
äëÿ ñèí òå òàç ²² ñòðóê òóð íî ãî êëà ñó òèï óï³çíà âàí íÿ òÐÍÊ – ç
ïðî òè ëåæ íî ãî áîêó, òîá òî ç áîêó âàð³àáåëü íî ãî ñòåá ëà ³ âå ëè êî¿
áî ðî çåí êè àê öåï òîð íî ãî ñòåá ëà. ², íà ðåøò³, ÒèðÐÑ íà â³äì³íó â³ä
êà íîí³÷íî ãî äëÿ ôåð ìåíò³â ² êëà ñó òèïó ìຠòèï óï³çíà âàí íÿ
òÐÍÊ, âëàñ òè âèé ñèí òå òà çàì ²² êëà ñó.
Êëþ ÷îâ³ ñëî âà: òÐÍÊ äðó ãî ãî òèïó, äîâ ãà âàð³àáåëü íà ã³ëêà,
àì³íî à öèë-òÐÍÊ ñèí òå òà çà, óï³çíà âàí íÿ òÐÍÊ, êîì ïëåê ñè
àì³íî à öèë-òÐÍÊ ñèí òå òà çè.
Ì. À. Òó êà ëî, À. Ä. ßðåì ÷óê, Î. Ï. Êî âà ëåí êî, È. À. Êðèê ëè âûé,
Î. È. Ãóä çå ðà
Óçíà âà íèå òÐÍÊ ñ äëèí íîé âà ðè à áåëü íîé âåò êîé
àìè íî à öèë-òÐÍÊ ñèí òå òà çà ìè
Ðå çþ ìå
 êëåò êàõ ýó êà ðè î òîâ òÐÍÊ òðåõ ñïå öè ôè÷ íîñ òåé – òÐÍÊSer,
òÐÍÊLeu è òÐÍÊTyr – èìå þò äëèí íóþ âà ðè à áåëü íóþ âåò êó äëè íîé
320
TUKALO M. A. ET AL.
11–20 íóê ëå î òè äîâ (2-ÿ ãðóï ïà òÐÍÊ) â îò ëè ÷èå îò ÷å òû ðåõ èëè
ïÿòè íóê ëå î òè äîâ 1-é ãðóï ïû òÐÍÊ. Ñóì ìè ðî âà íû ðå çóëü òà òû
íà øèõ èñ ñëå äî âà íèé ñòðóê òóð íûõ îñíîâ óçíà âà íèÿ è äèñ êðè ìè íà -
öèè òÐÍÊ 2-é ãðóï ïû ñå ðèë-, òè ðî çèë- è ëåé öèë-òÐÍÊ ñèí òå òà -
çà ìè èç Thermus thermophiles (ÑåðÐÑ, ÒèðÐÑ è ËåéÐÑ), ïî ëó ÷åí-
íûå ìå òî äà ìè ðåí òãå íîâ ñêîé êðèñ òàë ëîã ðà ôèè è õè ìè ÷åñ êîé ìî-
äè ôè êà öèè òÐÍÊ â ðàñ òâî ðå. Íà ñå ãî äíÿ êðèñ òàë ëè ÷åñ êàÿ ñòðóê -
òó ðà èç âåñ òíà äëÿ âñåõ òðåõ êîì ïëåê ñîâ àìè íî à öèë-òÐÍÊ ñèí -
òå òàç ñ ñî îò âå òñòâó þ ùè ìè òÐÍÊ 2-é ãðóï ïû, ðàç íûå òèïû óç-
íà âà íèÿ êî òî ðûõ îá ñóæ äà þò ñÿ â îá çî ðå.  ÷àñ òíîñ òè, îñî áåí íîå
âíèìà íèå óäå ëå íî ðå çóëü òà òàì àíà ëè çà óçíà âà íèÿ ãî ìî ëî ãè÷ íûìè
ñèí òå òà çà ìè õà ðàê òåð íûõ ÷åðò ïðî ñòðà íñòâåí íîé ñòðóê òó ðû
òÐÍÊ 2-é ãðóï ïû. Ó òÐÍÊSer, òÐÍÊLeu è òÐÍÊTyr îðèåí òà öèÿ
äëèí íîé âà ðè à áåëü íîé âåò âè îò íî ñè òåëü íî îñíîâ íî ãî òåëà òÐÍÊ
îò ëè ÷à åò ñÿ è êîí òðî ëè ðó åò ñÿ ðàç íîé óïà êîâ êîé êî ðî âîé ÷àñòè
ìî ëå êó ëû.  ñëó ÷àå ÑåðÐÑ N-êîí öå âîé, à â ñëó ÷àå ÒèðÐÑ – C-êîí -
öåâîé äî ìå íû ñâÿ çû âà þò ñÿ ñ îïðå äå ëåí íû ìè ñòðóê òó ðà ìè äëèí íûõ
âà ðè à áåëü íûõ âå òîê ãî ìî ëî ãè÷ íûõ ÐÍÊ, óçíà âàÿ òåì ñà ìûì óíè -
êàëü íóþ ñòðóê òóð íóþ ôîð ìó òÐÍÊ. Êî ðî âàÿ ÷àñòü òÐÍÊLeu èìå-
åò íå ñêîëü êî ñëî åâ íå î áû÷ íûõ ïàð îñíî âà íèé, âû ÿâ ëåí íûõ ïðè èçó-
÷å íèè êðèñ òàë ëîã ðà ôè ÷åñ êîé ñòðóê òó ðû êîì ïëåê ñà òÐÍÊLeu ñ
ËåéÐÑ èç T. thermophiles è ïðè èñ ñëå äî âà íèè ñâî áîä íîé òÐÍÊ â
ðàñ òâî ðå ìå òî äîì õè ìè ÷åñ êîé ìî äè ôè êà öèè ñ èñ ïîëü çî âà íè åì
ñïå öè ôè ÷åñ êèõ ðå à ãåí òîâ.  êðèñ òàë ëîã ðà ôè ÷åñ êîé ñòðóê òó ðå
êîì ïëåê ñà ËåéÐÑ–òÐÍÊLeu óíè êàëü íîå ñòðî å íèå D-ñòåá ëÿ óçíà -
åò ñÿ Ñ-êîí öå âûì äî ìå íîì ËåéÐÑ è ýòè äàí íûå õî ðî øî ñî ãëà ñó -
þò ñÿ ñ ðå çóëü òà òà ìè, ïî ëó ÷åí íû ìè â ðàñ òâî ðå. ËåéÐÑ ñâîéñò-
âåí êà íî íè ÷åñ êèé äëÿ ñèí òå òàç ² ñòðóê òóð íî ãî êëàñ ñà òèï óçíà -
âà íèÿ òÐÍÊ – ñî ñòî ðî íû D-ñòåá ëÿ è ìà ëîé áî ðîç äêè àê öåï òîð -
íî ãî ñòåá ëÿ. Äëÿ ÑåðÐÑ òàê æå õà ðàê òåð íûé êà íî íè ÷åñ êèé äëÿ
ñèí òå òàç ²² ñòðóê òóð íî ãî êëàñ ñà òèï óçíà âà íèÿ òÐÍÊ – ñ ïðî -
òè âî ïî ëîæ íîé ñòî ðî íû, ò. å. ñî ñòî ðî íû âà ðè à áåëü íî ãî ñòåá ëÿ
è áîëü øîé áî ðîç äêè àê öåï òîð íî ãî ñòåá ëÿ. È, íà êî íåö, ÒèðÐÑ â
îò ëè ÷èå îò êà íî íè ÷åñ êî ãî äëÿ ôåð ìåí òîâ ² êëàñ ñà òèïà èìå åò
òèï óçíà âà íèÿ òÐÍÊ, ïðè ñó ùèé ñèí òå òà çàì ²² êëàñ ñà.
Êëþ ÷å âûå ñëî âà: òÐÍÊ âòî ðî ãî òèïà, äëèí íàÿ âà ðè à áåëü íàÿ
âåò êà, àìè íî à öèë-òÐÍÊ ñèí òå òà çà, óçíà âà íèå òÐÍÊ, êîì ïëåê -
ñû àìè íî à öèë-òÐÍÊ ñèí òå òà çû.
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