Документ взят из кэша поисковой машины. Адрес оригинального документа : http://kodomo.fbb.msu.ru/~smivic/Projects/1NZJ/downloads/Smirnova_Cr1.doc Дата изменения: Sun Mar 15 18:27:02 2009 Дата индексирования: Sun Apr 5 16:21:02 2009 Кодировка: koi8-r

Зачетное задание по 1 блоку 2 семестра
Смирнова Виктория

15.03.2009



Название моего белка - Глутамил-Q тРНК(Asp) синтетаза. Это фермент
бактерии Escherichia coli (штамм K12), который катализирует тРНК-
независимую активацию глутамата в присутствии АТФ и последующий перенос
глутамата на тРНК (Asp). Глутамат переносится на 2-амино-5-(4,5-дигидрокси-
2-циклопентен-1-ил) часть квеуозина в 34 позицию тРНК(Asp) QUC-антикодона.
Не переносит глутамат ни на тРНК(Glu), ни на тРНК(Gln).

Белок кодируется геном yadB.

В молекуле белка - 1 цепь в 298 аминокислот, его молекулярная масса -
33601.



Аминокислотная последовательность в fasta-формате:



>sp|P27305|GLUQ_ECOLI Glutamyl-Q tRNA(Asp) synthetase OS=Escherichia coli
(strain K12) GN=gluQ PE=1 SV=5

MTDTQYIGRFAPSPSGELHFGSLIAALGSYLQARARQGRWLVRIEDIDPPREVPGAAETI

LRQLEHYGLHWDGDVLWQSQRHDAYREALAWLHEQGLSYYCTCTRARIQSIGGIYDGHCR

VLHHGPDNAAVRIRQQHPVTQFTDQLRGIIHADEKLAREDFIIHRRDGLFAYNLAVVVDD

HFQGVTEIVRGADLIEPTVRQISLYQLFGWKVPDYIHLPLALNPQGAKLSKQNHAPALPK

GDPRPVLIAALQFLGQQAEAHWQDFSVEQILQSAVKNWRLTAVPESAIVNSTFSNASC

(скачать)



В третичной структуре белка не проглядывается какого-либо явного
разделения на субъединицы, нет характерной для какой-либо функции формы.





Изображение третичной структуры белка (из RasMol). Атомная модель.





































Во вторичной структуре белка представлены 13 альфа-спиралей, 3 бета-
поворота и 10 неструктурированных участков.



Остовная модель (на изображении, созданном с помощью программы RasMol
малиновым цветом выделены альфа-спирали, желтым - бета-структуры.)





















В третичной структуре белка есть несколько сайтов связывания. Четыре
аминокислотных остатка (3 цистеина и 1 тирозин) участвуют в связывании с
ионом металла - цинком.



Взаимное разположение иона и контактирующих с ним остатков.



































Так же в белке есть сайты связывания глутамата (3 остатка) и АТФ (1
остаток).



Литература и ссылки по моему белку.



Ссылки на записи про мой белок в различных БД:



1. Cтраница белка на сайте UniProt -
http://www.uniprot.org/uniprot/P27305; текстовый файл с теми же данными
- http://www.uniprot.org/uniprot/P27305.txt (AC - P27305; P75662; Id -
GLUQ_ECOLI).

2. PDB-файл - http://www.pdb.org/pdb/files/1nzj.pdb (Id - 1NZJ; 2ZLZ).

3. Страницы в других поисковых системах по базе данных SwissProt: SRS;
MRS.





Статьи непосредственно о моем белке:


[1]"A minimalist glutamyl-tRNA synthetase dedicated to aminoacylation of
the tRNAAsp QUC anticodon."

Blaise M., Becker H.D., Keith G., Cambillau C., Lapointe J., Giege R.,
Kern D.

Nucleic Acids Res. 32:2768-2775(2004)

Escherichia coli encodes YadB, a protein displaying 34% identity with the
catalytic core of glutamyl-tRNA synthetase but lacking the anticodon-
binding domain. We show that YadB is a tRNA modifying enzyme that
evidently glutamylates the queuosine residue, a modified nucleoside at the
wobble position of the tRNA(Asp) QUC anticodon. This conclusion is
supported by a variety of biochemical data and by the inability of the
enzyme to glutamylate tRNA(Asp) isolated from an E.coli tRNA-guanosine
transglycosylase minus strain deprived of the capacity to exchange
guanosine 34 with queuosine. Structural mimicry between the tRNA(Asp)
anticodon stem and the tRNA(Glu) amino acid acceptor stem in prokaryotes
encoding YadB proteins indicates that the function of these tRNA modifying
enzymes, which we rename glutamyl-Q tRNA(Asp) synthetases, is conserved
among prokaryotes.


[2]"An aminoacyl-tRNA synthetase-like protein encoded by the Escherichia
coli yadB gene glutamylates specifically tRNAAsp."

Dubois D.Y., Blaise M., Becker H.D., Campanacci V., Keith G., Giege R.,
Cambillau C., Lapointe J., Kern D.

Proc. Natl. Acad. Sci. U.S.A. 101:7530-7535(2004)

The product of the Escherichia coli yadB gene is homologous to the N-
terminal part of bacterial glutamyl-tRNA synthetases (GluRSs), including
the Rossmann fold with the acceptor-binding domain and the stem-contact
fold. This GluRS-like protein, which lacks the anticodon-binding domain,
does not use tRNA(Glu) as substrate in vitro nor in vivo, but
aminoacylates tRNA(Asp) with glutamate. The yadB gene is expressed in wild-
type E. coli as an operon with the dksA gene, which encodes a protein
involved in the general stress response by means of its action at the
translational level. The fate of the glutamylated tRNA(Asp) is not known,
but its incapacity to bind elongation factor Tu suggests that it is not
involved in ribosomal protein synthesis. Genes homologous to yadB are
present only in bacteria, mostly in Proteobacteria. Sequence alignments
and phylogenetic analyses show that the YadB proteins form a distinct
monophyletic group related to the bacterial and organellar GluRSs (alpha-
type GlxRSs superfamily) with ubiquitous function as suggested by the
similar functional properties of the YadB homologue from Neisseria
meningitidis.


[3]"A truncated aminoacyl-tRNA synthetase modifies RNA."

Salazar J.C., Ambrogelly A., Crain P.F., McCloskey J.A., Soell D.

Proc. Natl. Acad. Sci. U.S.A. 101:7536-7541(2004)

Aminoacyl-tRNA synthetases are modular enzymes composed of a central
active site domain to which additional functional domains were appended in
the course of evolution. Analysis of bacterial genome sequences revealed
the presence of many shorter aminoacyl-tRNA synthetase paralogs. Here we
report the characterization of a well conserved glutamyl-tRNA synthetase
(GluRS) paralog (YadB in Escherichia coli) that is present in the genomes
of >40 species of proteobacteria, cyanobacteria, and actinobacteria. The
E. coli yadB gene encodes a truncated GluRS that lacks the C-terminal
third of the protein and, consequently, the anticodon binding domain.
Generation of a yadB disruption showed the gene to be dispensable for E.
coli growth in rich and minimal media. Unlike GluRS, the YadB protein was
able to activate glutamate in presence of ATP in a tRNA-independent
fashion and to transfer glutamate onto tRNA(Asp). Neither tRNA(Glu) nor
tRNA(Gln) were substrates. In contrast to canonical aminoacyl-tRNA,
glutamate was not esterified to the 3'-terminal adenosine of tRNA(Asp).
Instead, it was attached to the 2-amino-5-(4,5-dihydroxy-2-cyclopenten-1-
yl) moiety of queuosine, the modified nucleoside occupying the first
anticodon position of tRNA(Asp). Glutamyl-queuosine, like canonical Glu-
tRNA, was hydrolyzed by mild alkaline treatment. Analysis of tRNA isolated
under acidic conditions showed that this novel modification is present in
normal E. coli tRNA; presumably it previously escaped detection as the
standard conditions of tRNA isolation include an alkaline deacylation step
that also causes hydrolysis of glutamyl-queuosine. Thus, this aminoacyl-
tRNA synthetase fragment contributes to standard nucleotide modification
of tRNA.


[4]"The Escherichia coli YadB gene product reveals a novel aminoacyl-tRNA
synthetase like activity."

Campanacci V., Dubois D.Y., Becker H.D., Kern D., Spinelli S., Valencia
C., Pagot F., Salomoni A., Grisel S., Vincentelli R., Bignon C., Lapointe
J., Giege R., Cambillau C.

J. Mol. Biol. 337:273-283(2004)

In the course of a structural genomics program aiming at solving the
structures of Escherichia coli open reading frame products of unknown
function, we have determined the structure of YadB at 1.5A using molecular
replacement. The YadB protein is 298 amino acid residues long and displays
34% sequence identity with E.coli glutamyl-tRNA synthetase (GluRS). It is
much shorter than GluRS, which contains 468 residues, and lacks the
complete domain interacting with the tRNA anticodon loop. As E.coli GluRS,
YadB possesses a Zn2+ located in the putative tRNA acceptor stem-binding
domain. The YadB cluster uses cysteine residues as the first three zinc
ligands, but has a weaker tyrosine ligand at the fourth position. It
shares with canonical amino acid RNA synthetases a major functional
feature, namely activation of the amino acid (here glutamate). It differs,
however, from GluRSs by the fact that the activation step is tRNA-
independent and that it does not catalyze attachment of the activated
glutamate to E.coli tRNAGlu, but to another, as yet unknown tRNA. These
results suggest thus a novel function, distinct from that of GluRSs, for
the yadB gene family.



[5]"Crystal structure of glutamyl-queuosine tRNAAsp synthetase complexed
with L-glutamate: structural elements mediating tRNA-independent
activation of glutamate and glutamylation of tRNAAsp anticodon."

Blaise M., Olieric V., Sauter C., Lorber B., Roy B., Karmakar S., Banerjee
R., Becker H.D., Kern D.

J. Mol. Biol. 381:1224-1237(2008)

Glutamyl-queuosine tRNA(Asp) synthetase (Glu-Q-RS) from Escherichia coli
is a paralog of the catalytic core of glutamyl-tRNA synthetase (GluRS)
that catalyzes glutamylation of queuosine in the wobble position of
tRNA(Asp). Despite important structural similarities, Glu-Q-RS and GluRS
diverge strongly by their functional properties. The only feature common
to both enzymes consists in the activation of Glu to form Glu-AMP, the
intermediate of transfer RNA (tRNA) aminoacylation. However, both enzymes
differ by the mechanism of selection of the cognate amino acid and by the
mechanism of its activation. Whereas GluRS selects l-Glu and activates it
only in the presence of the cognate tRNA(Glu), Glu-Q-RS forms Glu-AMP in
the absence of tRNA. Moreover, while GluRS transfers the activated Glu to
the 3' accepting end of the cognate tRNA(Glu), Glu-Q-RS transfers the
activated Glu to Q34 located in the anticodon loop of the noncognate
tRNA(Asp). In order to gain insight into the structural elements leading
to distinct mechanisms of amino acid activation, we solved the three-
dimensional structure of Glu-Q-RS complexed to Glu and compared it to the
structure of the GluRS.Glu complex. Comparison of the catalytic site of
Glu-Q-RS with that of GluRS, combined with binding experiments of amino
acids, shows that a restricted number of residues determine distinct
catalytic properties of amino acid recognition and activation by the two
enzymes. Furthermore, to explore the structural basis of the distinct
aminoacylation properties of the two enzymes and to understand why Glu-Q-
RS glutamylates only tRNA(Asp) among the tRNAs possessing queuosine in
position 34, we performed a tRNA mutational analysis to search for the
elements of tRNA(Asp) that determine recognition by Glu-Q-RS. The analyses
made on tRNA(Asp) and tRNA(Asn) show that the presence of a C in position
38 is crucial for glutamylation of Q34. The results are discussed in the
context of the evolution and adaptation of the tRNA glutamylation system.







Статьи по секвенированию генома Escherichia coli K-12, в том числе и
гена моего белка:



[1]"Systematic sequencing of the Escherichia coli genome: analysis of
the 2.4-4.1 min (110,917-193,643 bp) region."

Fujita N., Mori H., Yura T., Ishihama A.

Nucleic Acids Res. 22:1637-1639(1994)



[2]"The complete genome sequence of Escherichia coli K-12."

Blattner F.R., Plunkett G. III, Bloch C.A., Perna N.T., Burland V., Riley
M., Collado-Vides J., Glasner J.D., Rode C.K., Mayhew G.F., Gregor J.,
Davis N.W., Kirkpatrick H.A., Goeden M.A., Rose D.J., Mau B., Shao Y.

Science 277:1453-1474(1997)


[3]"Highly accurate genome sequences of Escherichia coli K-12 strains
MG1655 and W3110."

Hayashi K., Morooka N., Yamamoto Y., Fujita K., Isono K., Choi S., Ohtsubo
E., Baba T., Wanner B.L., Mori H., Horiuchi T.

Mol. Syst. Biol. 2:E1-E5(2006)


[4]"Identification and characterization of a new Escherichia coli gene
that is a dosage-dependent suppressor of a dnaK deletion mutation."

Kang P.J., Craig E.A.

J. Bacteriol. 172:2055-2064(1990)