Tài liệu Báo cáo Y học: Novel complexes of mammalian translation elongation factor eEF1AÆGDP with

Novel complexes of mammalian translation elongation factor

eEF1AÆGDP with uncharged tRNA and aminoacyl-tRNA synthetase

Implications for tRNA channeling

Zoya M. Petrushenko, Tatyana V. Budkevich, Vyacheslav F. Shalak, Boris S. Negrutskii

and Anna V. El’skaya

Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine, Kiev, Ukraine

Multimolecular complexes involving the eukaryotic

elongation factor 1A (eEF1A) have been suggested to play

an important role in the channeling (vectorial transfer) of

tRNA during protein synthesis [Negrutskii, B.S. & El’skaya,

A.V. (1998) Prog. Nucleic Acids Res. Mol. Biol. 60, 47–78].

Recently we have demonstrated that besides performing its

canonical function of forming a ternary complex with GTP

and aminoacyl-tRNA, the mammalian eEF1A can produce

a noncanonical ternary complex with GDP and uncharged

tRNA [Petrushenko, Z.M., Negrutskii, B.S., Ladokhin,

A.S., Budkevich, T.V., Shalak, V.F. & El’skaya, A.V. (1997)

FEBS Lett. 407, 13–17]. The [eEF1AÆGDPÆtRNA] complex

has been hypothesized to interact with aminoacyl-tRNA

synthetase (ARS) resulting in a quaternary complex where

uncharged tRNA is transferred to the enzyme for amino￾acylation. Here we present the data on association of the

[eEF1AÆGDPÆtRNA] complex with phenylalanyl-tRNA

synthetase (PheRS), e.g. the formation of the above

quaternary complex detected by the gel-retardation and

surface plasmon resonance techniques. To estimate the

stability of the novel ternary and quaternary complexes of

eEF1A the fluorescence method and BIAcore analysis were

used. The dissociation constants for the [eEF1AÆGDPÆ

tRNA] and [eEF1AÆGDPÆtRNAPheÆPheRS] complexes were

found to be 20 nM and 9 nM, respectively.We also revealed a

direct interaction of PheRS with eEF1A in the absence of

tRNAPhe (Kd ¼ 21 nM). However, the addition of tRNAPhe

accelerated eEF1AÆGDP binding to the enzyme. A possible

role of these stable novel ternary and quaternary complexes

of eEF1AÆGDP with tRNA and ARS in the channeled

elongation cycle is discussed.

Keywords: translation elongation factor; macromolecular

complexes; tRNA channeling; eukaryotic protein synthesis;

BIAcore analysis.

Aminoacyl-tRNA synthetase (ARS) and eEF1A are the

proteins that advance the translation elongation cycle. ARS

binds ATP, an amino acid and tRNA to produce aminoacyl￾tRNA. The molecules of eEF1A bind GTP and aminoacyl￾tRNA, and deliver the latter to the A site of a translating

ribosome. The main steps of protein biosynthesis are similar

in all living organisms. However, some peculiarities of the

higher eukaryotic translation have been revealed, among

which a compartmentalization of the translation apparatus

is of particular importance. There is an increasing body of

evidence for special structural organization of the protein

synthesis machinery in the higher eukaryotic cells. The

existence of multimolecular complexes of ARS [1], initiation

factors [2] and eEF1 [3,4], ribosome–ARS interactions [5–7],

and the association of translation components with cyto￾skeletal framework [8] are among the important signs of the

protein synthesis compartmentalization. Moreover, detailed

fluorescence-based measurements of translation in living

dendrites have visualized the mammalian protein synthesis

compartments in situ [9].

An important mechanism to put into effect the potential

advantages of the compartmentalization is thought to be a

channeling (vectorial transfer) of aminoacyl-tRNA/tRNA

from ARS to the elongation factor, ribosome and back to

ARS without dissociation into the surrounding medium

[10,11]. The channeling influences positively the transla￾tional efficiency because the number of nonspecific

searches is diminished, the effective concentrations of

translational components are increased and the leakage of

important compounds to another metabolic processes is

hampered [12]. The channeling is a mechanism operating

by the formation of intermediate complexes between

subsequent participants of the metabolic pathway. Deut￾scher and coauthors revealed that aminoacyl-tRNA and

tRNA were never free in the cytoplasm of the eukaryotic

cell [10–12]. ARS and eEF1A are supposed to play a main

role in the tRNA sequestering during the mammalian

translation [13].

Several examples of the functional interaction of eEF1A

with ARS resulting in the activation of the latter have been

described [4,14,15]. While the stimulation of the valyl-tRNA

Correspondence to A. V. El’skaya, Department of Translation

Mechanisms, Institute of Molecular Biology and Genetics,

150, Zabolotnogo Str., Kiev 03143 Ukraine.

Fax: +38 044 2660759, Tel.: +38 044 2660749,

E-mail: [email protected]

Abbreviations: ARS, aminoacyl-tRNA synthetase;

eEF1A, eukaryotic translation elongation factor 1A (formerly EF-1a);

EF1A, prokaryotic translation elongation factor 1A (formerly

EF-Tu); FITC, fluorescein isothiocyanate isomer I; GMP-PNP,

guanosine-5¢-(b,c-imido)triphosphate; PheRS, phenylalanyl-tRNA

synthetase; RU, resonance unit.

(Received 10 May 2002, revised 11 July 2002,

accepted 13 August 2002)

Eur. J. Biochem. 269, 4811–4818 (2002) FEBS 2002 doi:10.1046/j.1432-1033.2002.03178.x