Tài liệu Báo cáo khoa học: Functional effects of deleting the coiled-coil motif in Escherichia coli

Functional effects of deleting the coiled-coil motif

in Escherichia coli elongation factor Ts

Henrik Karring1

, Asgeir Bjo¨ rnsson2

, Søren Thirup1

, Brian F. C. Clark1 and Charlotte R. Knudsen1

1

Department of Molecular Biology, Aarhus University, Denmark; 2

deCODE Genetics, Inc., Reykjavik, Iceland

Elongation factor Ts (EF-Ts) is the guanine nucleotide￾exchange factor for elongation factor Tu (EF-Tu) that is

responsible for promoting the binding of aminoacyl￾tRNA to the mRNA-programmed ribosome.The struc￾ture of the Escherichia coli EF-Tu–EF-Ts complex reveals

a protruding antiparallel coiled-coil motif in EF-Ts, which

is responsible for the dimerization of EF-Ts in the crystal.

In this study, the sequence encoding the coiled-coil motif

in EF-Ts was deleted from the genome in Escherichia coli

by gene replacement.The growth rate of the resulting

mutant strain was 70–95% of that of the wild-type strain,

depending on the growth conditions used.The mutant

strain sensed amino acid starvation and synthesized the

nucleotides guanosine 5¢-diphosphate 3¢-diphosphate and

guanosine 5¢-triphosphate 3¢-diphosphate at a lower

cell density than the wild-type strain.Deletion of the

coiled-coil motif only partially reduced the ability of

EF-Ts to stimulate the guanine nucleotide exchange in

EF-Tu.However, the concentration of guanine nucleo￾tides (GDP and GTP) required to dissociate the mutant

EF-Tu–EF-Ts complex was at least two orders of mag￾nitude lower than that for the wild-type complex.The

results show that the coiled-coil motif plays a significant

role in the ability of EF-Ts to compete with guanine

nucleotides for the binding to EF-Tu.The present results

also indicate that the deletion alters the competition bet￾ween EF-Ts and kirromycin for the binding to EF-Tu.

Keywords: elongation factor Ts; elongation factor Tu;

guanine nucleotide exchange; kirromycin; (p)ppGpp.

Elongation factor Tu (EF-Tu) and elongation factor Ts

(EF-Ts) are proteins known from the classical model of the

elongation cycle of protein synthesis in prokaryotes.EF-Tu,

which is a highly conserved G-protein, is active in the GTP￾bound form (EF-Tu–GTP) and inactive in the GDP-bound

form (EF-Tu–GDP).The equilibrium dissociation con￾stants for EF-Tu–GDP and EF-Tu–GTP are 1 · 10)9 and

5 · 10)8 M, respectively [1].The active EF-Tu–GTP binds

aminoacyl-tRNA (aa-tRNA) and promotes the binding of

the aa-tRNA to the A-site of the mRNA-programmed

ribosome.Upon codon recognition by a cognate ternary

complex (EF-Tu–GTP–aa-tRNA), the ribosomal GTPase

centre stimulates the GTPase activity of EF-Tu and the

bound GTP is hydrolysed.The inactive EF-Tu–GDP is

released from the ribosome and recycled to the active EF￾Tu–GTP by the exchange of GDP with GTP [2].Stimula￾tion of the guanine nucleotide release in EF-Tu by EF-Ts [3]

is required as the dissociation of GDP is otherwise very slow

(2 · 10)3 s

)1

) [1,4].I n vivo, the binding of GTP to the binary

EF-Tu–EF-Ts complex is favoured owing to the ninefold

higher concentration of GTP (0.9 mM) than GDP (0.1 mM)

[5].The activation of EF-Tu is completed by the dissociation

of EF-Ts from EF-Tu–GTP.The equilibrium governing

EF-Tu is further driven to the GTP-bound state by the

formation of EF-Tu–GTP–aa-tRNA.Previous studies have

indicated the existence of a structural isomerization in the

EF-Tu–GDP–EF-Ts complex from a high- to a low-affinity

nucleotide binding conformation [1,6,7].According to the

results published by Gromadski et al.[1], the structures

of the binary EF-Tu–EF-Ts complex and the nucleotide￾bound ternary complexes are different.

EF-Ts in Escherichia coli is encoded by a single gene (tsf)

located in the rpsB-tsf operon of the chromosome.The

elongation factor consists of 282 residues and has a

molecular mass of 30.3 kDa [8]. The structure of the E. coli

EF-Tu–EF-Ts complex (Fig.1) reveals that EF-Ts is an

elongated molecule containing four domains: the N-ter￾minal domain; the core domain; the dimerization domain;

and the C-terminal module [9].The dimerization domain

(residues 180–228), which consists of a-helices 9, 10 and 11,

is inserted in subdomain C of the core domain and contains

the protruding antiparallel coiled-coil motif (helices 10 and

11, residues 187–203 and 208–226) responsible for the

dimerization of EF-Ts in the crystal.In the crystal of E. coli

EF-Tu–EF-Ts, a quaternary complex, formed by two

molecules of each of the elongation factors, is observed.

The coiled-coil motifs of each of the two EF-Ts molecules

form strong intimate contacts with each other, and therefore

the tetramer is best designated as [EF-Ts]2)2EF-Tu.

However, the stoichiometry of the E. coli EF-Tu–EF-Ts

Correspondence to C.R.Knudsen, Department of Molecular Biology,

Aarhus University, Gustav Wieds Vej 10c, DK-8000 Aarhus C,

Denmark.Fax: + 45 8612 3178, Tel.: + 45 8942 5036,

E-mail: [email protected]

Abbreviations: aa-tRNA, aminoacyl-tRNA; CBD, chitin binding

domain; EF-G, elongation factor G; EF-Ts, elongation factor

Ts; EF-Tsmt, mitochondrial EF-Ts; EF-Tu, elongation factor Tu;

LB, Luria–Bertani; MCS, multiple cloning site; ppGpp, guanosine

5¢-diphosphate 3¢-diphosphate; pppGpp, guanosine 5¢-triphosphate

3¢-diphosphate; (p)ppGpp, ppGpp and pppGpp.

(Received 10 June 2003, revised 26 August 2003,

accepted 8 September 2003)

Eur. J. Biochem. 270, 4294–4305 (2003) FEBS 2003 doi:10.1046/j.1432-1033.2003.03822.x