Mutation of Thr445 and Ile500 of initiation factor 2 G-domain affects Escherichia coli growth rate at low temperature.
The Escherichia coli protein synthesis initiation factor IF2 is a member of the large family of G-proteins. Along with translational elongation factors EF-Tu and EF-G and translational release factor RF-3, IF2 belongs to the subgroup of G-proteins that are part of the prokaryotic translational apparatus. The roles of IF2 and EF-Tu are similar: both promote binding of an aminoacyl-tRNA to the ribosome and hydrolyze GTP. In order to investigate the differences and similarities between EF-Tu and IF2 we have created point mutations in the G-domain of IF2, Thr445 to Cys, Ile500 to Cys, and the double mutation. Threonine 445 (X1), which corresponds to cysteine 81 in EF-Tu, is well conserved in the DX1X2GH consensus sequence that has been proposed to interact with GTP. The NKXD motif, in which X is isoleucine 500 in IF2, corresponds to cysteine 137 in EF-Tu, and is responsible for the binding of the guanine ring. The recombinant mutant proteins were expressed and tested in vivo for their ability to sustain growth of an Escherichia coli strain lacking the chromosomal copy of the infB gene coding for IF2. All mutated proteins resulted in cell viability when grown at 42 degrees C or 37 degrees C. However, Thr445 to Cys mutant showed a significant decrease in the growth rate at 25 degrees C. The mutant proteins were overexpressed and purified. As observed in vivo, a reduced activity at low temperature was measured when carrying out in vitro ribosome dependent GTPase and stimulation of ribosomal fMet-tRNAfMet binding.
Larigauderie
G
G
Department of Molecular and Structural Biology, Aarhus University, 8000, Aarhus C, Denmark.
Laalami
S
S
Nyengaard
N R
NR
Grunberg-Manago
M
M
Cenatiempo
Y
Y
Mortensen
K K
KK
Sperling-Petersen
H U
HU
D000595:Amino Acid Sequence; D002455:Cell Division; D017124:Conserved Sequence; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development*; D040061:Eukaryotic Initiation Factor-5; D020558:GTP Phosphohydrolases / Q000378:metabolism; D005816:Genetic Complementation Test; D006160:Guanosine Triphosphate / Q000378:metabolism; D007532:Isoleucine; D009154:Mutation; D010448:Peptide Initiation Factors / Q000235:genetics* / Q000378:metabolism*; D017354:Point Mutation; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D013696:Temperature; D013912:Threonine
D040061:Eukaryotic Initiation Factor-5; D010448:Peptide Initiation Factors; D007532:Isoleucine; D013912:Threonine; D006160:Guanosine Triphosphate; D020558:GTP Phosphohydrolases