Mutation of the conserved Gly94 and Gly126 in elongation factor Tu from Escherichia coli. Elucidation of their structural and functional roles.
All guanine-nucleotide-binding proteins cycle between an inactive, GDP-bound and an active, GTP-bound conformation whereby they function as molecular switches. Elongation factor Tu from Escherichia coli is used as a model for defining residues important in the switch mechanism. Gly94 and Gly126 were separately mutated to alanine residues to study their role in the switch mechanism. The mutant proteins are denoted [G94A]EF-Tu and [G126A]EF-Tu, respectively. Both mutations affect the affinities for guanine nucleotides considerably, resulting in a decrease in the characteristic preference for GDP over GTP. Furthermore the [G94A]EF-Tu mutant possesses an increased GTPase activity. The aminoacyl-tRNA affinity is much reduced for [G94A]EF-Tu, as reflected in an increase of the dissociation rate constant for the ternary complex by a factor of 40. Surprisingly, however, both mutants in their GDP forms have a low, but significant affinity for aminoacyl-tRNA, which is not seen for the wild-type elongation factor Tu. The mutants only exhibit minor changes compared to the wild type with respect to in vitro translation of a poly(U) messenger.
Knudsen
C R
CR
Institute of Chemistry, Aarhus University, Denmark.
Kjaersgård
I V
IV
Wiborg
O
O
Clark
B F
BF
D001483:Base Sequence; D004926:Escherichia coli / Q000737:chemistry; D020963:GTP Phosphohydrolase-Linked Elongation Factors / Q000378:metabolism; D006153:Guanosine Diphosphate / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D008969:Molecular Sequence Data; D009154:Mutation; D010444:Peptide Elongation Factor Tu / Q000737:chemistry* / Q000502:physiology; D014176:Protein Biosynthesis; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D013329:Structure-Activity Relationship
D012346:RNA, Transfer, Amino Acyl; D006153:Guanosine Diphosphate; D006160:Guanosine Triphosphate; D020963:GTP Phosphohydrolase-Linked Elongation Factors; D010444:Peptide Elongation Factor Tu