Quantitative assessment of EF-1alpha.GTP binding to aminoacyl-tRNAs, aminoacyl-viral RNA, and tRNA shows close correspondence to the RNA binding properties of EF-Tu.
A ribonuclease protection assay was used to determine the equilibrium dissociation constants (Kd) for the binding of various RNAs by wheat germ EF-1alpha.GTP. Aminoacylated fully modified tRNAs and unmodified tRNA transcripts of four specificities (valyl, methionyl, alanyl, and phenylalanyl) from higher plants or Escherichia coli were bound with Kd values between 0.8 and 10 nM. A valylated 3'-fragment of turnip yellow mosaic virus RNA, which has a pseudoknotted amino acid acceptor stem, was bound with affinity similar to that of Val-tRNAVal. Uncharged tRNA and initiator Met-tRNAMet from wheat germ, RNAs that are normally excluded from the ribosomal A site in vivo, bound weakly. The discrimination against wheat germ initiator Met-tRNAMet was almost entirely due to the 2'-phosphoribosyl modification at nucleotide G64, since removal resulted in tight binding by EF-1alpha.GTP. A 44-nucleotide RNA representing a kinked acceptor/T arm obtained by in vitro selection to bacterial EF-Tu formed an Ala-RNA.EF-1alpha.GTP complex with a Kd of 29 nM, indicating that much of the binding affinity for aminoacylated tRNA is derived from interaction with the acceptor/T half of the molecule. The pattern of tRNA interaction observed for EF-1alpha (eEF1A) therefore closely resembles that of bacterial EF-Tu (EF1A).
Dreher
T W
TW
Department of Microbiology, Oregon State University, Corvallis, Oregon 97331-3804, USA.
Uhlenbeck
O C
OC
Browning
K S
KS
D000595:Amino Acid Sequence; D001483:Base Sequence; D006160:Guanosine Triphosphate / Q000378:metabolism*; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D010444:Peptide Elongation Factor Tu / Q000378:metabolism*; D012346:RNA, Transfer, Amino Acyl / Q000737:chemistry / Q000378:metabolism*; D012367:RNA, Viral / Q000737:chemistry / Q000378:metabolism*; D016601:RNA-Binding Proteins / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid; D014908:Triticum / Q000378:metabolism
D012346:RNA, Transfer, Amino Acyl; D012367:RNA, Viral; D016601:RNA-Binding Proteins; D006160:Guanosine Triphosphate; D010444:Peptide Elongation Factor Tu