Interaction between eukaryotic initiation factors 1A and 5B is required for efficient ribosomal subunit joining.
Eukaryotic initiation factor 5B (eIF5B) is a GTPase that facilitates joining of the 60 S ribosomal subunit to the 40 S ribosomal subunit during translation initiation. Formation of the resulting 80 S initiation complex triggers eIF5B to hydrolyze its bound GTP, reducing the affinity of the factor for the complex and allowing it to dissociate. Here we present a kinetic analysis of GTP hydrolysis by eIF5B in the context of the translation initiation pathway. Our data indicate that stimulation of GTP hydrolysis by eIF5B requires the completion of early steps in translation initiation, including the eIF1- and eIF1A-dependent delivery of initiator methionyl-tRNA to the 40 S ribosomal subunit and subsequent GTP hydrolysis by eIF2. Full activation of GTP hydrolysis by eIF5B requires the extreme C terminus of eIF1A, which has previously been shown to interact with the C terminus of eIF5B. Disruption of either isoleucine residue in the eIF1A C-terminal sequence DIDDI reduces the rate constant for GTP hydrolysis by approximately 20-fold, whereas changing the aspartic acid residues has no effect. Changing the isoleucines in the C terminus of eIF1A also disrupts the ability of eIF5B to facilitate subunit joining. These data indicate that the interaction of the C terminus of eIF1A with eIF5B promotes ribosomal subunit joining and possibly provides a checkpoint for correct complex formation, allowing full activation of GTP hydrolysis only upon formation of a properly organized 80 S initiation complex.
Acker
Michael G
MG
Department of Biophysics and Biophysical Chemistry, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Shin
Byung-Sik
BS
Dever
Thomas E
TE
Lorsch
Jon R
JR
D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D004926:Escherichia coli / Q000235:genetics; D015977:Eukaryotic Initiation Factor-1 / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D039642:Eukaryotic Initiation Factors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005454:Fluorescence Polarization; D020558:GTP Phosphohydrolases / Q000032:analysis / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D006868:Hydrolysis; D007532:Isoleucine / Q000378:metabolism; D007700:Kinetics; D008715:Methionine / Q000378:metabolism; D008954:Models, Biological; D010442:Peptide Chain Initiation, Translational; D010761:Phosphorus Radioisotopes; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D012343:RNA, Transfer / Q000378:metabolism; D012270:Ribosomes / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D013462:Sulfur Radioisotopes
D015977:Eukaryotic Initiation Factor-1; D039642:Eukaryotic Initiation Factors; D010761:Phosphorus Radioisotopes; D012333:RNA, Messenger; D013462:Sulfur Radioisotopes; C451459:eukaryotic initiation factor-5B; D007532:Isoleucine; D006639:Histidine; D006160:Guanosine Triphosphate; D012343:RNA, Transfer; D008715:Methionine; D020558:GTP Phosphohydrolases