ATP hydrolysis by initiation factor 4A is required for translation initiation in Saccharomyces cerevisiae.
Saccharomyces cerevisiae translation initiation factor eIF-4A, an RNA helicase of the Asp-Glu-Ala-Asp (DEAD) box protein family, was mutated in the putative ATP binding site and expressed in Escherichia coli. Mutant proteins with alanine at position 66 replaced by glycine [eIF-4A(A66G)] or valine [eIF-4A(A66V)] were purified from Escherichia coli extracts and analyzed in vitro for activity in ATP crosslinking, ATP hydrolysis, RNA helicase, and translation assays. The results show that in vitro ATP hydrolysis activity, RNA helicase activity, and translation activity of eIF-4A correlate with in vivo activity of the factor. Whereas eIF-4A(A66G) showed wild-type activity in all assays, eIF-4A(A66V) was active in ATP crosslinking but inactive in ATP hydrolysis and RNA helicase assays. In vitro translation was supported by wild-type eIF-4A and eIF-4A(A66G) but not by eIF-4A(A66V). The results show that, for their translation, the majority of mRNAs from Saccharomyces cerevisiae including an mRNA with the initiator AUG positioned 8 nucleotides downstream of the cap structure require eIF-4A that is able to hydrolyze ATP.
Blum
S
S
Institut für Biochemie und Molekularbiologie, Universität Bern, Switzerland.
Schmid
S R
SR
Pause
A
A
Buser
P
P
Linder
P
P
Sonenberg
N
N
Trachsel
H
H
D000255:Adenosine Triphosphate / Q000378:metabolism*; D000595:Amino Acid Sequence; D001483:Base Sequence; D003062:Codon / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D039601:Eukaryotic Initiation Factor-4A; D005822:Genetic Vectors; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010442:Peptide Chain Initiation, Translational; D010448:Peptide Initiation Factors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D014176:Protein Biosynthesis; D020365:RNA Helicases; D012316:RNA Nucleotidyltransferases / Q000235:genetics / Q000378:metabolism*; D012331:RNA, Fungal / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*
D003062:Codon; D009838:Oligodeoxyribonucleotides; D010448:Peptide Initiation Factors; D012331:RNA, Fungal; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D039601:Eukaryotic Initiation Factor-4A; D012316:RNA Nucleotidyltransferases; D020365:RNA Helicases