Evidence that the 5'-untranslated leader of mRNA affects the requirement for wheat germ initiation factors 4A, 4F, and 4G.
The efficiency of translation of alfalfa mosaic virus (AMV) RNA 4, barley alpha-amylase (B alpha A) mRNA, and two chimeric mRNAs, AMV 4-B alpha A and B alpha A-AMV 4 (in which the 5' leader sequences of the two mRNAs were interchanged), was measured in an S30 extract from wheat germ and a fractionated system from wheat germ in which translation could be made dependent upon initiation factor (eIF) 3, 4A, 4F, or 4G. In the S30 system, AMV RNA 4 and the chimeric mRNA AMV 4-B alpha A are translated much more efficiently than B alpha A mRNA and the chimeric mRNA B alpha A-AMV 4. When the S30 system was supplemented with high amounts of purified eIF-3, eIF-4A, eIF-4F, and eIF-4G, B alpha A and B alpha A-AMV 4 mRNAs were translated as efficiently as AMV RNA 4 and AMV 4-B alpha A mRNA. These findings indicated that the mRNAs containing the B alpha A leader sequence required higher amounts of one or more of the initiation factors (eIF-3, eIF-4A, eIF-4F, and eIF-4G) for efficient translation. Determination of the amounts of the initiation factors required for translation in the fractionated system showed that AMV RNA 4 required 2-4-fold lower amounts of eIF-3, eIF-4A, eIF-4F, and eIF-4G than did B alpha A mRNA. Replacement of the B alpha A leader sequence with that of AMV RNA 4 decreased the amounts of eIF-4A, eIF-4G, and eIF-3 required, but did not affect the amount of eIF-4F required. Replacement of the AMV RNA 4 leader sequence with that of B alpha A mRNA increased the amounts of eIF-4F, eIF-4G, and eIF-3 required, but did not affect the amount of eIF-4A required. These data strongly suggest that the amounts of the factors required are affected not only by the 5' leader itself but also by interactions between the 5' leader and a region(s) of the mRNA 3' to the initiation codon.
Browning
K S
KS
Department of Chemistry, University of Texas, Austin 78712-1096.
Lax
S R
SR
Humphreys
J
J
Ravel
J M
JM
Jobling
S A
SA
Gehrke
L
L
D002213:Capsid / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel; D039621:Eukaryotic Initiation Factor-3; D039601:Eukaryotic Initiation Factor-4A; D039562:Eukaryotic Initiation Factor-4F; D039603:Eukaryotic Initiation Factor-4G; D000455:Medicago sativa; D009029:Mosaic Viruses / Q000235:genetics*; D010448:Peptide Initiation Factors / Q000494:pharmacology / Q000502:physiology*; D010944:Plants / Q000235:genetics; D014176:Protein Biosynthesis / Q000187:drug effects; D021382:Protein Sorting Signals / Q000235:genetics*; D012333:RNA, Messenger / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics*; D014908:Triticum; D000516:alpha-Amylases / Q000235:genetics
D039621:Eukaryotic Initiation Factor-3; D039562:Eukaryotic Initiation Factor-4F; D039603:Eukaryotic Initiation Factor-4G; D010448:Peptide Initiation Factors; D021382:Protein Sorting Signals; D012333:RNA, Messenger; D012367:RNA, Viral; D039601:Eukaryotic Initiation Factor-4A; D000516:alpha-Amylases