Initiation factors eIF4A and C1 from wheat germ and the formation of mRNA X ribosome complexes

Abstract

The binding of ribosomes to mRNA is analyzed in a fractionated system from wheat germ with [3H]uridine-labeled poly(A)+ RNA prepared from germinating wheat embryos. The reaction requires factors eIF3, eIF4C, and eIF5; Met-tRNA and the Met-tRNA binding system; either GTP or GMP-PNP; ATP; and factors C1 and eIF4A. These requirements are identical to those previously found to be necessary for formation of ribosome X Met-tRNAMeti complexes, with the exception of ATP, and factors C1 and eIF4A. The function of factors C1 and eIF4A is therefore specifically related to the mRNA attachment reaction. The presence of GTP in the mRNA binding reaction results in the formation of 80 S ribosome complexes, while with GMP-PNP only 40 S ribosome complexes are formed. Ribosome binding to native reovirus RNA in the fractionated wheat germ system is similar to the reaction with poly(A)+ RNA, strongly requiring ATP and factors C1 and eIF4A. Binding to inosine-substituted reovirus RNA, however, is only partially dependent upon ATP, and both the ATP-dependent and the ATP-independent binding reactions strongly require factor C1 and are substantially stimulated by factor eIF4A. The ATP-independent reaction is inhibited by pm7GDP, has a strong requirement for Met-tRNAMeti, and the 40 S ribosome complex is stable to RNase. These results indicate that the ATP-independent binding of ribosomes to inosine-substituted reovirus RNA proceeds through the normal initiation process. They further suggest that neither factor C1 nor eIF4A function exclusively to unwind mRNA secondary structure. Since eIF4A is required for the ATP-independent binding to inosine mRNA, and at the same time interacts with ATP in the reaction with ATP-requiring mRNAs, this factor may have two roles in protein chain initiation, one related to the mRNA X ribosome interaction, and one related to the function of ATP.