Samarendra N. Seal

[11] Protein chain initiation in wheat embryo

Publisher Summary Wheat embryo extracts contain four soluble factors that are required for the incorporation of radioactive amino acids into protein in response to plant viral mRNA. Two of these factors function in initiation and two in elongation. This chapter describes the resolution of the factors from wheat embryo supernatant and some of the characteristics of the initiation reactions.

Reactivity of ribosomally bound methionyl-tRNA with puromycin and the locus of pactamycin inhibition of chain initiation

Abstract Methionyl tRNA binding to wheat embryo ribosomes is catalyzed either by two initiation factors, or by one initiation factor together with a transfer factor. The bound Met-tRNA is reactive with puromycin, however, only when the binding reaction is catalyzed by the two initiation factors. If pactamycin is added to the binding reaction, the bound Met-tRNA is again unreactive with puromycin. These results suggest a two-step sequence for Met-tRNA binding, with puromycin reactivity occurring only as a consequence of the second step. Pactamycin appears to be a specific inhibitor of the reaction converting bound Met-tRNA to puromycin reactivity.

Inhibition of noncapped mRNA translation by the cap analogue, 7-methylguanosine-5′-phosphate☆

Abstract The cap analogue, 7-methylguanosine-5′-phosphate (pm7G), inhibits the translation of the noncapped STNV (satellite tobacco necrosis virus) RNA and CPMV (cowpea mosaic virus) RNA in the in vitro wheat germ protein synthesizing system. While the translation of some capped mRNAs is inhibited more strongly by the analogue, other capped mRNAs have a level of sensitivity similar to that of the noncapped RNAs. Evidence is presented demonstrating that the effect of the analogue is exerted at a cap binding site even when it is inhibiting noncapped mRNAs. These results therefore indicate that the cap binding site of the translational system is either part of or is closely linked to another mRNA binding component, this component being specific for a site on the mRNA other than the 5′ cap. The observations also suggest caution in the use of pm7G inhibition to indicate the presence of a 5′ cap on a particular mRNA.

Initiation factors elF4A and C1 from wheat germ and the formation of mRNA · 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 · Met-tRNAiMet 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-tRNAiMet, 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 · ribosome interaction, and one related to the function of ATP.

Functional Characterization of the Initiation Factors of Wheat Germ

Protein biosynthesis is one of the first biological processes “activated” by exposure of wheat embryos to water (1). A major facet of the “activation” process is the attachment of ribosomes to preformed mRNA (2), and the reaction can, in part, be reproduced in vitro (3). In further studies it was found that the ribosome attachment reaction could be carried out with a number of eucaryotic mRNAs, in particular with plant viral RNAs (4, 5, 6). Utilizing particularly TMV-RNA, we have studied aspects of the initiation reaction in vitro and the subsequent sections summarize the current status of these studies.