Isolation and characterization of factors from wheat germ that exhibit eukaryotic initiation factor 4B activity and overcome 7-methylguanosine 5'-triphosphate inhibition of polypeptide synthesis.

Abstract

Three highly purified preparations (preparations I, II-1, and 11-2) have been obtained from wheat germ and shown to support in vitro polypeptide synthesis directed by capped or uncapped mRNAs in a eukaryotic initiation factor 4B (eIF-4B)-deficient system. The three preparations differ, however, in polypeptide composition and in the ability to overcome the inhibitory effect of 7-methylguanosine 5'-triphosphate (m7GTP) on in vitro polypeptide synthesis. Preparation I contains two polypeptides (Mr = 80,000 and 28,000), which are present in a 1:1 molar ratio and are associated in a high molecular weight complex. Preparation II-1 contains two major polypeptides (Mr = 220,000 and 26,000) and preparation II-2 also contains two major polypeptides (Mr = 110,000 and 26,000). Preparations II-1 and 11-2 are high molecular weight complexes; neither contains detectable amounts of a Mr 80,000 or a Mr 50,000 component. Preparations 11-1 and II-2 both overcome m7GTP inhibition, whereas preparation I does not. These findings raise several questions with regard to the identity of eIF-4B and its relationship to cap recognition factors.

Plant Cap-binding Complexes Eukaryotic Initiation Factors eIF4F and eIFISO4F

“…Interestingly, eIFiso4F displays sigmoidal behavior with some mRNA templates such as ␤-hemoglobin mRNA (shown in Fig. 4) or yeast polysomal mRNA (9). Other plant mRNA templates also display this behavior in vitro (data not shown), and this behavior may reflect a difference in the interaction of some mRNAs with eIFiso4F compared with eIF4F.…”

“…The amount of eIF4F present in wheat germ extracts is about 5-10 times less compared with eIFiso4F (21). eIFiso4F consists of two subunits, eIFiso4G and eIFiso4E, and has activities in vitro similar to those of eIF4F (9,22). The eIFiso4G subunit contains an eIF4E binding site and the two HEAT domains found in plant eIF4G, but has lost most of the N-terminal region compared with eIF4G (see Fig.…”

“…Plants contain a second form of eIF4F not found in other eukaryotes, eIFiso4F (9,20). The amount of eIF4F present in wheat germ extracts is about 5-10 times less compared with eIFiso4F (21).…”

“…eIF4E is a small protein of ϳ24 kDa that directly binds the m 7 G cap and facilitates initiation events. Mammalian eIF4F complexes contain eIF4A, the prototype of the DEAD box helicase family (7,8); however, plant eIF4A is loosely associated and is easily removed during purification (9). eIF4G is a multifunctional protein that is an important structural platform for the assembly or nucleation of several initiation factors (eIF4A, eIF3, eIF4B, eIF5, and poly(A)-binding protein) and interaction with the 40 S ribosome during the initiation process (10,11).…”

The Phosphorylation State of Poly(A)-binding Protein Specifies Its Binding to Poly(A) RNA and Its Interaction with Eukaryotic Initiation Factor (eIF) 4F, eIFiso4F, and eIF4B

“…eIF4G, the large subunit of eIF4F, serves as a platform for the interaction and assembly of multiple initiation factors, including eIF4E, eIF4A (whose RNA helicase and ATPase activities are required for unwinding RNA secondary structure), and eIF3 (responsible for 40 S ribosomal subunit recruitment) (6 -8). In addition to eIF4F, plants contain an isoform called eIFiso4F composed of eIFiso4E and eIFiso4G (9). A direct interaction between PABP and eIF4G or eIFiso4G has been demonstrated in plants, animals, and yeast (10 -14).…”