Abstract
A new protein with translational activity has been identified on the basis of its ability to stimulate translation in an in vitro globin synthesis assay deficient in eukaryotic initiation factor (eIF) 4B and eIF4F. This protein has been purified to greater than 80% homogeneity from rabbit reticulocyte lysate and has been given the name eIF4H. eIF4H was shown to stimulate the in vitro activities of eIF4B and eIF4F in globin synthesis, as well as the in vitro RNA-dependent ATPase activities of eIF4A, eIF4B, and eIF4F. Three tryptic fragments of eIF4H yielded amino acid sequences that were 100% identical to a human sequence found in the GeneBankTM that codes for a previously uncharacterized protein (HUMORFU_1). The calculated molecular weight of the protein encoded by this sequence, its predicted cyanogen bromide fragmentation, and calculated isoelectric point are all consistent with those determined experimentally for eIF4H. Also, the presence of an RNA recognition motif within HUMORFU_1 suggests that eIF4H may interact with mRNA. We conclude that this newly characterized protein, eIF4H, functions to stimulate the initiation of protein synthesis at the level of mRNA utilization, and is encoded by the gene for HUMORFU_1.
Highlights
From the Departments of Biochemistry and §Veteran’s Affairs Medical Center and of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4935
Purification of a New Translation Factor, eIF4H—During the purification of eIF4B and eIF4F from rabbit reticulocyte lysate, an additional protein possessing translational activity was identified on the basis of its ability to stimulate protein translation in an in vitro globin synthesis assay deficient in eIF4B and eIF4F (Fig. 1)
We have described a novel translational activity that stimulates protein synthesis in a reconstituted reticulocyte lysate system
Summary
From the Departments of Biochemistry and §Veteran’s Affairs Medical Center and of Medicine, Case Western Reserve University, Cleveland, Ohio 44106-4935. A new protein with translational activity has been identified on the basis of its ability to stimulate translation in an in vitro globin synthesis assay deficient in eukaryotic initiation factor (eIF) 4B and eIF4F. The steps involving delivery of the mRNA to the ribosome and positioning of the first initiating methionyl-tRNA (Met-tRNAi) at the AUG start codon is termed initiation This process involves at least 11 protein. The other major step in the initiation of protein synthesis involves binding of the first aminoacyl-tRNA, Met-tRNAi, to the 40 S ribosomal subunit. This is facilitated by the translation factors eIF1A,2 eIF2, and eIF3. The ribosome is ready to begin the elongation phase of protein synthesis
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