Translation of phage Qbeta mRNA: a test of the two-site model for ribosomal function.
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Biomedical subjects
Publications and source records attributed to P Leder.
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This report describes a convenient scheme for the further purification of an E. coli enzyme which is required for the translocation step in protein biosynthesis. The homogeneous enzyme translocase appears to be a relatively large monomeric protein, having a molecular weight of approximately 72,000, and acts in a catalytic fashion during protein synthesis. It is also one of the major soluble macromolecular constituents of rapidly growing E. coli, comprising more than 2 per cent of the protein in ribosome-free extracts. Further, the rate of in vitro protein synthesis is linearly dependent upon the concentration of the pure enzyme until approximately one molecule of translocase is present per ribosome in reaction mixtures.
A purified preparation of translocase, one of several enzymes required for protein biosynthesis, has been used to prepare a specific anti-translocase antibody. This antibody provides an extremely useful tool not only for detecting the enzyme independent of its activity, but for inhibiting the translocase-mediated reaction and, thus, protein biosynthesis. Though the antibody very rapidly inhibits elongation of the peptide chain, it fails to effect initiation, binding, or peptide bond formation, which strongly suggests that translocase is not a direct participant in these reactions. It also arrests translation of the defined tricodon AUG(U(6)) at the second codon, permitting formation only of the dipeptide, fMet-Phe, rather than the tripeptide, fMetPhe-(Phe)(2), which is formed in the presence of control serum. We have shown previously that the third codon becomes available only in the presence of translocase, thereby defining the site of action of the antibody. The antibody also has been used to demonstrate that translocase is antigenically distinct from the other proteins required for initiation and protein synthesis in E. coli.
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Cloned segments of mouse chromosomal DNA provide direct evidence for the somatic rearrangement of kappa variable and constant region genes in antibody producing cells. This rearrangement apparently affects only one member of an allelic pair of light chain genes.