Ribosome salt wash

Increasing evidence suggests that Met-tRNAf is also the initiator tRNA in eukaryotic systems (C35, G16). The failure of previous experiments to demonstrate the role of this Met-tRNAf for the in vitro protein synthesis is probably due to the lack of protein initiation factors. Mi, Ma, and Ms, which are present in a ribosomal salt-wash protein fraction (P24, S35, S36). The most recent experiments by Anderson and co-workers (C34, C35) show that the Met-tRNAf binds the initiation factors Ml and Ms to form an initiation complex with messenger RNA. The binding of this complex requires CTP and Mg + ions. A methionyl-valine dipeptide production is the next step in the biosynthesis of the chain the synthesis of this bond requires Mg + ions, an additional initiation... 

In order to stimulate fmet incorporation by cell-free extracts, crude preparations of initiation factors prepared from the 0.5 M KGI wash of HeLa cell ribosomes were added to the protein-synthesizing reactions. The ribosomal salt wash from uninfected cells stimulated total fmet incorporation about two-fold at the concentrations utilized, and stimulated initiation at both sites eq ially. Ribosomal salt wash prepared from infected cells also stimulated total fmet incorporation to about the same extent as the uninfected cell preparation however, SDS-PAGE analysis of the fmet-labeled products revealed that only synthesis of the smaller polypeptide was increased. No stimulation of NCVPla synthesis occurred (Knauert and Ehrenfeld, submitted). 

The experiments with poliovirus-infected cell extracts and ribosomal salt washes focused attention on those initiation factors which were involved with steps surrounding the recognition and binding of mRNA to the ribosome. Two different approaches were applied to identify the factor(s) which was responsible for the failure of in-... 

The initial identification of a cap-binding protein resulted from experiments by Sonenberg and Shatkin (1977), who developed a crosslinking assay to detect polypeptides which were in physical proximity to the 5 ends of capped mRNAs. Reovirus mRNA was synthesized containing H-label in the methyl groups of the 5 cap. The mRNA was oxidized with NaI04 so as to convert the 2, 3 -cw-diol of the 5 -terminal m G to a reactive dialdehyde, and the oxidized mRNA was then incubated with ribosomal salt wash from rabbit reticulocytes or from Ehrlich ascites cells (Sonenberg et al., 1978). The... 

Lee et al. (1983) attempted to directly measure the effect of mRNA secondary structure on its reaction with cap-binding proteins. The 50,000- and 80,000-dalton, ATP/Mg -dependent CBPs in crude rabbit reticulocyte ribosomal salt wash showed the usual ATP-de-pendence for crosslinking to the oxidized cap structure of native reovirus mRNA. However, when inosine-substituted mRNA was used, specific crosslinking of these polypeptides occurred in the absence of ATP/Mg ". These results were contradicted in a subsequent report (Tahara et al., 1983) which demonstrated that ATP was required for crosslinking purified eIF-4A and 4B to oxidized, inosine-substituted mRNA as well as for authentic ribosome binding by the denatured RNA. The reason for the discrepant results by these two laboratories has not yet been elucidated. Perhaps small amounts of ATP were... 

Efforts to identify the inhibitory component in infected cell ribosomal salt wash yielded a protein of Mr 33,0(X) daltons, which comigrated with the SFV capsid protein on SDS-polyacrylamide gels and which reacted with antibodies raised against viral capsid protein (H. Van Steeg, personal communication). This protein, purified from infected cell ribosomal salt wash, selectively inhibited translation in vitro of host and early viral mRNA, but had no effect on translation of late viral mRNA or EMC virus RNA. The mechanism by which SFV capsid protein interferes with translation initiation of some mRNAs is not understood, nor is it clear how SFV late mRNA eludes the inhibition, or what role cap recognition plays in this scheme. However, the overall strategies followed by SFV and poliovirus may be quite similar, despite differences in underlying mechanisms. 

The reovirus-induced transition of the host cell machinery from cap-dependent to cap-independent translation may be analogous to the modification of the translational machinery by poliovirus. Poliovirus causes a marked inhibition of host cell protein synthesis, soon after infection. Poliovirus mRNAs, which are naturally uncapped (Nomoto et al., 1976 Hewlett et al., 1976), are preferentially translated. Although capped host cell mRNA remains structurally intact and can be translated in vitro, it does not enter into initiation complexes (Leibowitz and Penman, 1971 Kaufman et al., 1976 Fernan-dez-Munoz and Darnell, 1976). In vitro studies indicate that poliovirus inhibits the initiation of cellular protein synthesis by inactivating an initiation factor. In a cell-free translation system, the ribosomal salt wash from poliovirus-infected cells does not stimulate translation of capped host mRNAs but does stimulate the translation of poliovirus... 

In recent studies, Lemieux and Millward (Zarbl and Millward, 1983) have examined fractionated translation system lysates for their ability to stimulate the translation of uncapped reovirus mRNAs in unfractionated lysates prepared from uninfected cells. Fractions from infected but not uninfected cells could stimulate this translation, indicating that a viral factor stimulates the translation. The bulk of the stimulation activity was present equally in the S-200 supernate and the ribosomal salt wash. The specific activity of the stimulatory factor was greatest in the ribosomal salt wash, that appears to be enriched for the a3 outer capsid polypeptide. A monoclonal antibody to a3 could specifically inhibit the translation of uncapped reovirus mRNAs in lysates prepared from infected cells. 

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