Translation of MS2 RNA in vitro in the Absence of Initiation Factor IF‐3

Abstract

An Escherichia coli cell‐free translational system, deprived of initiation factor IF‐3, has been used to study the role of the factor in protein synthesis. In this system, 30‐S ribosomal subunits are preincubated together with MS2 phage RNA in a small volume in the presence of 10 mM Mg(Ac)2; the missing components required for protein synthesis are then added and assembly of elongating ribosomes is allowed to occur. This stepwise assembly process prermits formation of functional complexes which can carry out protein synthesis in the complete absence of IF‐3. The translational products, obtained in the absence of IF‐3, have been analysed and shown to be similar to those synthesized in the presence of the factor. The main product observed is the phage coat protein.

Comparison of initiation of protein synthesis in procaryotes, eucaryotes, and organelles.

“…Most of the evidence from procaryotic systems indicates that recognition of initiation sites in mRNA is an intrinsic property of the ribosome, as first revealed by Lodish's mixing experiments with ribosomes and factors from E. coli and Bacillus stearothermophilus (274,275; see discussion below). In support of that conclusion, studies using washed (i.e., factor-free) ribosomes from E. coli revealed that the low level of binding obtained in the absence of IF-3 occurs at the authentic initiation sites in coliphage mRNAs (458,553). Nevertheless, bacterial initiation factors-particularly IF-3-influence the relative utilization of initiation sites by differentially stabilizing the binding of ribosomes to mRNAs.…”

“…Several of the factors bind to both free ribosomal subunits and mRNA, but it has been difficult to evaluate the functional significance of the latter complexes. Bacterial component IF-3, for example, binds to phage mRNA (209), but the preformed complex of IF-3 and coliphage MS2 RNA was inert when subsequently tested for translation in the presence of anti-IF-3 antibodies (553). Several eucaryotic initiation faetors also bind to mRNA, including eIF-2 (216) and eIF-3 (182,512); however, neither of those interactions is specific for mRNA (70,181,512).…”

In vitro characterisation of the MS2 RNA polymerase complex reveals host factors that modulate emesviral replicase activity

“…Furthermore, our results suggest a direct competition for the 3′ end between the replicase and IF3 because increasing concentrations of either the replicase or the substrate RNA compensated for the inhibitory effect of IF3. Notably, MS2 gene expression was also shown to be completely independent from IF3, unlike for E. coli host proteins 53 . Thus, replication and translation of MS2 genomes seem to be well adapted to conditions under which IF3 levels are minimal, such as it was reported for E. coli cells that have reached stationary phase 54 .…”

Circular dichroism study of Escherichia coli initiation factor 3 binding to nucleic acids

“…[for reviews see Lodish (1976), Grunberg-Manago & Gros (1977), and Revel (1977)], although under certain conditions this protein has been found to be dispensable (Zipori et al, 1978). The degree of initiation stimulation by IF3 appears to correlate with the extent of secondary structure present in the initiation region of the mRNA, regardless of whether the mRNA is synthetic or naturally occurring (Lodish, 1976;Revel, 1977).…”