The continued recycling of ribosomes during protein synthesis in rabbit reticulocyte lysates at 370 requires an initiation factor whose activity is rapidly lost in the absence of added heme. Partially purified factor (i) fully maintains the polysomes; (ii) inhibits the association of 40S and 60S ribosomal subunits into single ribosomes; (iii) promotes the quantitative entry of added 60S subunits into polysomes; (iv) allows the accumulation of ribosomal subunits, instead of single ribosomes, when initiation is blocked with aurin tricarboxylate; and (v) is absolutely required for the binding of globin messenger RNA to ribosomes.These properties suggest that this mammalian initiation factor functions analogously to bacterial IF-3. In addition, the translational control of globin synthesis by heme is exerted, directly or indirectly, through this factor.Ribosomes in growing bacteria and in the cytoplasm of yeast frequently undergo exchange of their large and small ribosomal subunits (1, 2), apparently by dissociation after each passage over messenger RNA and reformation from a pool of ribosomal subunits that continuously recycle through polysomes (3,4). When protein synthesis slows down, synthetically inactive single ribosomes (lacking mRNA and growing polypeptide chains) accumulate at the expense of polysomes in both prokaryotic and eukaryotic cells. Bacterial 30S and 50S ribosomal subunits possess an extremely high affinity for each other and readily associate to form single ribosomes (5); this association is specifically inhibited by initiation factor IF-3 (5, 6). We have proposed that this is the mechanism by which bacterial IF-3 maintains the recycling or ribosomal subunits through polysomes and prevents their accumulation as single ribosomes (5).We have asked if an analogous factor operates in the mammalian ribosome cycle, and, if so, whether it plays any role in exerting translational control. Rabbit reticulocytes are particularly suitable to answer these questions, because in a cellfree system prepared from them, ribosomes can recycle more than fifty times over endogenous messenger RNA (7); furthermore, when this system is incubated at 280 or higher, initiation of protein synthesis (mostly globin) decreases drastically after 5 min, and polysomes disappear, unless heme is added (7-11).Our finding is that rabbit reticulocytes indeed contain a factor activity that regulates the flow of ribosomal subunits through the ribosome cycle in a manner strictly analogous to bacterial IF-3. This factor, tentatively named IF-3rr, is absolutely required for the binding of globin mRNA to ribosomes.Abbreviation: rr, rabbit reticulocyte.In the absence of added heme, reticulocyte ribosomes fail to recycle through polysomes, unless IF-3rr is added. Directly or indirectly, therefore, translational control of reticulocyte protein synthesis by heme must be exerted through this factor. RESULTS Control of reticulocyte ribosome cycle by the factorThe sedimentation distribution of ribosomes in a 32P-labeled rabbit reticulocyte l...

Inhibition of protein synthesis in rabbit reticulocyte lysates by double-stranded RNA is caused by the inactivation of IF-3, an initiation factor required for the recycling of ribosomes and for their binding to messenger RNA. The evidence for this is that (i) the inhibition can be overcome by addition of exogenous IF-3; (ii) doublestranded RNA inactivates stoichiometric amounts of IF-3; (iii) double-stranded RNA forms a complex with IF-3; and (iv) double-stranded DNA, which lacks inhibitory activity, also binds to IF-3, but with a much lower affinity than double-stranded RNA. It is concluded that doublestranded RNA inhibits cellular protein synthesis by tightly completing with IF-3. It is suggested that IF-3 normally recognizes a double-stranded region in messenger RNA.

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