Unwinding protein specific for mRNA translation fractionated together with rabbit reticulocyte initiation factor 3 complex.
Experiments with a rabbit reticulocyte cell-free system dependent on the addition of initiation factor 3 (eIF-3) and mRNA were carried out. In this system, using ribosomal subunits, AUG(U)(n) can direct polyphenylalanine synthesis in the absence of eIF-3 at 3 mM MgCl(2). Globin mRNA was not translated under similar conditions; its translation requires the addition of eIF-3. Moreover, the maximal rate of globin synthesis was achieved when the molar ratio of eIF-3 to ribosomes was approximately 1. This was taken to indicate that some ribosomal proteins were fractionated with eIF-3 and functioned in reconstitution of salt-washed ribosomes. In our system, almost all ribosomes were active, as evident from the fact that all were found in polysomes when analyzed at the time of linear incorporation, and the molar ratio of ribosomes to mRNA was maintained at 4:1. When AUG(U)(n) was hybridized with poly(A), it could not direct polyphenylalanine synthesis with or without eIF-3 and was a potent inhibitor of the translation of globin mRNA in the presence of eIF-3. When poly(A) containing 10% U was hybridized with AUG(U)(n) and added to the cell-free system, addition of eIF-3 promoted polyphenylalanine synthesis to about 80% of control. Moreover, eIF-3 was seen to shift significantly the melting temperature of globin and synthetic double-stranded RNA. These observations suggest that extraction of ribosomes with 0.5 M KCl may release a ribosomal protein that fractionates with eIF-3. This protein may function in unwinding or melting the secondary structure of mRNA and thus facilitate translation.
D000818:Animals; D001798:Blood Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002458:Cell Fractionation; D005914:Globins / Q000096:biosynthesis; D007700:Kinetics; D009691:Nucleic Acid Denaturation; D010448:Peptide Initiation Factors; D011132:Polyribosomes / Q000378:metabolism / Q000648:ultrastructure; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism*; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism; D013696:Temperature
D001798:Blood Proteins; D010448:Peptide Initiation Factors; D012333:RNA, Messenger; D005914:Globins
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