Protein Synthesis in Rabbit Reticulocytes: ASSAYS, PURIFICATION, AND PROPERTIES OF DIFFERENT RIBOSOMAL FACTORS AND THEIR ROLES IN PEPTIDE CHAIN INITIATION

Abstract

Abstract Protein synthesis initiation was studied using preincubated reticulocyte ribosomes, and the transfer of methionine from Met-tRNAfmet into the NH2-terminal positions of the polypeptides synthesized in response to poly[r(U-G)] and poly[r(A-U-G)] messengers was measured. Methionine transfer reactions in the above systems were dependent on the addition of 0.5 m KCl wash of reticulocyte ribosomes (I fraction). The I fraction was further purified by DEAE-cellulose column chromatography. At least three protein factors (IF-1, IF-2, IF-3) that eluted from the column stimulated both poly[r(U-G)]- and poly[r(A-U-G)]-directed methionine transfer reactions. However, the ability of these factors to catalyze the methionine transfer reactions in response to poly[r(U-G)] and poly[r(A-U-G)] messengers (containing two different initiation codons GUG and AUG) was significantly different. Also, these three factors when added together catalyzed more than additive transfer of methionine in response to poly[r(U-G)] messenger but not in response to poly[r(A-U-G)] messenger. A protein factor also found in the crude I fraction that eluted similarly to IF-1 on DEAE-cellulose chromatography, bound Met-tRNAfmet in the presence of GTP. The complex formed was quantitatively retained on Millipore filter and was assayed accordingly. The complex formation was specific for reticulocyte Met-tRNAfmet. Escherichia coli Met-tRNAfmet and f-Met-tRNAfmet also form similar complexes with IF-1; however, this complex formation was not GTP dependent. A preformed Met-tRNAfmet (reticulocyte) complex was more active in methionine transfer reactions than free Met-tRNAfmet (reticulocyte), indicating that this complex formation is an essential step in peptide chain initiation. Another protein factor (Fx) was obtained by re-extraction of the 0.5 m KCl-washed ribosomes with 1 m KCl. This factor bound Met-tRNAfmet and other aminoacyl tRNAs in the absence of GTP. As was the IF-1 complex, the aminoacyl tRNAs-Fx complexes were retained on Millipore filters and were assayed accordingly. The Fx fraction possibly binds unspecifically to different RNA species as the binding of Met-tRNAfmet to Fx was inhibited by addition of uncharged crude reticulocyte tRNA, poly(r-U), poly(r-A), and poly(r-U):poly(r-A). The Fx fraction strongly inhibited protein synthesis in response to poly[r(U-G)], poly[r(A-U-G)], and poly(r-U) messengers. With poly(r-U) messenger tested, the inhibition of polyphenylalanine synthesis by Fx fraction was overcome by increasing the poly(r-U) concentration.