Puromycin-Peptide Bond Formation with Reticulocyte Initiation Factors M ₁ and M ₂

Abstract

The ability to form a "peptide" bond between various forms of Met-tRNA or Phe-tRNA and puromycin has been studied in the reticulocyte cell-free system. When Met-tRNAF, fMet-tRNAF, or N-acetylPhetRNA are used as substrate at low Mg++ concentration (3 mM), reticulocyte initiation factors M1 and M, (M2A + M2B) are required for puromycin-peptide synthesis. In contrast to bacterial systems, this reaction is also stimulated by the elongation factor Ti. When Met-tRNAM or Phe-tRNA is used as substrate, there is no M-factor requirement for the puromycin reaction; T, is absolutely required, and the reaction is stimulated by T2. These studies indicate that reticulocyte factors Ml and M2 may function in part by placing the initiator tRNA into the P site. The detailed mechanism for mammalian initiation, however, may be more complex than that for bacterial systems.In previous studies, we have reported the isolation and identification of specific enzyme factors required for the initiation of reticulocyte cell-free protein synthesis (1-3 (24)(25)(26)(27). In the present studies with the reticulocyte cell-free system, we find that reticulocyte initiation factors Ml and M2 are required to synthesize puromycin peptides from reticulocyte Met-tRNAF, fMet-tRNAF or N-acetylPhe-tRNA, but little activity is obtained in the absence of elongation factors.In contrast, when Met-tRNAm or Phe-tRNA is used as a substrate, the optimum Mg++ concentration is higher, and only elongation factors are required for puromycin-peptide synthesis. From these studies, it appears that the mammalian initiation factors are capable of placing initiator tRNA into the peptidyl site and that the basic mechanism for mammalian initiation is similar to that in bacteria, although the details of the reaction sequence in mammalian systems appear to be more complex. METHODS The preparation of reticulocyte ribosomes washed with 0.5 M KCl, elongation factors T1 and T2, and initiation factors M1 and M2 was as reported (3,4), except that T, and T2 were chromatographed on phosphocellulose as well as on DEAEcellulose and Sephadex G-150. Initiation factor M2 has been subdivided into two components-M2A, the major peak of M2 activity eluting from DEAE-cellulose at 0.26 M KCI, and M2B, the minor peak of M2 activity eluting from DEAE-cellulose at 0.32 M KCl. tRNAmet and tRNAmet were isolated from crude reticulocyte tRNA by reversed-phase chromatography (RPC-IV) (28). tRNAmet could be 14.1% acylated with either reticulocyte or E. coli synthetase and was more than 98% formylated with crude E. coli transformylase by the method of Caskey et al. (5). tRNAmet could be 4.7% acylated by reticulocyte synthetase, but only 0.1% by E. coli synthetase, and was 2% formylated by E. coli transformylase.

Function of Met‐tRNAMetf and Met‐tRNAMet in Peptide‐Chain Elongation in Cell‐Free Systems from Mouse‐Liver and Ascites‐Tumor Cells

“…No conclusive explanation can, a t present, be offered for this finding which is in agreement with data presented by other authors [5]. One might speculate, however, that in mammalian systems, the triplet associates preferentially with the ribosomal P-site to which Met-tRNAfMet is transferred by the initiation factors [22,30]. I n this case, apparently, the codon would not become available to MettRNAMet transferred to the ribosomal A-site and a stimulating effect of the triplet on the T-factorcatalyzed binding of either Met-tRNAMet should not be observed.…”

“…This result could possibly be explained by the binding of Met-tRNAf Met to both ribosomal sites, with T factor responsible for transferring MettRNAfMet to the A-site and the ribosomal wash fraction bringing the same tRNA species to the peptidy1 site. Alternatively, the additive effects of T factor and ribosomal wash on the binding of MettRNAfMet could reflect an assistant role of T factor in chain initiation similar to the enhancing effect of T factor on the formation of an initiation complex as observed by Shafritz et al in a cell-free system from rabbit reticulocytes [22]. The fact that an additive effect of T factor and ribosomal wash was not found with formylated initiator tRNA seems to favour the first explanation.…”

Initial Dipeptide Formation in Hemoglobin Biosynthesis

“…Although it is not yet possible to define the exact role of each M factor in tRNA binding to ribosomes, the present studies, together with other data from this laboratory (2,19,28), suggest that Ml + M2 confer specific recognition of Met-tRNAF species to ribosomes containing endogenous messenger RNA, as well as to ribosomes with artificial template. This recognition appears to take place on the small ribosomal subunit in both the endogenous mRNA system and the artificial AUG-template system.…”

Requirement for GTP in the Initiation Process on Reticulocyte Ribosomes and Ribosomal Subunits

“…Fusidic acid and aurintricarboxylic acid were the gifts of H. Weissbach and A. Grollman, respectively. The assays for aminoacyl-tRNA binding, peptide-bond formation with puromycin, and cell-free poly(U)-dependent protein synthesis were also as reported (4,7,8).…”

“…The preparation of washed reticulocyte ribosomes, elongation factors T, and T2, initiation factors M1 and M2(AB), and acylated reticulocyte tRNA was as reported (4,(7)(8)(9), except that M1 was purified through phosphocellulose as well as DEAE-cellulose and Sephadex G-150 chromatography. The separation of M2 into two components, M2A (which elutes at the void volume on Sephadex G-200 chromatography) and M2B (which has a molecular weight of 20,000-40,000), will be reported elsewhere.…”