ABSTRACT
The ribosome and initiation factor requirements for translation of rabbit-reticulocyte hemoglobin mRNA on rabbit reticulocyte ribosomes, reticulocyte ribosomal subunits, and liver ribosomes have been studied. Excellent synthesis of globin chains from exogenous mRNA in the fractionated cell-free system has been achieved. There is a near absolute requirement for each of the initiation factors, M(1), M(2), and M(3) (as well as for the supernatant proteins) for the translation of exogenous mRNA. Liver microsomal wash will partially replace reticulocyte factors M(1) and M(2), but will not replace the requirement for reticulocyte factor M(3). Rabbit liver ribosomes and rabbit reticulocyte ribosomes are equally active in their ability to support the translation of exogenous hemoglobin mRNA.
Subject(s)
Hemoglobins/biosynthesis , Liver/metabolism , RNA, Messenger/metabolism , Reticulocytes/metabolism , Ribosomes/metabolism , Animals , Cell-Free System , Chromatography, DEAE-Cellulose , Globins/biosynthesis , Liver/cytology , Microsomes, Liver/metabolism , Peptide Chain Initiation, Translational , RNA, Messenger/biosynthesis , RabbitsABSTRACT
The requirement for GTP in the initiation process on reticulocyte ribosomes and ribosomal subunits has been examined by studying Met-tRNA(F) binding, ribosome-dependent [gamma-(32)P]GTP hydrolysis, and peptide-bond formation with puromycin. Met-tRNA(F) binding can be obtained with the methylene analogue, 5'-guanylylmethylene diphosphonate, as well as GTP, and it is not inhibited by fusidic acid or several other inhibitors of protein synthesis. This reaction can be performed with the 40S subunit and has the same requirements as the Met-tRNA(F)-binding reaction with washed ribosomes. Ribosome-dependent [gamma-(32)P]GTP hydrolysis can be obtained with the initiation factor M(2A) using either washed ribosomes or the 40S subunit. This reaction is also not significantly inhibited by fusidic acid. Peptide-bond formation between puromycin and Met-tRNA(F), however, is inhibited by fusidic acid, and does not occur if the methylene analogue of GTP is substituted for GTP. These data suggest that the binding of the initiator tRNA to the 40S subunit does not require the hydrolysis of GTP, but that at least one GTP hydrolysis event must occur after Met-tRNA(F) binding in order for the first peptide bond to be formed.
Subject(s)
Guanosine Triphosphate/metabolism , Peptide Biosynthesis , Reticulocytes/metabolism , Ribosomes/metabolism , Anti-Bacterial Agents/pharmacology , Autoradiography , Carbon Isotopes , Fusidic Acid/pharmacology , Guanine Nucleotides/metabolism , Hydrolysis , Methionine/metabolism , Peptide Chain Elongation, Translational , Peptide Chain Initiation, Translational , Protein Binding , Puromycin/metabolism , RNA, Transfer , Reticulocytes/cytology , TritiumSubject(s)
Hemoglobins , RNA, Messenger , Ribosomes , Thalassemia , Animals , Base Sequence , Cell-Free System , Globins/physiology , Humans , Methods , RNA, Messenger/physiology , RNA, Ribosomal , Rabbits , Rats , Thalassemia/etiologyABSTRACT
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-tRNA(F), fMet-tRNA(F), or N-acetylPhe-tRNA are used as substrate at low Mg(++) concentration (3 mM), reticulocyte initiation factors M(1) and M(2) (M(2A) + M(2B)) are required for puromycin-peptide synthesis. In contrast to bacterial systems, this reaction is also stimulated by the elongation factor T(1). When Met-tRNA(M) or Phe-tRNA is used as substrate, there is no M-factor requirement for the puromycin reaction; T(1) is absolutely required, and the reaction is stimulated by T(2). These studies indicate that reticulocyte factors M(1) and M(2) 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.
