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

Previous data demonstrated that reovirus mRNA, synthesized in vitro with the particulate RNA transcriptase of reovirus cores, efficiently directs the synthesis of polypeptides in vitro. The ability to synthesize reovirus messenger RNA (mRNA) in vitro in quantity (1-4) is useful for a study of the mechanism of protein initiation in mammalian systems. In eukaryotic cells there exist two major methionyl-tRNA species (5-10) that have been identified, by several criteria, as MettRNAF and Met-tENAx. As for the bacterial initiator tRNA, eukarytic Met-tRNAF is an initiator of protein synthesis in eukaryotic cell-free systems (8, 11-18) and, in contrast to Met-tRNAm, can be acylated and formylated by bacterial enzymes (7,8,13,(16)(17)(18)(19)(20) In the studies described here, we observed that purified rat liver Met-tRNAF, but not rat liver Met-tRNAm, formed an initiation complex with ribosomal subunits that was dependent upon the presence of reovirus mRNA. The mRNAdependent complex reacted with puromycin to form methionyl-puromycin in the same manner as the initiation complexes formed in the presence of poly(A,G,U) or the initiator triplet AUG. METHODSThe growth of mouse fibroblast L-929 cells, infection by reovirus (Dearing strain, Type 3), the preparation of reovirus RNA transcriptase, and the synthesis in vitro of reovirus mRNA have been described (4).Ribosomal Subunits from L-929 Cells. All operations were performed at 0-40, unless otherwise indicated. In a standard preparation, 109 L-929 cells were harvested during exponential growth, and extracts were prepared by homogenization in 15 ml of 10 mM Tris HCl (pH 7.5)-10 mM KCl-1.5 mM Mg-C12-1 mM dithiothreitol as described (4). The extract was brought to 30 mM Tris HOl (pH 7.5)-130 mM KCI-6 mM MgCl2-3 mM dithiothreitol (TKM buffer). The mitochondria were removed by centrifugation. The extract was supplemented with 20 amino acids, ATP, GTP, creatine phosphate, phosphocreatine kinase, and 1 mM puromycin, and incubated at 370 for 50 min. The reaction mixture was made 0.8% in deoxycholate and centrifuged overnight at 160,000 X g through a cushion of 68% sucrose in TKM buffer. The pellet was suspended in 3 ml of 50 mM triethanolamine-HCl (pH 7.6)-500 mM KCl-1.5 mM MgCl2-1 mM puromycin, incubated at 00 for 10 min and at 370 for 10 min, and then centrifuged at 200 in 10-30% sucrose gradients in 50 mM triethanolamine-HCl (pH 7.6)-500 mM KCI-5 mM MgCl2 for 5.5 hr at 64,000 X g (22). The gradients were monitored for A20, and the 40S and 60S subunit areas were separately collected and centrifuged at 160,000 X g overnight through a cushion of 40% sucrose in TKM buffer. The subunit pellets were each resuspended in TKM buffer containing 20% glycerol and could be stored at 00 for 2 weeks without significant loss of activity.Preparation of Rat Liver [*S1]Met-tRNAF and [n5S]MettRNAm. Rat liver tRNA (110 mg) was fractionated by chromatography on BD-cellulose, as described for wheat germ tRNA by Leis and Keller (9). The location of the two major tRNAMet peaks in the A20 profile ...

Section: Discussionsupporting

confidence: 70%

Formation of a Mammalian Initiation Complex with Reovirus Messenger RNA, Methionyl-tRNA _F , and Ribosomal Subunits

Levin

Kyner

Acs

1972

“…For the Millipore filter-binding assays and the puromycin assays, IF-Ml was further purified by Sephadex G-200 chromatography (7). The IF-M2 preparation was not further fractionated; it contains both M2A and M2B (4,5). For all assays except the dipeptide assay on exogenous mRNA, IF-M3 was further purified by phosphocellulose chromatography (9).…”

Section: Methodsmentioning

confidence: 99%

“…In the synthesis of the artificial dipeptide, MetF-puromycin, on ApUpG, GTP is strictly required, since GDPCP will not substitute for GTP. Therefore, at least one GTP hydrolysis step is involved in the formation of this peptide bond (5 (Fig. 1, bottom, right).…”

Section: Initial Tripeptide Synthesismentioning

confidence: 99%

“…This strong association has enabled us to develop natural models of the individual steps during the initiation of globin synthesis on naturally occurring endogenous mRNA (2). In order to test the validity of the model reactions in predicting the requirements for mammalian initiation, we have compared the factor and energy requirements of the natural models that use endogenous (and exogenous) globin mRNA with the requirements of artificial models that use artificial templates (3)(4)(5).…”

mentioning

confidence: 99%

See 1 more Smart Citation

Initiation of Hemoglobin Synthesis: Comparison of Model Reactions That Use Artificial Templates with Those Using Natural Messenger RNA

Crystal

Anderson

1972

Partial reactions designed to study the individual steps in the initiation of protein synthesis may be divided into two groups: artificial models using artificial mRNA templates and natural models using naturally occurring mRNA. The requirements for each of the reticulocyte initiation factors, IF-M1, IF-M2, and IF-Ms, and for GTP are examined using these models in order to determine if either type of model is a valid representation of the events occurring in natural initiation.Binding of the initiator tRNA, Met-tRNAF, to endogenous mRNA requires IF-Ml, IF-M2, and GTP. A requirement for hydrolysis of GTP is not found when the artificial template ApUpG is used since GDPCP may be substituted for GTP. Met-tRNAF bound to the template ApUpG by IFMl + IF-M2 can form a peptide bond with the aminoacyltRNA analog, puromycin. Met-tRNAF bound by IF-Ml + IF-M2 to the initiator codon of natural globin mRNA, however, cannot form a peptide bond with either puromycin or valine-tRNA unless IF-M, is also present. The requirements for MetF-valine synthesis on exogenous globin mRNA are the same as the requirements on endogenous mRNA. Synthesis of the initial tripeptides of the a and j3 chains of rabbit globin, MetF-Val-Leu and MetF-Val-His, requires, in addition, leucyl-tRNA and histidyl-tRNA.It appears, therefore, that model systems that use natural messenger RNA can duplicate the factor and energy requirements of natural initiation, but that the model systems thus far studied that use artificial messengers as templates do not.The initiation of protein synthesis requires the formation of an "initiation complex," which includes a ribosome, mRNA, initiator tRNA, protein initiation factors, and an energy source. In order to understand this event, partial reactions have been developed that isolate the individual steps in the formation of the initiation complex. These partial reactions are of two types: "artificial models" and "natural models." Nucleic acid analogs of mRNA templates (e.g., artificial polynucleotides or individual codons), are used in artificial models, while naturally occurring mRNA is used as the template in natural models. In prokaryotes, the natural models have demonstrated that artificial models can provide valid concepts for some aspects of the initiation process (1).The initiation of protein synthesis in eukaryotes is similar to that in prokaryotes in that initiation factors, initiator tRNA, and GTP are required. Unlike association between naturally occurring mRNA and ribosomes in reticulocytes is unusually strong. This strong association has enabled us to develop natural models of the individual steps during the initiation of globin synthesis on naturally occurring endogenous mRNA (2). In order to test the validity of the model reactions in predicting the requirements for mammalian initiation, we have compared the factor and energy requirements of the natural models that use endogenous (and exogenous) globin mRNA with the requirements of artificial models that use artificial templates (3-5). MATERIALS A...

“…The data in Table 3 indicate that IF-M2B stimulates fMet-tRNAF binding by 40% in the presence of saturating concentrations of IF-M1, while IF-M2A has no effect. The stimulatory effect of IF-M2B is independent of GTP (Table 3), and is also observed at 10 mM Mg++ (data The role of GTP in the partial reactions of protein synthesis involving the reticulocyte initiation factors has been examined in previous publications (7,9). It is of interest to note here, however, that LeLong et al (15) have reported a separation of Escherichia coli initiation factor C (IF-2) into two components, "C1" and "C2", by DEAE-Sephadex chromatography.…”

mentioning

confidence: 98%

Separation of Reticulocyte Initiation Factor M ₂ Activity into Two Components

Shafritz

Prichard

Gilbert

et al. 1972

IF-M2, one of three initiation factors isolated by DEAE-cellulose chromatography from the 0.5 M KCl-wash fraction of rabbit reticulocyte ribosomes, has been separated by (3), reticulocyte elongation factors EF-1 and EF-2 (5), and the aminoacyltRNA species (8) were prepared as described.Preparation of IF-Ml. IF-Ml was purified from the 0.5 M KCl-wash fraction from ribosomes essentially as described previously (Table 3, (Fig. 1). 36 mg of IF-M2 protein in a 3-ml volume was chromatographed on a 1.5 X 86-cm Sephadex G-200 column equilibrated with 10 mM Tris * HCl (pH 7.5)-100 mM KC1-1 mM dithiothreitol-0.1 mM EDTA. Protein was eluted with the same buffer at a flow rate of 10 ml/hr; 2-ml fractions were collected. Activity