Studies on the initiation of protein synthesis in animal tissues

Kerwar, S.S.; Spears, Carlos; Weissbach, Herbert

doi:10.1016/0006-291x(70)90471-7

Cited by 59 publications

(23 citation statements)

References 11 publications

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“…Thus, the T-factor-induced association of both Met-tRNAMet species depends on the presence of a code-bearing polynucleotide, whereas the binding of formylated or unformylated initiator tRNA mediated by initiation factors functions equally well with just the codon ApUpG. In agreement with other observations [5] we found that neither T factor nor ribosomal wash stimulated the ribosomal attachment of MettRNAMet in the presence of the triplet.…”

Section: Binding Experimentssupporting

confidence: 91%

“…The second Met-tRNA species (Met-tRNAMet) present in eukaryotic cells was shown to be involved exclusively in peptide chain elongation, inserting methionine into internal positions of the nascent polypeptide chain [I -31. Studies on the enzymatic binding of both methionine-accepting tRNA species to ribosomes appeared to be in agreement with this concept [4,5].…”

mentioning

confidence: 59%

“…It had previously been reported [4] that the triplet ApUpG does not function in the binding of aminoacyl tRNAs to mammalian ribosomes promoted by T factor. I n contrast, ApUpG has been shown to stimulate the binding of Met-tRNAfMet to rabbit reticulocyte ribosomes in the presence of initiation factors [4,5]. I n view of these results it seemed warranted to repeat our binding experiments using the triplet ApUpG instead of poly(A,U,G,) As demonstrated in Table 2, ApUpG did not stimulate the Tfactor-induced binding of either Met-tRNAMet species to ribosomes.…”

Section: Binding Experimentsmentioning

confidence: 84%

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Function of Met‐tRNA^Met_f and Met‐tRNA^Met in Peptide‐Chain Elongation in Cell‐Free Systems from Mouse‐Liver and Ascites‐Tumor Cells

Drews

Grasmuk

Weil

1972

European Journal of Biochemistry

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The two methionine-accepting tRNA species from mouse ascites tumor cells were investigated with respect to their function in peptide chain elongation, using a mouse liver polysome system and a T-factor dependent ribosomal-binding assay. I n support of results published previously it was found that in a non-initiating cell-free system Met-tRNAfMet inserts methionine into internal positions of nascent peptide chains, even in the presence of Met-tRNAMet.Correspondingly, T factor from ascites tumor cells stimulated the binding of both methionineaccepting tRNAs to washed ribosomes. When Met-tRNAMet and Met-tRNAfMet were introduced together into the ribosomal binding assay, a strong preference for the T-factor-induced attachment of Met-tRNAMet became visible. Formylated initiator tRNA was not recognized by T factor Whereas the ribosomal binding of formylated or unformylated initiator tRNA promoted by initiation factors functioned equally well with poly(A,U,G) and the triplet ApUpG, the T-factor-induced attachment of both Met-tRNAMet species required the presence of the polynucleotide.Competition experiments with high concentration of Val-tRNAVa1 and Met-tRNAfMet revealed the occurrence of a partial mutual interference of these two tRNA species during chain elongation.The magnitude of this effect, however, was not sufficient to attribute the function of MettRNAtMet in chain elongation to miscoding. No competition was observed between Val-tRNAVa1 and Met-tRNAMet.Recent studies from several laboratories suggest that eukaryotic proteins, like the bacterial ones, are initiated by a specific methionine tRNA. This species (Met-tRNAfMet) can be formylated by transformylase from Escherichia coli and has been shown to respond to an ApUpG or GpUpG triplet located a t or close to the 5' end of synthetic polynucleotides. The second Met-tRNA species (Met-tRNAMet) present in eukaryotic cells was shown to be involved exclusively in peptide chain elongation, inserting methionine into internal positions of the nascent polypeptide chain [I -31. Studies on the enzymatic binding of both methionine-accepting tRNA species to ribosomes appeared to be in agreement with this concept [4,5].I n contrast, we recently demonstrated that in a cell-free system from mouse liver, Met-tRNAfMet Abbreviations. Met-tRNAf Met, methionyl-tRNA which can be converted enzymically to formyl-methionyl-tRNA; Met-tRNAMet, tRNA which cannot be formylated enzymically; poly(A,U,G), polyriboadenylic-uridylic-guanylic acid; ApUpG, adenyl-uridyl-guanosine ; GpUpG, guanyl-uridylguanosine; Met-Gly-Leu, methionyl-glycyl-leucine.Definition. A,,, unit, the quantity of material contained in 1 ml of a solution which has an absorbance of 1 at 260 nm, when measured in B I-cm path-length cell.is highly active in peptide chain elongation [6]. The possibility that the incorporation of methionine from Met-tRNAfMet was due to initiation of protein synthesis could be excluded by Edman degradation and cyanogen bromide cleavage of the synthesized material : both methods failed to demonst...

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Section: Binding Experimentssupporting

confidence: 91%

mentioning

confidence: 59%

Section: Binding Experimentsmentioning

confidence: 84%

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Function of Met‐tRNA^Met_f and Met‐tRNA^Met in Peptide‐Chain Elongation in Cell‐Free Systems from Mouse‐Liver and Ascites‐Tumor Cells

Drews

Grasmuk

Weil

1972

European Journal of Biochemistry

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“…We are very grateful to them tbr making available to us the radioactively labelled 4 S-RNA from their cell extracts for use as our source material. In common with file tRNA~ let from rabbit liver [2,3], guinea pig liver [5], mouse ascites turnout calls [ I ] and yeast [4], a methionine-accepting species from these cells can be both aminoacylated with methionine by purified E. col[ methionyl tRNA synthetase (a gift from Drs. C.J.…”

mentioning

confidence: 99%

Lack of Tpψp in loop IV of a mammalian initiator transfer RNA

Piper

Clark

1973

FEBS Letters

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Two classes of methionine-accepting transfer RNA have been characterised from the cytoplasm of mammalian cells. As in other eukaryotic systems, one of these (tRNA Met) is found to donate methionine into the internal posttions of protein cimins, whereas the other, the initiator (tRNA~-~et), acts as a specific donor for the N-terminal methionine residue [band in newly synthesized polypeptides [1][2][3]. MethionyltRNA~ let in the cytoplasm 0feukaryotes, unlike that orE. col[ and mitochondria, does not need to be formylated before it can donate methionine into the N-ternfinal positions of proteins. We are attempting to relate the structure of a eukaryotic initiator tRNA to its role in the cell with ~e aim of explaining the differences in its be!tar[our front the bacterial initiator tRNA. At present we are determining the nucleotide sequence of the tRbrA~ et from mou~ P3 myeloma ceils.Very recently the nucleotide sequence of the first eukaryotic initiator tRNA has been determined by Simsek and RajBhanda~ [4]. This yeast tl~NAf Met has a feature distinct from aU other tRNA's of known sequences functioning in protein biosynthesis: the common -TGpTp~pCp-sequence found in loop IV of the cloverleaf structure it replaced in yeast tRNA~ tot by the sequence -GpAp-UpCp-. Now we have also found the latter sequence in a mammalian initiator ti~NA replacing the common sequence confirming the notion that this region of the molecule is concerned with the special initiation role. Methods and resultsThe mouse P3 myelo~zqa ceils have been labeled with [32p]phosphate in tts~-'te culture in this laboratory in studies en the structure of myeloma faessenger RNA by Dzs. G.G. Brownlee and C. Milstein. We are very grateful to them tbr making available to us the radioactively labelled 4 S-RNA from their cell extracts for use as our source material. In common with file tRNA~ let from rabbit liver [2,3], guinea pig liver [5], mouse ascites turnout calls [ I ] and yeast [4], a methionine-accepting species from these cells can be both aminoacylated with methionine by purified E. col[ methionyl tRNA synthetase (a gift from Drs. C.J. Bruton and G. Koch) as welt as formylated by the b~L col[ tr=as-formylase [6l. This property of formylation by the bacterial enzyme has been used to identify t~te fractionated tRNA Met as an initiator tRNA~ let. The 32p-labelled mouse P3 cell tlC.NA~ let has been purified by chromatography on reverse phase RPC-5 columns 16], and characterized by twodimensional fractionation of the ribonuclea~ 6i-gestion fragments using the procedures developed by Brownlee et al. [7]. The oligonucleotide map yielded by RNAase T t digestion of the tRNA~f let is illustrated in fig. I. 14 oligonucleotides including two identified as the 5'-end (pApGp) and 3'-end (CpUpApCpCpA) oligonucleotides ate released from the sample by RNAase T t digestion. This corresponds to the number which might bc expected from a single species of tRNA and permits the approximate length of 75 nucleotides to be estimated front a comparison of the yields with the ...

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“…The results of Kerwar and Weissbach:in vitro [15] and of Brown in vivo [16] show that initiation is possible when methionine is replaced by its analogues ethionine and norleucine. In the work reported here we emphasize that methionine may also be replaced in vitro by structurally unrelated amino acids such as phenylalanine or valine.…”

Section: Discussionmentioning

confidence: 98%

Initiation of protein synthesis with mischarged tRNA^Met_f fromE. coli

et al. 1973

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Studies on the initiation of protein synthesis in animal tissues

Cited by 59 publications

References 11 publications

Function of Met‐tRNA^Met_f and Met‐tRNA^Met in Peptide‐Chain Elongation in Cell‐Free Systems from Mouse‐Liver and Ascites‐Tumor Cells

Function of Met‐tRNA^Met_f and Met‐tRNA^Met in Peptide‐Chain Elongation in Cell‐Free Systems from Mouse‐Liver and Ascites‐Tumor Cells

Lack of Tpψp in loop IV of a mammalian initiator transfer RNA

Initiation of protein synthesis with mischarged tRNA^Met_f fromE. coli

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Studies on the initiation of protein synthesis in animal tissues

Cited by 59 publications

References 11 publications

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

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

Lack of Tpψp in loop IV of a mammalian initiator transfer RNA

Initiation of protein synthesis with mischarged tRNAMetf fromE. coli

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Function of Met‐tRNA^Met_f and Met‐tRNA^Met in Peptide‐Chain Elongation in Cell‐Free Systems from Mouse‐Liver and Ascites‐Tumor Cells

Function of Met‐tRNA^Met_f and Met‐tRNA^Met in Peptide‐Chain Elongation in Cell‐Free Systems from Mouse‐Liver and Ascites‐Tumor Cells

Initiation of protein synthesis with mischarged tRNA^Met_f fromE. coli