Initiation of protein synthesis II a convenient assay for the ribosome-dependent synthesis of N-formyl-C14-methionylpuromycin

Leder, Philip; Bursztyn, Hela

doi:10.1016/0006-291x(66)90586-9

Cited by 350 publications

(54 citation statements)

References 7 publications

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“…The first step, which involves the binding of fMet-tRNA to the E. coli ribosomes, was an incubation for 30 min at 300. The reaction mixture contained, in a total volume of 250 ,ul: 50 mM Tris-acetate (pH 7.4), 10 (25). The fMet-tRNA -AUG. ribosome complex formed in the above incubation was then used as a substrate to study the effect of L7 and L12 on the release of formylmethionine from the ribosome.…”

Section: Methodsmentioning

confidence: 99%

The Requirement for Ribosomal Proteins L7 and L12 in Peptide-Chain Termination

Brot¹,

Tate

Caskey

et al. 1974

Proc. Natl. Acad. Sci. U.S.A.

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Proteins L7 and L12 from 50S ribosomal subunits of Escherichia coli are required for peptidechain termination. This termination process is inhibited by thiostrepton. Since both thiostrepton-treated ribosomes and those depleted of L7 and L12 have a markedly reduced ability to form release factor UA[3H]A ribosome complexes, the binding of release factors to the ribosome appears to be the primary site of inhibition.The ribosomal proteins L7 and L12 of Escherichia coli have been shown to be involved in the elongation factor Tu (EF-Tu)-dependent binding of aminoacyl-tRNA to ribosomes (1, 2) and in the elongation factor G (EF-G)-dependent hydrolysis of GTP (3-5). These same reactions are also inhibited by the antibiotic thiostrepton (6)(7)(8)(9)(10)(11)(12). In addition, as initially suggested by Hamel et al. (1), L7 and L12 are also required for the enzymatic binding of fMet-tRNA to ribosomest (13). Since these reactions all involve hydrolysis of the -y-phosphate of GTP and since L7 and L12 do not affect ribosomal peptidyl transferase activity (1), it appears that L7 and L12 might be required for reactions involving GTP hydrolysis dependent on a soluble factor and the 50S ribosomal subunit. The hydrolysis of GTP is required for peptide-chain termination in vitro, as studied with reticulocyte release factor (RF) and ribosomes (14). Although no such requirement has been demonstrated for peptide-chain termination in E. coli in vitro, a protein factor, RF-3, and guanine nucleotides are known to influence the ribosomal binding of E. coli termination factors RF-1 and RF-2 (15). Recently the binding of EF-G to E. coli ribosomes, which requires L7 and L12, was shown to inhibit the interaction of RF-1 and RF-2 with ribosomes (16). For these reasons the effects of the ribosomal proteins L7 and L12 and the antibiotic thiostrepton were studied with respect to their influence on peptide-chain termination. The present report provides evidence that L7 and L12 are required for peptidechain termination in E. coli and that this process is inhibited by thiostrepton. MATERIALS AND METHODSMaterials. Ammonium chloride-washed ribosomes were isolated from E. coli B as described (17), and ribosomal subunits were prepared from these ribosomes by the procedure of Pestka and Nirenberg (18). The ribosomal proteins L7 and L12 were extracted from the 50S ribosomal subunits with an Abbreviations: L7 and L12, 50S ribosomal proteins according to the nomenclature of Kaltschmidt and Wittmann (38); release factors, RF-1, RF-2, and RF-3; elongation factor Tu, EF-Tu; elongation factor G, EF-G.

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Section: Methodsmentioning

confidence: 99%

The Requirement for Ribosomal Proteins L7 and L12 in Peptide-Chain Termination

Brot¹,

Tate

Caskey

et al. 1974

Proc. Natl. Acad. Sci. U.S.A.

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“…The ionic conditions are given in the legends to the figures. Formylmethionylpuromycin formation was measured with the ethylacetate extraction procedure [20].…”

Section: In It Iation Assaysmentioning

confidence: 99%

The Mode of Action of Thiostrepton in the Initiation of Protein Synthesis

Naaktgeboren¹,

Roobol²,

Gubbens³

et al. 1976

Eur J Biochem

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The inhibition by thiostrepton of the initiation of protein synthesis is exerted at a different level from the inhibition of reactions mediated by EF‐Tu and EF‐G in the elongation of protein synthesis. The presence of thiostrepton on the 50‐S subunit completely prevents the binding of the EFT‐Tu ? GTP ? aa‐tRNA complex and EF‐G ? GTP complex to the 70‐S ribosome, resulting in cessation of protein synthesis at a concentration of 1 μM thiostrepton. On the other hand, during initiation thiostrepton impairs the coupling of the 50‐S subunit with the 30‐S initiation complex, indirectly causing inhibition of IF‐2‐dependent reactions. Impairment of the coupling is strongly influenced by the conditions of incubation. Siace formation of formylmethionylpuromycin and the IF‐2‐dependent GTP hydrolysis are inhibited to the same extent and recycling of IF‐2 can take place in the presence of thiostrepton, we conclude that the basic mechanism of inhibition of initiation differs from that of inhibition of elongation.

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“…Incubation was continued at 37°C for another 20 min after addition of 1 mM puromycin (Calbiochem), 60S subunits (6 pmoles) and 2 µg of the same 50%-70% ammonium sulphate (A.S.) RSW fraction as described above. The reaction mixture was then diluted with 0.2 M potassium phosphate at pH 8.0 and extracted with ethyl acetate as described (Leder and Bursztyn 1966). An aliquot of the ethyl acetate phase was counted by liquid scintillation.…”

mentioning

confidence: 99%

A prokaryotic-like mode of cytoplasmic eukaryotic ribosome binding to the initiation codon during internal translation initation of hepatitis C and classical swine fever virus RNAs

Pestova¹,

Shatsky²,

Fletcher³

et al. 1998

Genes Dev.

676

787

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Initiation of translation of hepatitis C virus and classical swine fever virus mRNAs results from internal ribosomal entry. We reconstituted internal ribosomal entry in vitro from purified translation components and monitored assembly of 48S ribosomal preinitiation complexes by toe-printing. Ribosomal subunits (40S) formed stable binary complexes on both mRNAs. The complex structure of these RNAs determined the correct positioning of the initiation codon in the ribosomal ''P'' site in binary complexes. Ribosomal binding and positioning on these mRNAs did not require the initiation factors eIF3, eIF4A, eIF4B, and eIF4F and translation of these mRNAs was not inhibited by a trans-dominant eIF4A mutant. Addition of Met-tRNA i Met , eIF2, and GTP to these binary ribosomal complexes resulted in formation of 48S preinitiation complexes. The striking similarities between this eukaryotic initiation mechanism and the mechanism of translation initiation in prokaryotes are discussed. Protein synthesis begins following assembly of an initiation complex in which the initiation codon of an mRNA and the anticodon of initiator tRNA are base-paired in the ribosomal ''P'' site. There are similarities and significant differences in the mechanisms of initiation complex formation in prokaryotes and eukaryotes. A universal characteristic is that initiation starts with separated ribosomal subunits.In prokaryotes, the small (30S) ribosomal subunit binds mRNA and initiator tRNA in random order to form a complex that then undergoes conformational rearrangement, promoting codon-anticodon base-pairing at the P site and joining of the large (50S) subunit (Gualerzi and Pon 1990). Ribosome binding results from interactions between the 30S subunit and multiple recognition elements in the mRNA, such as the Shine-Dalgarno sequence (McCarthy and Brimacombe 1994). It does not depend on initiation factors. Ribosome-binding sites can occur at any position within an mRNA and as a result many prokaryotic mRNAs are polycistronic.In contrast, the small (40S) ribosomal subunit in eukaryotes requires several eukaryotic initiation factors (eIFs), first to bind initiator tRNA (as a ternary complex with eIF2 and GTP) to form a 43S complex and then to bind mRNA to form a 48S complex (Merrick 1992). The most common mechanism for recruitment of an mRNA is mediated by its capped 5Ј end, which is bound by the eIF4E subunit of eIF4F and then by the 43S complex. Ribosomal binding to mRNA and scanning to the initiation codon require ATP hydrolysis and involve eIF4A, eIF4B, and eIF4F. Most mRNAs that use this mechanism of ribosomal binding are monocistronic because initiation is usually limited to the 5Ј-most AUG codon.

show abstract

Initiation of protein synthesis II a convenient assay for the ribosome-dependent synthesis of N-formyl-C14-methionylpuromycin

Cited by 350 publications

References 7 publications

The Requirement for Ribosomal Proteins L7 and L12 in Peptide-Chain Termination

The Requirement for Ribosomal Proteins L7 and L12 in Peptide-Chain Termination

The Mode of Action of Thiostrepton in the Initiation of Protein Synthesis

A prokaryotic-like mode of cytoplasmic eukaryotic ribosome binding to the initiation codon during internal translation initation of hepatitis C and classical swine fever virus RNAs

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