Hemin control of globin synthesis: An assay for the inhibitor formed in the absence of hemin and some characteristics of its formation

Maxwell, Charles R.; Kamper, C.; Rabinovitz, M.

doi:10.1016/0022-2836(71)90249-x

Cited by 135 publications

(39 citation statements)

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“…From the characterization of the complex described above, a function of ribosomal particles in the 80S complex seems to have been impaired, although it is uncertain whether such an accumulation is attributable to some components in the ribosomal wash. These findings, however, cannot be considered to be caused by the action of hemin-controlled repressor (Maxwell et al, 1971;Adamson et al, 1972;Gross and Rabinovitz, 1972) since the inhibition of globin synthesis under hemin-deficient conditions was shown to result from impaired binding of the initiator tRNA to 40S ribosomal subunits by hemin-controlled repressor (Legon et al, 1973;Balkow et al, 1973). By use of a highly purified preparation of the hemin-controlled repressor, more recently, it was further shown to be a 3':5'-cyclic AMP-independent protein kinase phosphorylating the small subunit of the initiation factor that forms a ternary complex with Met-tRNA1 and GTP (Kramer et al, 1976;Levin et al, 1976;Raunu and London, 1976).…”

mentioning

confidence: 90%

ACCUMULATION OF MET-tRNA-80S RIBOSOME COMPLEX IN A CELL-FREE SYSTEM OF GLOBIN SYNTHESIS

Takemoto¹,

Yoshida²

1977

JJMSB

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confidence: 90%

ACCUMULATION OF MET-tRNA-80S RIBOSOME COMPLEX IN A CELL-FREE SYSTEM OF GLOBIN SYNTHESIS

Takemoto¹,

Yoshida²

1977

JJMSB

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“…The reaction mixtures, containing 100-300 x lo3 counts/min of 35S, were analysed on gradients and the total radioactivity associated with the 40-S subunit peak was calculated as in lysate 45 2 300 37 lysate 37 2 4160 37 lyaate 45 2 4370 45 lysate 37 2 4185 45 lysate 45 2 3800 tion. The haemin-controlled repressor [8] is a possible candidate for this inhibitor. Addition of homologous 0.5 M KC1 ribosomal washes to native 40-S subunits generally increased the level of [35S]Met-tRNAf binding, by the subunits, 5 -20-Sold.…”

Section: -S-subunit Initiation Complexmentioning

confidence: 99%

“…Moreover, addition of optimal amounts of control initiation factors, contained in the 0.5 M KCl ribosomal wash of unincubated lysates, to the 45 "C preheated lysates [2,6] or of a mixture of partially purified factors, including the Met-tRNAf binding factor, to preheated KCIwashed ribosomes and reticulocyte supernatant [7], fail to restore their rate of protein synthesis to the control level when subsequently assayed at 30-37 T. Incubation in the absence of haemin on the other hand results in the formation of a soluble inhibitor [4,8], identified as a protein kinase [9,10], which phosphorylates the small subunit of the initiation factor eIF-2; this inhibition can be overcome by the addition of large amounts of initiation factors [l I], notably eIF-2 [12-141. However, the question of whether the two processes, i.e.…”

mentioning

confidence: 99%

Reduced Formation of Initiation Complexes between Met-tRNAf and 40-S Ribosomal Subunits in Rabbit Reticulocyte Lysates Incubated at Elevated Temperatures. Activity of the Met-tRNAf Binding Factor

et al. 1978

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Rabbit reticulocyte lysates preincubated at 42 -45 "C and subsequently assayed for protein synthesis at 37 "C show (a) a decrease in their rate of polypeptide chain initiation and (b) decreased Met-tRNAf binding to native 40-S subunits and 80-S ribosomes. Similarly, the amount of [3sS]Met-tRNAf . 40-S-subunit initiation complexes formed in vitro with native 40-S subunits isolated from supraoptimally heated lysates is only 10 -30 of that formed with native 40-S subunits prepared from control lysates preincubatedat 0 "C or 37 "C. This decrease is not due to reduced formation of Met-tRNAr ternary complex or to increased destruction of this complex, since we find that 0.5 M KC1 ribosomal washes extracted from lysates preincubated at 0 "C, 37 "C, or 43-45 C have almost identical activities in Met-tRNAf . GTP . eIF-2 ternary complex formation and Met-tRNAf hydrolysis, whether assayed at 37 "C or 45 "C. Similarly, the results of binding assays of [35S]Met-tRNAf to 40-S subunits performed at both temperatures show that the control and heated washes, added at either limiting or optimal amounts, stimulate almost equally the binding of Met-tRNAf to (a) salt-washed derived 40-S subunits and (b) native 40-S subunits, prepared from control or supraoptimally heated lysates. Moreover the factor-directed binding of Met-tRNAf by the derived 40-S subunits is not affected by the presence of either the control or heated postribosomal supernatant. We conclude that at elevated temperatures there is no irreversible inactivation of the Met-tRNAf binding factor for ternary complex formation and Met-tRNAf binding to 40-S subunits in vitro and discuss some possible explanations for the lowered Met-tRNAf binding observed in vivo.Incubation of rabbit reticulocyte lysates at 42 -45 "C leads to a rapid decrease in their rate of protein synthesis with a concomittant breakdown of polysomes and a reduction in the amount of [35S]Met-tRNAf . 40-S-ribosomal-subunit initiation complex formed [1,2]. This and other evidence implicates a lesion in polypeptide chain initiation which superficially resembles that seen in lysates incubated without haemin [3 -51. We have already presented evidence that the haemin-independent high-temperature-sensitive lesion in polypeptide chain initiation, although mediated by a soluble inhibitor which resembles the haemincontrolled repressor, does not lead to any apparent unit, the quantity of material contained in 1 ml of a solution which has an absorbance of 1 at 260 nm when measured in a l-cm pathlength cell.Enzyme. Creatine kinase (EC 2.7.3.2).l?efinirion.inactivation of initiation factors when assayed at 30-37 "C (see preceding paper [6]). Moreover, addition of optimal amounts of control initiation factors, contained in the 0.5 M KCl ribosomal wash of unincubated lysates, to the 45 "C preheated lysates [2,6] or of a mixture of partially purified factors, including the Met-tRNAf binding factor, to preheated KCIwashed ribosomes and reticulocyte supernatant [7], fail to restore their rate of protein synthesis to the cont...

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“…Lysates of reticulocytes obtained from phenylhydrazine-treated rabbits were prepared as described (13 (pH 7.6)-10 mM KCl-1.5 mM magnesium acetate]. An aliquot (400 Ml) was layered onto 11.5 ml of a 15-30% (w/w) sucrose gradient containing the above buffer and centrifuged for 150 min at 40 in a Spinco SW41 rotor at 40,000 rpm.…”

Section: Preparation Of a Fraction Withmentioning

confidence: 99%

“…The gradients were displaced with 40% (w/w) sucrose at 80 ml/hr, and the absorbance of the effluent at 260 nm was monitored with a Cary 14 spectrophotometer. The remaining portion was used for determination of [14C]leucine incorporation into protein as described (13).…”

Section: Preparation Of a Fraction Withmentioning

confidence: 99%

Factor-Promoted Dissociation of Free Ribosomes in a Rabbit Reticulocyte Lysate System: Inhibition and Requirement for an Energy Source

Mizuno

Rabinovitz

1973

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

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A factor that promoted the dissociation of ribosomes into ribosomal subunits in a complete globin-synthesizing system was isolated from rabbit reticulocyte ribosomes. The factor stimulated both globin synthesis and the association of the small ribosomal subunit with polyribosomes. The dissociation of free ribosomes by the factor could be followed as an independent reaction in the presence of a high concentration of cycloheximide, which inhibited translation and the resulting accumulation of runoff subunits. This reaction required an energy source and was inhibited by P-(5'-guanylyl)-methylenebisphosphonate. These data suggest that energy generated by hydrolysis of GTP may be a requirement for the dissociation reaction. Aurintricarboxylic acid inhibited the factor-mediated dissociation of free ribosomes but not the factor-mediated preservation of subunits formed on peptide-chain termination.In mammalian cells or cell preparations engaged in protein synthesis, the ribosomes of polyribosomes dissociate into subunits after completion of translation. These subunits undergo exchange with a pool of ribosomal subunits (1-5). Single ribosomes, however, do not readily enter polyribosomes nor do their components exchange with the pool of ribosomal subunits (1-6). When initiation of protein synthesis is decreased, single ribosomes accumulate at the expense of polyribosomes, but essentially a constant amount of subunits is maintained. This change in the ribosome cycle is reversible; single ribosomes return to the polyribosome component and again engage in protein synthesis (7-9). These observations indicate the existence of a mechanism through which free single ribosomes dissociate into subunits which then function in the initiation of peptide-chains.We report here that a fraction that contains an initiation factor (IF) from rabbit reticulocyte ribosomes has dissociation factor (DF) activity for these ribosomes in a complete protein-synthesizing system. This factor also promotes the engagement of the dissociated subunits in globin synthesis. Under conditions of active protein synthesis, it is not possible to discriminate between a role of DF in preservation of subunits released from polyribosomes after peptide chain termination and active dissociation of the free ribosome pool, which is formed by combination of such subunits. Differential inhibition of each of the above reactions is described, and an energy source (or GTP) requirement for dissociation of free ribosomes is established. This factor is shown to participate Abbreviations: DF, ribosomal dissociation factor; IF, peptide chain initiation factor; GDP-CH2P, P-(5'-guanylyl)methylenebisphosphonate. 787 both in the dissociation of free ribosomes and in the preservation of subunits formed after peptide chain termination. METHODS Preparation of a Fraction with

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Hemin control of globin synthesis: An assay for the inhibitor formed in the absence of hemin and some characteristics of its formation

Cited by 135 publications

References 10 publications

ACCUMULATION OF MET-tRNA-80S RIBOSOME COMPLEX IN A CELL-FREE SYSTEM OF GLOBIN SYNTHESIS

ACCUMULATION OF MET-tRNA-80S RIBOSOME COMPLEX IN A CELL-FREE SYSTEM OF GLOBIN SYNTHESIS

Reduced Formation of Initiation Complexes between Met-tRNAf and 40-S Ribosomal Subunits in Rabbit Reticulocyte Lysates Incubated at Elevated Temperatures. Activity of the Met-tRNAf Binding Factor

Factor-Promoted Dissociation of Free Ribosomes in a Rabbit Reticulocyte Lysate System: Inhibition and Requirement for an Energy Source

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