Regulation of protein synthesis in rabbit reticulocyte lysates by the heme-regulated protein kinase

R. S. Ranu, I. M. London, A. Das, A. Dasgupta, A. Majumdar, R. Ralston, Reena Roy, N. K. Gupta

Research output: Contribution to journalArticle

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Abstract

Protein synthesis in reticulocytes and their lysates is regulated by heme. In heme deficiency a heme-regulated translational inhibitor (HRI) that blocks initiation of polypeptide chains is activated. HRI is a protein kinase (ATP: protein phosphotransferase, EC 2.7.2.37) that specifically phosphorylates the 38,000-dalton subunit of the Met-tRNAf(Met) binding factor (IF), which forms a ternary complex with Met-tRNAf(Met) and GTP, a finding that suggests that the inhibition by HRI involves the phosphorylation of IF. We have investigated the effect of HRI in the partial reactions of protein chain initiation in which the IF-promoted binding of Met-tRNAf(Met) to 40S ribosomal subunits is enhanced by another initiation factor [ternary complex dissociation factor (TDF)] and AUG. The results show that HRI at very low concentrations markedly inhibits the binding of Met-tRNAf(Met) to 40S subunits. The inhibitory effect of HRI requires ATP. Under these conditions HRI phosphorylates only the 38,000-dalton subunit of IF. The TDF preparations not only promote the binding of the ternary complex to 40S subunits but also promote the dissociation of the ternary complex in the presence of 5mM Mg2+ at 0°. The preincubation of purified IF alone with low concentrations of HRI and ATP does not significantly affect its capacity to form the ternary complex; however, the TDF-promoted dissociation of the ternary complex is inhibited. The non hydrolyzable analog adenosine 5'-[β, γ-imido]triphosphate does not substitute for ATP. These findings suggest that phosphorylation causes a conformational modification in IF, whih results in inhibition of the interaction between the ternary complex and TDF that is required for the binding of the ternary complex to 40S subunits.

Original languageEnglish (US)
Pages (from-to)745-749
Number of pages5
JournalUnknown Journal
Volume75
Issue number2
DOIs
StatePublished - Jan 1 1978

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Reticulocytes
Heme
Protein Kinases
Rabbits
Ternary Complex Factors
Proteins
Adenosine Triphosphate
Eukaryotic Small Ribosome Subunits
Phosphorylation
Peptide Initiation Factors
Guanosine Triphosphate
Adenosine
Peptides

All Science Journal Classification (ASJC) codes

  • General

Cite this

Ranu, R. S., London, I. M., Das, A., Dasgupta, A., Majumdar, A., Ralston, R., ... Gupta, N. K. (1978). Regulation of protein synthesis in rabbit reticulocyte lysates by the heme-regulated protein kinase. Unknown Journal, 75(2), 745-749. https://doi.org/10.1073/pnas.75.2.745
Ranu, R. S. ; London, I. M. ; Das, A. ; Dasgupta, A. ; Majumdar, A. ; Ralston, R. ; Roy, Reena ; Gupta, N. K. / Regulation of protein synthesis in rabbit reticulocyte lysates by the heme-regulated protein kinase. In: Unknown Journal. 1978 ; Vol. 75, No. 2. pp. 745-749.
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abstract = "Protein synthesis in reticulocytes and their lysates is regulated by heme. In heme deficiency a heme-regulated translational inhibitor (HRI) that blocks initiation of polypeptide chains is activated. HRI is a protein kinase (ATP: protein phosphotransferase, EC 2.7.2.37) that specifically phosphorylates the 38,000-dalton subunit of the Met-tRNAf(Met) binding factor (IF), which forms a ternary complex with Met-tRNAf(Met) and GTP, a finding that suggests that the inhibition by HRI involves the phosphorylation of IF. We have investigated the effect of HRI in the partial reactions of protein chain initiation in which the IF-promoted binding of Met-tRNAf(Met) to 40S ribosomal subunits is enhanced by another initiation factor [ternary complex dissociation factor (TDF)] and AUG. The results show that HRI at very low concentrations markedly inhibits the binding of Met-tRNAf(Met) to 40S subunits. The inhibitory effect of HRI requires ATP. Under these conditions HRI phosphorylates only the 38,000-dalton subunit of IF. The TDF preparations not only promote the binding of the ternary complex to 40S subunits but also promote the dissociation of the ternary complex in the presence of 5mM Mg2+ at 0°. The preincubation of purified IF alone with low concentrations of HRI and ATP does not significantly affect its capacity to form the ternary complex; however, the TDF-promoted dissociation of the ternary complex is inhibited. The non hydrolyzable analog adenosine 5'-[β, γ-imido]triphosphate does not substitute for ATP. These findings suggest that phosphorylation causes a conformational modification in IF, whih results in inhibition of the interaction between the ternary complex and TDF that is required for the binding of the ternary complex to 40S subunits.",
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Ranu, RS, London, IM, Das, A, Dasgupta, A, Majumdar, A, Ralston, R, Roy, R & Gupta, NK 1978, 'Regulation of protein synthesis in rabbit reticulocyte lysates by the heme-regulated protein kinase', Unknown Journal, vol. 75, no. 2, pp. 745-749. https://doi.org/10.1073/pnas.75.2.745

Regulation of protein synthesis in rabbit reticulocyte lysates by the heme-regulated protein kinase. / Ranu, R. S.; London, I. M.; Das, A.; Dasgupta, A.; Majumdar, A.; Ralston, R.; Roy, Reena; Gupta, N. K.

In: Unknown Journal, Vol. 75, No. 2, 01.01.1978, p. 745-749.

Research output: Contribution to journalArticle

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T1 - Regulation of protein synthesis in rabbit reticulocyte lysates by the heme-regulated protein kinase

AU - Ranu, R. S.

AU - London, I. M.

AU - Das, A.

AU - Dasgupta, A.

AU - Majumdar, A.

AU - Ralston, R.

AU - Roy, Reena

AU - Gupta, N. K.

PY - 1978/1/1

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N2 - Protein synthesis in reticulocytes and their lysates is regulated by heme. In heme deficiency a heme-regulated translational inhibitor (HRI) that blocks initiation of polypeptide chains is activated. HRI is a protein kinase (ATP: protein phosphotransferase, EC 2.7.2.37) that specifically phosphorylates the 38,000-dalton subunit of the Met-tRNAf(Met) binding factor (IF), which forms a ternary complex with Met-tRNAf(Met) and GTP, a finding that suggests that the inhibition by HRI involves the phosphorylation of IF. We have investigated the effect of HRI in the partial reactions of protein chain initiation in which the IF-promoted binding of Met-tRNAf(Met) to 40S ribosomal subunits is enhanced by another initiation factor [ternary complex dissociation factor (TDF)] and AUG. The results show that HRI at very low concentrations markedly inhibits the binding of Met-tRNAf(Met) to 40S subunits. The inhibitory effect of HRI requires ATP. Under these conditions HRI phosphorylates only the 38,000-dalton subunit of IF. The TDF preparations not only promote the binding of the ternary complex to 40S subunits but also promote the dissociation of the ternary complex in the presence of 5mM Mg2+ at 0°. The preincubation of purified IF alone with low concentrations of HRI and ATP does not significantly affect its capacity to form the ternary complex; however, the TDF-promoted dissociation of the ternary complex is inhibited. The non hydrolyzable analog adenosine 5'-[β, γ-imido]triphosphate does not substitute for ATP. These findings suggest that phosphorylation causes a conformational modification in IF, whih results in inhibition of the interaction between the ternary complex and TDF that is required for the binding of the ternary complex to 40S subunits.

AB - Protein synthesis in reticulocytes and their lysates is regulated by heme. In heme deficiency a heme-regulated translational inhibitor (HRI) that blocks initiation of polypeptide chains is activated. HRI is a protein kinase (ATP: protein phosphotransferase, EC 2.7.2.37) that specifically phosphorylates the 38,000-dalton subunit of the Met-tRNAf(Met) binding factor (IF), which forms a ternary complex with Met-tRNAf(Met) and GTP, a finding that suggests that the inhibition by HRI involves the phosphorylation of IF. We have investigated the effect of HRI in the partial reactions of protein chain initiation in which the IF-promoted binding of Met-tRNAf(Met) to 40S ribosomal subunits is enhanced by another initiation factor [ternary complex dissociation factor (TDF)] and AUG. The results show that HRI at very low concentrations markedly inhibits the binding of Met-tRNAf(Met) to 40S subunits. The inhibitory effect of HRI requires ATP. Under these conditions HRI phosphorylates only the 38,000-dalton subunit of IF. The TDF preparations not only promote the binding of the ternary complex to 40S subunits but also promote the dissociation of the ternary complex in the presence of 5mM Mg2+ at 0°. The preincubation of purified IF alone with low concentrations of HRI and ATP does not significantly affect its capacity to form the ternary complex; however, the TDF-promoted dissociation of the ternary complex is inhibited. The non hydrolyzable analog adenosine 5'-[β, γ-imido]triphosphate does not substitute for ATP. These findings suggest that phosphorylation causes a conformational modification in IF, whih results in inhibition of the interaction between the ternary complex and TDF that is required for the binding of the ternary complex to 40S subunits.

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