Protein synthesis in rabbit reticulocytes: Requirements for Met-tRNAf·40S preinitiation complex formation with AUG-codon and physiological mRNAs

Roy, R.; Nasrin, Nargis; Ahmad, Mir F.; Gupta, Naba K.

doi:10.1016/0006-291x(84)91249-x

Cited by 11 publications

(2 citation statements)

References 10 publications

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“…and Co-eIF-2C were determined using a Bio-Rad protein assay with BSA as the standard and values for Mr(eIF-2) of 144 000 and Mr(CO-eIF-2C) of 600 000. The use of the nonhydrolyzable GTP analogue, GMP-PNP, has been reported to stabilize the ternary complex (Roy et al, 1984) and was used in lieu of GTP for the spectroscopic assays.…”

Section: Methodsmentioning

confidence: 99%

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Thermodynamic Characterization of the Cooperativity of 40S Complex Formation during the Initiation of Eukaryotic Protein Synthesis

Parkhurst¹,

Hileman²,

Saha³

et al. 1994

Biochemistry

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The first step in mammalian protein synthesis is the formation of the 40S initiation complex, composed of the 40S ribosomal subunit (R), mRNA (M, here, a 10-mer oligoribonucleotide analogue containing the initiation codon), and the quaternary complex (Q, composed of eIF-2, GTP, Met-tRNA(fMet), and the ancillary protein factor Co-eIF-2C). The interdependence of the binding of R, M, and Q in forming the 40S complex is currently unclear. We have determined the thermodynamic parameters that characterize these interactions. The binary constants for R+M and Q+M were determined spectroscopically, measuring changes in the anisotropy of the fluorescence emission of 3'-fluorescein labeled M. The other binary constant, for Q+R, and the ternary constant were determined from Millipore filtration assays using radiolabeled Met-tRNA(fMet). The association constants for the binary reactions were as follows: Ka(Q,M) < or = 0.14 x 10(6) M-1, Ka(R,M) = 1.78 x 10(6) M-1, and Ka(Q,R) = 0.94 x 10(6) M-1. The binding of Q to R.M was markedly greater than that of Q to R [Ka(Q,R.M)/Ka(Q,R) > 62]. High cooperativity for this interaction occurs in either a single-site model or in lattice models for the binding of M to R. Data obtained using five other RNA 10-mers, each with the sequence altered at the AUG codon, suggest that this cooperativity is AUG dependent. The data are consistent with a scheme in which mRNA and Q bind independently to the 40S ribosome, but when the AUG codon is properly aligned with Q, a conformational change results in a 2.4 kcal/mol stabilization of the complex.

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Section: Methodsmentioning

confidence: 99%

“…The Met-tRNA^MOS complex then scans the 5'noncoding sequence in mRNA until it reaches the initiation codon. In contrast, Gupta and co-workers, using purified peptide chain initiation factors, demonstrated that Met-tRNA^61 binding to the 40S ribosomal subunit was dependent on mRNA (Gupta et al, 1975;Roy et al, 1984Roy et al, , 1988.…”

mentioning

confidence: 91%

Thermodynamic Characterization of the Cooperativity of 40S Complex Formation during the Initiation of Eukaryotic Protein Synthesis

Parkhurst¹,

Hileman²,

Saha³

et al. 1994

Biochemistry

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Roles of Eukaryotic Initiation Factor 2 and Eukaryotic Initiation Factor 2 Ancillary Protein Factors in Eukaryotic Protein Synthesis Initiation

Gupta¹,

Ahmad²,

Chakrabarti³

et al. 1987

Translational Regulation of Gene Expression

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Mechanism of peptide chain initiation in animal cells: A reevaluation

Gupta

Chakrabarti

1986

Mol Cell Biochem

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Despite numerous reports from several laboratories, the precise mechanism of peptide chain initiation in animal cells and the roles of different factors involved in this process are not clear. The most widely accepted mechanism for peptide chain initiation is that proposed several years ago by Staehelin, Anderson, Hersey and coworkers. However, results reported from several laboratories differ significantly from the results on which the above mechanism is based. In this article, we have analyzed these different reports and their significance on the proposed mechanism of peptide chain initiation. Also, we have proposed an alternative mechanism based on the results reported from our laboratory.

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Protein synthesis in rabbit reticulocytes: Requirements for Met-tRNAf·40S preinitiation complex formation with AUG-codon and physiological mRNAs

Cited by 11 publications

References 10 publications

Thermodynamic Characterization of the Cooperativity of 40S Complex Formation during the Initiation of Eukaryotic Protein Synthesis

Thermodynamic Characterization of the Cooperativity of 40S Complex Formation during the Initiation of Eukaryotic Protein Synthesis

Roles of Eukaryotic Initiation Factor 2 and Eukaryotic Initiation Factor 2 Ancillary Protein Factors in Eukaryotic Protein Synthesis Initiation

Mechanism of peptide chain initiation in animal cells: A reevaluation

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