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Requirement for GTP in the initiation process on reticulocyte ribosomes and ribosomal subunits
Abstract:
The requirement for GTP in the initiation process on reticulocyte ribosomes and ribosomal subunits has been examined by studying Met-tRNA(F) binding, ribosome-dependent [gamma-(32)P]GTP hydrolysis, and peptide-bond formation with puromycin. Met-tRNA(F) binding can be obtained with the methylene analogue, 5'-guanylylmethylene diphosphonate, as well as GTP, and it is not inhibited by fusidic acid or several other inhibitors of protein synthesis. This reaction can be performed with the 40S subunit and has the same requirements as the Met-tRNA(F)-binding reaction with washed ribosomes. Ribosome-dependent [gamma-(32)P]GTP hydrolysis can be obtained with the initiation factor M(2A) using either washed ribosomes or the 40S subunit. This reaction is also not significantly inhibited by fusidic acid. Peptide-bond formation between puromycin and Met-tRNA(F), however, is inhibited by fusidic acid, and does not occur if the methylene analogue of GTP is substituted for GTP. These data suggest that the binding of the initiator tRNA to the 40S subunit does not require the hydrolysis of GTP, but that at least one GTP hydrolysis event must occur after Met-tRNA(F) binding in order for the first peptide bond to be formed.
Insights
Guanosine triphosphate (GTP) is essential for protein synthesis initiation. GTP hydrolysis is not required for initiator tRNA binding to the 40S ribosomal subunit, but is necessary for subsequent peptide bond formation.
Area of Science:
- Molecular Biology
- Biochemistry
- Cell Biology
Background:
- Guanosine triphosphate (GTP) plays a critical role in protein synthesis initiation.
- Understanding the precise role of GTP hydrolysis in ribosome function is key to deciphering translation regulation.
Purpose of the Study:
- To investigate the specific requirements of GTP in the initiation of protein synthesis on reticulocyte ribosomes.
- To differentiate the roles of GTP binding versus GTP hydrolysis in initiator tRNA (Met-tRNA(F)) binding and subsequent peptide bond formation.
Main Methods:
- Studied Met-tRNA(F) binding to ribosomes and ribosomal subunits.
- Assessed ribosome-dependent [gamma-(32)P]GTP hydrolysis.
- Examined peptide-bond formation using puromycin and Met-tRNA(F).
- Utilized GTP analogues and protein synthesis inhibitors like fusidic acid.
Main Results:
- Met-tRNA(F) binding to the 40S subunit occurs with GTP or its non-hydrolyzable analogue, 5'-guanylylmethylene diphosphonate, and is fusidic acid-insensitive.
- Ribosome-dependent GTP hydrolysis requires initiation factor M(2A) and is largely fusidic acid-insensitive.
- Peptide bond formation is inhibited by fusidic acid and does not occur with the GTP analogue, indicating a requirement for GTP hydrolysis.
Conclusions:
- Initiator tRNA binding to the 40S ribosomal subunit does not necessitate GTP hydrolysis.
- At least one GTP hydrolysis event is required post-Met-tRNA(F) binding for the formation of the first peptide bond during protein synthesis initiation.