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Activity of methoxyamine-modified f2 RNA in initiation and elongation steps of protein synthesis
Abstract:
1. Messenger activity of phage f2 RNA modified with methoxyamine under non-denaturing conditions was studied in E. coli-free system. The incorporation of amino acids into phage polypeptides was decreased, and the synthesis of phage-specific proteins was diminished. The RNA replicase synthesis was more affected than synthesis of coat protein. The impaired messenger activity of the methoxyamine-modified f2 RNA was due to the blocking of elongation process by modified cytosines present in RNA chain. 2. Specificity of f2 RNA to stimulate ribosomal binding predominantly at the coat protein initiation site was not affected by methoxyamine-treatment, as demonstrated by unchanged binding of f[3H]Met-tRNA and [14C]alanyl-tRNA to ribosomes. 3. Unfolding of f2 RNA molecule on treatment with methoxyamine in the presence of guanidine-HCl resulted in a significant increase of RNA capacity to direct fMet-tRNA binding to ribosomes. Sucrose-density gradient profiles revealed the formation of polysome-like initiation complexes indicating that ribosomes were able to bind at many hitherto inaccessible initiation codons in RNA molecules. fMet-tRNA bound to ribosomes in the presence of unfolded RNA was found to be fully reactive with puromycin.
Insights
Methoxyamine modification of phage f2 RNA blocks protein synthesis elongation by altering cytosines. Unfolding the RNA with guanidine-HCl restores translation initiation, revealing hidden codons.
Area of Science:
- Molecular Biology
- Virology
- Biochemistry
Background:
- Phage f2 RNA serves as a model for studying messenger RNA translation.
- Methoxyamine is a chemical agent used to modify nucleic acids.
- Understanding RNA structure-function relationships is crucial for molecular biology.
Purpose of the Study:
- To investigate the effect of methoxyamine modification on phage f2 RNA's messenger activity.
- To determine how methoxyamine affects protein synthesis and RNA structure.
- To explore the impact of RNA unfolding on translation initiation.
Main Methods:
- Studying messenger activity in an E. coli-free system.
- Analyzing amino acid incorporation and phage-specific protein synthesis.
- Assessing ribosomal binding specificity using labeled tRNAs.
- Employing sucrose-density gradient centrifugation to study initiation complexes.
Main Results:
- Methoxyamine modification decreased amino acid incorporation and phage protein synthesis, particularly RNA replicase.
- The impaired messenger activity was attributed to modified cytosines blocking elongation.
- Ribosomal binding specificity at the coat protein initiation site remained unaffected.
- Unfolding the RNA with guanidine-HCl significantly increased its capacity for translation initiation, revealing new initiation codons.
Conclusions:
- Methoxyamine modification of phage f2 RNA inhibits protein synthesis elongation by altering cytosine bases.
- RNA structural changes induced by methoxyamine can hinder translation.
- Unfolding the RNA molecule can expose previously inaccessible initiation sites, restoring translation capacity.