Activity of methoxyamine-modified f2 RNA in initiation and elongation steps of protein synthesis.
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.
D002474:Cell-Free System; D055598:Chemical Phenomena; D002621:Chemistry; D003090:Coliphages; D004926:Escherichia coli / Q000378:metabolism*; D006898:Hydroxylamines; D009690:Nucleic Acid Conformation; D010441:Peptide Chain Elongation, Translational; D010442:Peptide Chain Initiation, Translational; D011131:Polyribonucleotides; D014176:Protein Biosynthesis; D011691:Puromycin / Q000494:pharmacology; D012333:RNA, Messenger / Q000378:metabolism*; D012343:RNA, Transfer / Q000378:metabolism; D012367:RNA, Viral / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism; D014764:Viral Proteins / Q000096:biosynthesis
D006898:Hydroxylamines; D011131:Polyribonucleotides; D012333:RNA, Messenger; D012367:RNA, Viral; D014764:Viral Proteins; D011691:Puromycin; D012343:RNA, Transfer