Properties of tRNA species modified in the 3'-terminal ribose moiety in an eukaryotic ribosomal system

Abstract

Phe-tRNAPhe species modified on the 3'-terminal ribose residue were investigated for their ability to participate in individual steps of the elongation cycle using eukaryotic ribosomes from reticulocytes. None of the Phe-tRNAs used, namely Phe-tRNAPhe-C-C-3'dA, Phe-tRNAPhe-C-C-3'-NH2A, and Phe-tRNAPhe-C-C-Aoxi-red, can function in the overall process. All modified Phe-tRNAPhe species can be bound nonenzymatically to ribosomes. Phe-tRNAPhe-C-C-3'NH2A exhibits exceptionally high binding at low Mg2+ concentration compared with Phe-tRNAPhe-C-C-A binding. Ac-Phe-tRNAPhe species prepared from the three modified tRNAs, when bound to the donor site, were devoid of donor activity. The enzymatic binding of both Phe-tRNAPhe-C-C-3'dA and Phe-tRNAPhe-C-C-3'NH2A is less efficient than that of Phe-tRNAPhe-C-C-A but these Phe-tRNAPhe species have acceptor activity. Phe-tRNAPhe-C-C--Aoxi-red is not a substrate for the EF-I promoted binding reaction and has no acceptor activity. The nonaminoacylated species, tRNAPhe-C-C-A, tRNAPhe-C-C-3'dA, and tRNAPhe-C-C-3'NH2A, bind to the ribosome to a larger extent than the corresponding aminoacylated tRNAs, both in the presence and in the absence of poly(U). Peptidyl-tRNAPhe-C-C-3'dA bound to the donor site cannot activate the acceptor site for EF-I promoted binding of Phe-tRNAPhe as does peptidyl-tRNAPhe-C-C-A. Further, it was observed that a correct codon-anticodon interaction influences the recognition of the 3' terminus of tRNA. Specificity of eukaryotic ribosomes for the 2'- and/or 3'-aminoacylated tRNA species is discussed and compared with the properties of Escherichia coli system.

Activity of the 2' and 3' isomers of aminoacyl-tRNA in the in vitro peptide elongation on E. coli ribosomes

“…Since two isomeric deoxy-tRNAsLys were used in our investigations, the role of the missing hydroxyl group during peptide transfer, translocation, and binding of aminoacyl-tRNA could also be approached in this study. The results presented here are an extension of experiments performed previously and reported by Chinali et al (1974) and Baksht et al (1976). In contrast to the previous studies where heterologous in vitro systems in which the components were derived from different organisms were used, in this paper experiments are described which were performed in a homologous E. coli system.…”

“…A pair of aminoacylated deoxy-tRNA species could be prepared by enzymatic tyrosylation of tRNATyr-CpCp2,dA1 *and tRNATyr-CpCp3'dA (Sprinzl et al, 1977a), but the lack of the corresponding mRNA hindered the investigation of the activity of these tRNAs in the in vitro translation. Previous work on this field was therefore performed with Phe-tRNAPhe-CpCp3'dA (Chinali et al, 1974;Baksht et al, 1976) and with aminoacylated tRNA fragments derived from the 3' end of tRNA, which can be prepared by chemical synthesis (Bhuta et al, 1981; Krayevsky & Kukhanova, 1979, and references therein).…”

Properties of phenylalanine transfer ribonucleic acid with modified 3′-terminal end in protein biosynthesis using a rabbit reticulocyte cell-free system: effect of the replacement of cytidine residues from the CpCpA end of tRNA by 5-iodocytidine or 2-thiocytidine

“…The 3'-terminus of tRNA plays an important role in the binding of both aminoacyland peptidyl-tRNA to the ribosomal acceptor and donor sites (1). tRNA species modified at well defined sites within this nucleotide sequence are, therefore, useful for the study of ribosome-tRNA interactions (2)(3)(4)(5)(6)(7). In this communication we report the results we obtained with yeast Phe-tRNAPhe-Cpi5CpA, Phe-tRNA-i5Cpi5CpA and Phe-tRNAPhe_Cps2CpA, respectively, in a ribosomal system derived from rabbit reticulocytes.…”

Isomeric aminoacyl-tRNAs are both bound by elongation factor Tu.

“…Although aminoacyl-tRNA is thought to consist of a mixture of 2'-and 3'-O-aminoacyl species which equilibrate rapidly, the specificity generally associated with enzymic processes makes it not unlikely that single isomers of tRNA may be utilized exclusively for certain of the partial reactions of protein biosynthesis.' The rapid equilibration of the two isomers of aminoacyl-tRNAs has precluded a determination of positional specificities involved in such transformations, but data of this type are now accessible by the use of certain modified tRNAs, e.g., those terminating in 2'-and 3'-deoxyadenosine (1)(2)(3)(4)(5)(6). Results obtained from the study of suchmodified tRNAs have suggested, e.g., that tRNAs are aminoacylated exclusively on a single (2' or 3') OH group (7)(8)(9).…”

“…Results obtained from the study of suchmodified tRNAs have suggested, e.g., that tRNAs are aminoacylated exclusively on a single (2' or 3') OH group (7)(8)(9). The use of isomeric phenylalanyl-tRNAs has also permitted the study of positional specificities involved in the binding of (N-acetyl)phenylalanyl-tRNA to the ribosomal A-and P-sites and in participation of phenylalanyl-tRNA as an acceptor in the peptidyltransferase reaction (2,4,5).…”