Chain initiation during polypeptide synthesis in cell-free bacterial systems programmed with a plant viral messenger: Initiation with N-acetylated aminoacyl-tRNAs on adjacent codons

Abstract

As a part of a more extensive program dealing with the initiation of polypeptide synthesis in cell-free systems of Escherichia coli directed by plant viral RNAs and bacteriophage RNA, the role of N-acetylated aminoacyl-tRNAs in this process has been studied. The viral messenger was RNA derived from the top component a of alfalfa mosaic virus (AMV-RNA). The primary interaction between AMV-RNA and E. coli ribosomes is very selective. Only three species of aminoacyl-tRNA: phenylalanyl-tRNA, isoleucyl-tRNA, and valyl-tRNA can bind to the ribosome-AMV-RNA complex. Two of these species, when N-acetylated, can function as polypeptide chain initiator: N-acetylphenylalanyl-tRNA and N-acetylisoleucyl-tRNA. By contrast N-acetylvalyl-tRNA does not bind to the ribosome-mRNA complex and cannot act as chain initiator either. Analysis of the biosynthetic product formed with N-acetyl- 14C-phenylalanyl-tRNA revealed that N-acetylphenylalanine is incorporated into the N-terminal structure of the product. Deacetylation appeared to be very limited under the conditions of incorporation. Aminoacyl-tRNA species, which might respond to codons overlapping the two adjacent ones in the hexanucleotide coding for phenylalanine and isoleucine, do not bind to the mRNA-ribosome complex and presumably cannot function as chain initiator when N-acetylated. This has been confirmed experimentally for N-acetylleucyl-tRNA. Ribosomal initiation factors and GTP, which at low Mg 2+ concentration are essential for binding of N-acetylphenylalanyl-tRNA to the ribosomes programmed with poly U, do not promote the binding of the tRNA or N-acetylisoleucyl-tRNA to the AMV-RNA-ribosome complex. It is concluded that chain initiation in the presence of AMV-RNA can occur by means of two different mechanisms: one requires low Mg 2+ concentrations, initiation factors, GTP and N-formylmethionyl-tRNA (F-met-tRNA); the other can make use of N-acetylaminoacyl-tRNAs at relatively high Mg 2+ concentrations in the absence of initiation factors and GTP. The implications in connection with correct translation are discussed.