Internal initiation of polyuridylic acid translation in bacterial cell-free system

Abstract

The task of the present work was to answer the question: is the free 5'-end needed for effective translation of a model polyribonucleotide template - polyuridylic acid - in a bacterial (E. coli) cell-free system? For this purpose, the template activities of the original polyuridylic acid with its free 5'-end and the polyuridylic acid with blocked 5'-end were compared in the bacterial cell-free translation system. To block the 5'-end, the cytidylic oligodeoxyribonucleotide with fluorescein residue at its 5'-end and uridylic oligoribonucleotide sequence at its 3'-end, schematically described as FAM(dC)10(rU)50, was covalently attached (ligated) to the 5'-end of the template polyuridylic acid. It was shown that the efficiency of polyphenylalanine synthesis on the 5'-blocked template and on the polyuridylic acid with free 5'-end was virtually the same. It was concluded that bacterial ribosomes are capable of effectively initiating translation at the polyuridylic sequence independently of the 5'-end of template polyribonucleotide, i.e. via an internal initiation mechanism, in the absence of a Shine-Dalgarno sequence and AUG start codon.