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Internal Initiation of Polyuridylic Acid Translation in Bacterial Cell-Free System

E. A. Sogorin, S. Ch. Agalarov, and A. S. Spirin*

Institute of Protein Research, Russian Academy of Sciences, ul. Institutskaya 4, 142290 Pushchino, Moscow Region, Russia; fax: +7 (495) 514-0218; E-mail: spirin@vega.protres.ru; sultan@vega.protres.ru

* To whom correspondence should be addressed.

Received September 4, 2013
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.
KEY WORDS: polyuridylic acid, translation initiation, polyphenylalanine synthesis, T4 RNA ligase, cell-free translation

DOI: 10.1134/S0006297913120055