In Vitro Synthesis of Protein in Microbial Systems

The first useful systems for cell-free protein synthesis were developed in the Zamecnik laboratory, where it was shown that peptide synthesis takes place on the ribosome and requires ATP, GTP, and tRNA (1). Building on this, Nirenberg and his collaborators operationally separated the mRNA from the ribosome by showing that polyuridylic acid added to ribosomes stimulates polyphenylalanine synthesis (2). This discovery was a first step towards the solution of the genetic coding mechanism for proteins. Many other laboratories have contributed to understanding the biochemistry of protein synthesis. In spite of all the progre�s that has been made several aspects of the process are perplexing even in the bacterium Escherichia coli. The biochemistry of protein synthesis has recently been reviewed (3). This review is devoted to studies involving synthesis of complete proteins in cell-free systems. In the same year that Niremberg began his synthetic messenger studies aimed at elucidating the genetic code, the first complete protein, the coat protein of coliphage f2, was made in a cell-free extract of E. coli (4). Since then a number of viral RNAs have been used as messengers to prime the synthesis of complete proteins. By contrast, bacterial messengers have resisted translation until recent­ ly, because of the difficulty in isolating bacterial messengers from whole cells and partly because bacterial messengers are probably more prone to translation as they are being synthesized. To simulate whole cell conditions more closely, our laboratory began studies aimed at cell-free synthesis starting from the DNA. In 1967 DeVries and I (5) demonstrated synthesis of a portion of the {J­ galactosidase enzyme in a DNA-stimulated messenger-directed system. Subse­ quently a number of laboratories have studied the DNA-dependent RNA­ directed synthesis of both viral and bacterial proteins in E. coli extracts.