Abstract
The chapter describes a yeast in vitro system analogous to the E. coli S-30 systems. This provides a useful base for studies on transfer RNA (tRNA) function, inhibitors, ionic conditions, and supplementation with heterologous components, and hopefully for further development of the system. Polyphenylalanine synthesis operates with extremely high fidelity and does not show some of the elongation defects of some other systems nor the dependence on high Mg 2+ concentrations that can occur in highly purified system. The results from studies on, for example, inhibitor mechanisms in highly purified systems do not always agree with those obtained in vivo or in crude cell-free systems. Thus crude cell-free systems have a role in confirming the results obtained from better resolved systems, particularly where confirmation in vivo is not feasible. Yeast strain, growth and harvesting conditions, breakage method, buffers, protective agents, and freezing technique all have critical requirements for the successful preparation of an active cell-free system.
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