Incorporation of amino acids into protein by cell-free extracts of penicillium chrysogenum

Summary The preparation of ribosomal particles from P. chrysogenum has been described. After correction to 20°C and extrapolation to infinite dilution, they had sedimentation coefficients of approximately 80S, 60 S, and 40 S. The washed ribosomes plus purified supernatant fraction, KCl and sucrose incorporated C¹⁴-l-amino acids into protein. There was no stimulation of incorporation by ATP, GTP, CTP, UTP, or Mg ions. Incorporation was inhibited by streptomycin and chloramphenicol but not by ribonuclease.The amino acid incorporating system from P. chrysogenum did not resemble bacterial and yeast systems in many respects and reasons for this are discussed.     

Protein synthesis in tomato fruit locule tissue: The sites of synthesis and the pathway of carbon into protein

Summary The net increase in total protein during development of tomato fruit locule tissue has been further investigated with respect to the sites of protein synthesis within the cells. The results point to the plastids as being the major sites of protein synthesis. This is supported by radioisotope experiments in which labelled carbon from bicarbonate has been show to appear in protein at a higher rate than exogenously supplied amino acid. Isolated plastids incorporate carbon from bicarbonate, glucose and pyruvate into protein, and it is suggested that the pathway of carbon into protein involves photosynthetic pathways. The results are discussed with reference to possible differences between protein synthesis in plastids, and protein synthesis in the cytoplasm of plant cells.     

Control of cell-free protein synthesis by amino acids: effects on tRNA charging

In order to resolve the observation that addition of glutamine and glutamate appears to be of particular importance in enhancing the activity of a cell-free protein synthesis system derived from rat liver (Manchester and Tyobeka, 1980), we have measured the KM of the aminoacyl-tRNA synthetases towards amino acids and the extent of aminoacylation of tRNA under the conditions of our earlier experiments. During incubation of the cell-free system in the presence of an amino acid mixture the extent of acylation to tRNA of 15 amino acids studied showed no clear change from initial time values. When incubation took place in the absence of added amino acids, however, the levels of glutamate and glutamine bound to their appropriate tRNAs dropped more rapidly and to lower levels than for other amino acids except tryptophan. The pronounced drop for these two amino acids does not seem to result from an abnormally high KM value for the synthetases towards the respective amino acids, nor an abnormally low Vmax, but probably from the fact that the amounts of glutamyl and glutaminyl-tRNA in the cell-free system are comparatively low.     

Incorporation of non-natural amino acids into proteins

Chemical and biological diversity of protein structures and functions can be widely expanded by position-specific incorporation of non-natural amino acids carrying a variety of specialty side groups. After the pioneering works of Schultz's group and Chamberlin's group in 1989, noticeable progress has been made in expanding types of amino acids, in finding novel methods of tRNA aminoacylation and in extending genetic codes for directing the positions. Aminoacylation of tRNA with non-natural amino acids has been achieved by directed evolution of aminoacyl-tRNA synthetases or some ribozymes. Codons have been extended to include four-base codons or non-natural base pairs. Multiple incorporation of different non-natural amino acids has been achieved by the use of a different four-base codon for each tRNA. The combination of these novel techniques has opened the possibility of synthesising non-natural mutant proteins in living cells.     

Translational incorporation of multiple unnatural amino acids in a cell-free protein synthesis system

Nature uses 20 canonical amino acids as the standard building blocks of proteins; however, the incorporation of unnatural amino acids (Uaas) can endow polypeptide sequences with new structural and functional features. Although aminoacyl-tRNA synthetases (aaRSs) can accept an array of Uaas in place of their natural counterparts, Uaas generally are charged to tRNAs with substantially lower efficiencies. This particularly makes it difficult to incorporate multiple Uaas into a protein sequence. In this study, we discuss the use of a cell-free protein synthesis system as a versatile platform for the efficient incorporation of multiple Uaas into proteins. Taking advantage of the open nature of cell-free protein synthesis that allows flexible manipulation of its ingredients, we explored the application of Uaas in 10 mM range of concentrations to kinetically overcome the low affinity of aaRSs towards unnatural amino acids. Supplementation of recombinant aaRSs was also investigated to further increase the Uaa-tRNA pools. As a result, under the modified reaction conditions, as many as five different Uaas could be incorporated into a single protein without compromising the yield of protein synthesis.