Continuous-exchange protein-synthesizing systems.

Protein synthesis in cell-free systems is an emerging technology already competing with in vivo expression methods. In this chapter the basic principles of continuous-exchange protein synthesizing systems, and protocols for Escherichia coli and wheat germ translation and transcription-translation systems are described. The ways to improve substrate supply in cell-free systems and mRNA design for eukaryotic system are discussed.

Ribosomal protein S1 induces a conformational change of the 30S ribosomal subunit.

A comparative study of the 30S ribosomal subunit in the complex with protein S1 and the subunit depleted of this protein has been carried out by the hot tritium bombardment method. Differences in exposure of some ribosomal proteins within the 30S subunit depleted of S1 and within the 30S-S1 complex were found. It was concluded that protein S1 binds in the region of the neck of the 30S ribosomal subunit inducing a conformational change of its structure.

Aspartyl-tRNA synthetase is the target of peptide nucleotide antibiotic Microcin C.

Microcin C is a ribosome-synthesized heptapeptide that contains a modified adenosine monophosphate covalently attached to the C-terminal aspartate. Microcin C is a potent inhibitor of bacterial cell growth. Based on the in vivo kinetics of inhibition of macromolecular synthesis, Microcin C targets translation, through a mechanism that remained undefined.

Effective cotranslational folding of firefly luciferase without chaperones of the Hsp70 family.

Molecular chaperones of the Hsp70 family (bacterial DnaK, DnaJ, and GrpE) were shown to be strictly required for refolding of firefly luciferase from a denatured state and thus for effective restoration of its activity. At the same time the luciferase was found to be synthesized in an Escherichia coli cell-free translation system in a highly active state in the extract with no chaperone activity. The addition of the chaperones to the extract during translation did not raise the activity of the enzyme.