Vyacheslav A. Kolb

Folding of firefly luciferase during translation in a cell-free system.

In vitro synthesis of firefly luciferase and its folding into an enzymatically active conformation were studied in a wheat germ cell‐free translation system. A novel method is described by which the enzymatic activity of newly synthesized luciferase can be monitored continuously in the cell‐free system while this protein is being translated from its mRNA. It is shown that ribosome‐bound polypeptide chains have no detectable enzymatic activity, but that this activity appears within a few seconds after luciferase has been released from the ribosome. In contrast, the renaturation of denatured luciferase under identical conditions occurs with a half‐time of 14 min. These results support the cotranslational folding hypothesis which states that the nascent peptides start to attain their native tertiary structure during protein synthesis on the ribosome.

Ribosome-associated protein that inhibits translation at the aminoacyl-tRNA binding stage

We have recently isolated and characterized a novel protein associated with Escherichia coli ribosomes and named protein Y (pY). Here we show that the ribosomes from bacterial cells growing at a normal physiological temperature contain no pY, whereas a temperature downshift results in the appearance of the protein in ribosomes. The protein also appears in the ribosomes of those cells that reached the stationary phase of growth at a physiological temperature. Our experiments with cell‐free translation systems demonstrate that the protein inhibits translation at the elongation stage by blocking the binding of aminoacyl‐tRNA to the ribosomal A site. The function of the protein in adaptation of cells to environmental stress is discussed.

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. Here, we show that Microcin C is a subject of specific degradation inside the sensitive cell. The product of degradation, a modified aspartyl-adenylate containing an N-acylphosphoramidate linkage, strongly inhibits translation by blocking the function of aspartyl-tRNA synthetase.

Enzymatic activity of the ribosome-bound nascent polypeptide

Firefly luciferase was shown to be completely folded and thus enzymatically active immediately upon release from the ribosome [Kolb et al. (1994) EMBO J. 13, 3631–3637]. However, no luciferase activity was observed while full‐length luciferase was attached to the ribosome as a peptidyl‐tRNA, probably because the C‐terminal portion of the enzyme is masked by the ribosome and/or ribosome‐associated proteins. Here we have demonstrated that the ribosome‐bound enzyme acquires the enzymatic activity when its C‐terminus is extended by at least 26 additional amino acid residues. The results demonstrate that the acquisition of the final native conformation by a nascent protein does not need the release of the protein from the ribosome.

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. The abrupt arrest of translation by the addition of a translational inhibitor led to immediate cessation of the enzyme activity accumulation, indicating the cotranslational character of luciferase folding. The results presented suggest that the chaperones of the Hsp70 family are not required for effective cotranslational folding of firefly luciferase.

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. Correct folding of the synthesized protein is demonstrated and discussed in detail.

Attenuation-Based Dual-Fluorescent-Protein Reporter for Screening Translation Inhibitors

ABSTRACT A reporter construct was created on the basis of the transcription attenuator region of the Escherichia coli tryptophan operon. Dual-fluorescent-protein genes for red fluorescent protein and cerulean fluorescent protein were used as a sensor and internal control of gene expression. The sequence of the attenuator was modified to avoid tryptophan sensitivity while preserving sensitivity to ribosome stalling. Antimicrobial compounds which cause translation arrest at the stage of elongation induce the reporter both in liquid culture and on an agar plate. This reporter could be used for high-throughput screening of translation inhibitors.

Cell-free immunology: construction and in vitro expression of a PCR-based library encoding a single-chain antibody repertoire.

A novel cloning‐independent strategy has been developed to generate a combinatorial library of PCR fragments encoding a murine single‐chain antibody repertoire and express it directly in a cell‐free system. The new approach provides an effective alternative to the techniques involving in vivo procedures of preparation and handling large libraries of antibodies. The possible use of the described strategy in the ribosome display is discussed.

Synthesis and maturation of green fluorescent protein in a cell-free translation system

SummaryA cell-free translation system producing mature green fluorescent protein (GFP) can be a useful tool for studying the mechanism and kinetics of GFP chromophore formation, as well as for fast protein engineering. We report here that the mature GFP can be formed in the cell-free translation system from E.coli. The synthesis of GFP in the cell-free system reaches a plateau in 30 to 40 min whereas its maturation is completed in 4 h from the beginning of translation. The delay between the GFP synthesis and the chromophore formation in the cell-free system provides the possibility to isolate and to analyse maturation intermediates for elucidation of the modification pathway.

Folding of the firefly luciferase polypeptide chain with the immobilized C terminus

Refolding of firefly Photinus pyralis luciferase from a denatured state is a slow process; its rate and productivity depend on molecular chaperones of the Hsp70 family. In contrast, cotranslational folding of luciferase is fast and productive in the absence of chaperones. During cotranslational folding, the C termini of polypeptides are associated with ribosomes, massive particles. The question arises as to whether C-terminal immobilization on a massive particle promotes folding. To study this problem experimentally, luciferase was C-tagged with hexahistidine to allow its C-terminal immobilization of chelating Sepharose. Both immobilized and free chains of the urea-denatured enzyme refolded at the same rate. At the same time, immobilization led to a higher refolding yield owing to the prevention of intermolecular aggregation. Chaperones of the Hsp70 family promoted folding of both immobilized and free luciferase polypeptides. It was assumed that the high rate of cotranslational folding is not ensured by mere immobilization of the C terminus of the polypeptide, but is rather due to a favorable start conformation of the growing peptide in the peptidyltransferase center of the ribosome and/or the vectorial character of the folding, proceeding from the N to the C end during polypeptide synthesis.