Co-translational Folding of an Eukaryotic Multidomain Protein in a Prokaryotic Translation System

Vyacheslav A Kolb,Eugeny V Makeyev,Alexander S Spirin

doi:10.1074/jbc.m002030200

Vyacheslav A Kolb, Eugeny V Makeyev + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m002030200

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Abstract

Continuous monitoring of the enzymatic activity of newly synthesized firefly luciferase in Escherichia coli cell-free translation system was performed to record folding kinetics of this multidomain eukaryotic protein in the prokaryotic cytosol. Whereas in vitro refolding of denatured luciferase in prokaryotic cytosol occurred with a low yield of active enzyme and took about an hour, the enzyme acquired its native structure immediately upon release from the ribosome, as seen from the immediate halt of active luciferase accumulation upon blocking of translation with inhibitors. The nascent luciferase was also capable of acquiring the active conformation prior to release from the ribosome, when its C terminus was extended with a polypeptide segment. Specific enzymatic activity of the firefly luciferase was found to be equally high irrespective of whether this protein was synthesized in eukaryotic or prokaryotic translation systems. The data presented demonstrate the fundamental ability of prokaryotic cytosol to support effective co-translational protein folding in general and co-translational folding of multidomain proteins in particular.

Highlights

A hypothesis has been put forward according to which a shift from posttranslational to co-translational folding mechanisms has occurred during evolution [3]
In order to approach the problem, we examined the kinetics of folding of a multidomain protein, Photinus pyralis luciferase, during its synthesis in a bacterial cell-free translation system
We have found that the arrest of luciferase synthesis causes immediate cessation of active enzyme accumulation, suggesting that the enzyme acquires its native structure immediately upon release from the ribosome, without any delay for posttranslational folding

Summary

EXPERIMENTAL PROCEDURES

Plasmid Construction—The plasmid pT7 luc(NTPII) was constructed by cloning the luciferase-NPT II fusion sequence from the plasmid pTZ luc(NTPII) [16] into the NdeI site of the plasmid vector pT7-7 [18]. DNA template for synthesis of mRNA encoding full-length luciferase without a stop codon was prepared by polymerase chain reaction amplification of pT7 luc(NPT II) with 5Ј-TAATACGACTCACTATAGGG-3Ј (upstream) and 5ЈTACAATTTGGACTTTCCGCC-3Ј (downstream) primers. Translation in 30% wheat germ extract [21] was performed in the presence of 1.3 mM ATP, 0.25 mM GTP, 16 mM creatine phosphate (Roche Molecular Biochemicals), 0.05 mg/ml creatine kinase (Roche Molecular Biochemicals), and 80 ␮M each amino acid in buffer containing 2.25 mM Mg(OAc) 106 mM KOAc, 20 mM HEPES-KOH, pH 7.5, 0.1 mM EDTA, 0.25 mM spermidine (Fluka), and 3 mM DTT. The sample was placed in a luminometer cell at 25 °C, and a luciferase-catalyzed reaction was initiated by injection of 50 ␮l of a solution containing 20 mM HEPES-KOH, pH 7.5, 10 mM Mg(OAc) mM DTT, 0.2 mM EDTA, 0.2% Triton X-100 (Merck), 2 mM ATP, 0.2 mM luciferin, and 0.4 mM coenzyme A

RESULTS

Rabbit reticulocyte

DISCUSSION