[A new method to measure the functional activity of class-1 translation termination factor eRF1].
Termination of protein synthesis (hydrolysis of the last peptidyl-tRNA on the ribosome) takes place when the ribosomal A site is occupied simultaneously by one of the three stop codons and by a class-1 translation termination factor. The existing procedures to measure the functional activity of this factor both in vitro and in vivo have serious drawbacks, the main of which are artificial conditions for in vitro assays, far from those in the cell, and indirect evaluation of activity in in vivo systems. A simple reliable and sensitive system to measure the functional activity of class-1 translation termination factors could considerably expedite the study of the terminal steps of protein synthesis, at present remaining poorly known, especially in eukaryotes. We suggest a novel system to test the functional activity in vitro using native functionally active mRNA, rather than tri-, tetra-, or oligonucleotides as before. This mRNA is specially designed to contain one of the three terminating (stop) codons within the coding nucleotide sequence. Plasmids have been generated that carry the genes of suppressor tRNAs each of which is specific toward one of the three stop codons. They were shown to support normal synthesis of a reporter protein, luciferase, by reading through the stop codon within the coding mRNA sequence. We have demonstrated that human class-1 translation termination factor eRF1 is able to compete with suppressor tRNA for a stop codon and to completely prevent its suppressive effect at a sufficient concentration. Forms of eRF1 with point mutations in functionally essential regions have lower competitive ability, demonstrating the sensitivity of the method to the eRF1 structure. The enzymatic reaction catalyzed by the full-size reporter protein is accompanied by emission of light quanta. Therefore, competition between suppressor tRNA and eRF1 can be measured using a luminometer, and this allows precise kinetic measurements in a continuous automatic mode.
Mazur
A M
AM
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991 Russia.
Kholod
N S
NS
Seit Nebi
A S
AS
Kiselev
L L
LL
D000604:Amino Acyl-tRNA Synthetases / Q000235:genetics / Q000378:metabolism; D000818:Animals; D003062:Codon; D018388:Codon, Terminator; D006801:Humans; D008156:Luciferases / Q000235:genetics; D008967:Molecular Biology / Q000379:methods*; D016296:Mutagenesis; D010454:Peptide Termination Factors / Q000032:analysis* / Q000235:genetics / Q000378:metabolism*; D012343:RNA, Transfer / Q000378:metabolism; D012362:RNA, Transfer, Ser; D011817:Rabbits; D012156:Reticulocytes; D012694:Serine / Q000235:genetics
D003062:Codon; D018388:Codon, Terminator; C489002:ETF1 protein, human; D010454:Peptide Termination Factors; D012362:RNA, Transfer, Ser; D012694:Serine; D012343:RNA, Transfer; D008156:Luciferases; D000604:Amino Acyl-tRNA Synthetases