Cotranslational formation of active photoprotein obelin in a cell-free translation system: Direct ultrahigh sensitive measure of the translation course

Translation of apoobelin mRNA in a cell-free wheat germ translation system in the presence of coelenterazine and molecular oxygen results in cotransla tional formation of active photoprotein. Active obelin formation is recorde d by its luminescence, either direct in the translation mixture in the pres ence of coelenterazine and calcium ions or in aliquots from the translation mixture. In the second case translation is carried out with coelenterazine and EGTA. Registration of the translation course by luminescence of the sy nthesized product in both cases allows use of apoobelin mRNA at very low co ncentrations as an internal marker for immediate measure of protein biosynt hesis activity of in vitro translation systems. It is shown that the simult aneous translation of any other mRNA does not affect translation of photopr otein mRNAs under standard conditions. Continuous registration of luminesce nce in a cuvette of a liquid scintillation counter in photon-counting mode varies the time of signal accumulation in a wide temporal range, thus incre asing the numerical values of the recorded signals. Registration of photopr otein luminescence during translation can be used to obtain additional info rmation about the translation process, for example codon reading speed, abo ut protein folding, and about the formation of active proteins on ribosomes . (C) 1999 Academic Press.