Potassium salts influence the fidelity of mRNA translation initiation in rabbit reticulocyte lysates: unique features of encephalomyocarditis virus RNA translation.
It is widely assumed that in vitro translation of mRNA is more efficient in the presence of potassium acetate rather than KCl, that the optimum concentration of potassium acetate is higher than for KCl, and that uncapped RNAs exhibit a lower optimum salt concentration than capped mRNAs. When these assumptions were examined using several different mRNA species in four batches of rabbit reticulocyte lysate, some notable exceptions were found. The translation of encephalomyocarditis virus (EMCV) RNA exhibited a salt optimum unusually high for an uncapped mRNA, and was very much more efficient and accurate with KCl rather than potassium acetate. It was also unique in being strongly activated by low concentrations (5-10 mM) KSCN in the presence of 90 mM potassium acetate. For the translation of other uncapped RNAs (poliovirus RNA, cowpea mosaic virus (CPMV) M RNA and bacteriophage MS2 RNA) amino acid incorporation at the optimum potassium acetate level was significantly greater than could be achieved using KCl. However, KCl was found to be restrictive and potassium acetate permissive for the synthesis of abnormal products thought to arise from initiation at incorrect sites, with the result that KCl gave a product pattern closer to that observed in vivo. In the particular case of the reticulocyte lysate system, accurate translation therefore requires the use of KCl rather than potassium acetate, but the choice of salt was found to be less critical in cell-free extracts from HeLa or L-cells.
Jackson
R J
RJ
Department of Biochemistry, University of Cambridge, U.K.
D000818:Animals; D001435:Bacteriophages / Q000235:genetics; D002474:Cell-Free System; D004591:Electrophoresis, Polyacrylamide Gel; D004686:Encephalomyocarditis virus / Q000235:genetics*; D009029:Mosaic Viruses / Q000235:genetics; D011188:Potassium / Q000494:pharmacology*; D014176:Protein Biosynthesis / Q000187:drug effects*; D012333:RNA, Messenger / Q000187:drug effects*; D012367:RNA, Viral / Q000187:drug effects*; D011817:Rabbits; D012156:Reticulocytes / Q000187:drug effects*; D012492:Salts
D012333:RNA, Messenger; D012367:RNA, Viral; D012492:Salts; D011188:Potassium