Use of natural mRNAs in the cell-free protein-synthesizing systems of the moderate halophile Vibrio costicola

Abstract

In vitro protein synthesis was studied in extracts of the moderate halophile Vibrio costicola by using as mRNAs the endogenous mRNA of V. costicola and the RNA of the R17 bacteriophage of Escherichwa coli. Protein synthesis (amino acid incorporation) was dependent on the messenger, ribosomes, soluble cytoplasmic factors, energy source, and tRNAmet (in the R17 RNA system) and was inhibited by certain antibiotics. These properties indicated de novo protein synthesis. In the V. costicola system directed by R17 RNA, a protein of the same electrophoretic mobility as the major coat protein of the R17 phage was synthesized. Antibiotic action and the response to added tRNA4et showed that protein synthesis in the R17 RNA system, but not in the endogenous messenger system, absolutely depended on initiation. Optimal activity of both systems was observed in 250 to 300 mM NH4' (as glutamate). Higher salt concentrations, especially those with Cl-as anion, were generally inhibitory. The R17 RNA-directed system was more sensitive to Cl-ions than the endogenous system was. Glycine betaine stimulated both systems and partly overcame the toxic effects of Cl-ions. Both systems required Mg2+, but in lower concentrations than the polyuridylic acid-directed system previously studied. Initiation factors were removed from ribosomes by washing with 3.0 to 3.5 M NH4C1, concentrations about three times as high as that needed to remove initiation factors from E. coli ribosomes. Washing with 4.0 M NH4Cl damaged V. costicola ribosomes, although the initiation factors still functioned. Cl-ions inhibited the attachment of initiation factors to tRNAmet but had little effect on binding of initiation factors to R17 RNA.Polyuridylic acid [poly(U)]-directed incorporation of phenylalanine by cell extracts of the moderate halophile Vibrio costicola is inhibited by Cl-ions (9), which prevent ribosome binding to the artificial mRNA (3).There are a number of differences between a translation system directed by poly(U) and that directed by a natural mRNA. Poly(U) has no Shine-Dalgarno sequence, which normally plays an important role in the initial attachment of natural mRNAs to ribosomes. At MATERIALS AND METHODS Bacterial culture. V. costicola NRC 37001 was grown and harvested as described previously (9).Preparation of cellular extracts, ribosomes, and initiation factors. S-30 extract, that is, the cellular material remaining in the supernatant after centrifuging for 30 min at 30,000 x gmax (Beckman type 42.1 rotor), was prepared as described previously (9).The S-150 extract and the low-salt-washed ribosomes were obtained by centrifuging the S-30 extract at 150,000 x gmax for 3 h at 4°C. The soluble S-150 fraction and the ribosomal pellet were then dialyzed or washed and stored according to the procedure of Kamekura and Kushner (9).A different low-salt extraction buffer than the one described by Kamekura and Kushner (9) was used in the preparation of the cellular extracts and of the ribosomes. It contained 124 mM ammonium glutamate, 8 mM magnesium ace...