SENSITIVITY OF TRANSLATION BY BREVIBACTERIUM-LACTOFERMENTUM RIBOSOMESTO TYPE-1 AND TYPE-2 RIBOSOME-INACTIVATING PROTEINS

An active cell-free translation system was prepared from Brevibacteriu m lactofermentum, a Grampositive bacteria used in molecular cloning an d protein expression. The system contained high speed postribosomal su pernatant (S 370), purified ribosomes and a tRNA mixture from Escheric hia coil, and synthesized polyuridylic acid-directed polyphenylalanine once optimized for mono and divalent ions, time, and temperature. The translation system was evaluated for sensitivity to several translati onal inhibitors including several N-glycosidase ribosome-inactivating proteins (RIPs) isolated from plants. The pattern of inhibition by RIP s resembled that observed recently for Gram-negative bacteria such as Escherichia coli and Agrobacterium tumefaciens [Girbes et al., J. Bact eriol., 175, 6721-6724 (1993)]. A typical inhibitory type 1 RIP such a s cretin 2 promoted depurination of the rRNA, which upon treatment wit h acid aniline released a fragment of approximately 230 nucleotides. O n these grounds, we propose that bacterial ribosome sensitivity to pla nt RIPs depends on the bacterial ribosome-specific presence of protein recognition domains in the RIP present only in some RIP but not in ot hers.