Chloroplast-based in vitro translation systems

Abstract

Intact chloroplasts from 8-9 day old pea seedlings were capable of translating mRNA into proteins at high rates. Such metabolically active chloroplasts were lysed and fractionated further in order to develop a chloroplast-based in vitro translation system usable in the analysis of chloroplastic protein synthesis. Three in vitro systems were utilized for these studies: 1) lysed chloroplasts, 2) thylakoid bound polysomes supplemented with post-ribosomal supernatant of stroma, 3) low speed supernatant (30,000xg) alone. The chloroplast lysates and low speed supernatant (30,000xg) were capable of translating polyurydilic acid at high rates. Relatively low rate of short-lived translation,comparable to published data, was observed when translation was driven by endogenous mRNA. Addition of mRNA from phage MS2 to the system significantly inhibited translation. Results are discussed in relation to possible lack of re-initiation or Interaction with added RNA.