Cooperation between the chloroplast psbA 5-untranslated region and coding region is important for translational initiation: the chloroplast translation machinery cannot read a human viral gene coding region

Chloroplast mRNA translation is regulated by the 5-untranslated region (5-UTR). Chloroplast 5-UTRs also support translation of the coding regions of heterologous genes. Using an invitro translation system from tobacco chloroplasts, we detected no translation from a human immunodeficiency virus tat coding region fused directly to the tobacco chloroplast psbA 5-UTR. This lack of apparent translation could have been due to rapid degradation of mRNA templates or synthesized protein products. Replacing the psbA 5-UTR with the E.coli phage T7 gene 10 5-UTR, a highly active 5-UTR, and substituting synonymous codons led to some translation of the tat coding region. The Tat protein thus synthesized was stable during translation reactions. No significant degradation of the added tat mRNAs was observed after translation reactions. These results excluded the above two possibilities and confirmed that the tat coding region prevented its own translation. The tat coding region was then fused to the psbA 5-UTR with a cognate 5-coding segment. Significant translation was detected from the tat coding region when fused after 10 or more codons. That is, translation could be initiated from the tat coding region once translation had started, indicating that the tat coding region inhibits translational initiation but not elongation. Hence, cooperation/compatibility between the 5-UTR and its coding region is important for translational initiation. Significance Statement Chloroplast 5-UTRs were thought to support translation of any coding region, including those of foreign genes; however, this is not always the case. Here we use an in vitro translation system and the tat gene from HIV to dissect the requirements for chloroplast translation. We found that compatibility between the 5 UTR and coding region is important for translational initiation.