Small cis-acting sequences that specify secondary structures in a chloroplast mRNA are essential for RNA stability and translation

Nucleus-encoded proteins interact with cis-acting elements in chloroplast t ranscripts to promote RNA stability and translation. We have analyzed the s tructure and function of three such elements within the Chlamydomonas petD 5' untranslated region; petD encodes subunit IV of the cytochrome b(6)/f co mplex. These elements were delineated by linker-scanning mutagenesis, and R NA secondary structures were investigated by mapping nuclease-sensitive sit es in vitro and by in vivo dimethyl sulfate RNA modification. Element I spa ns a maximum of 8 nucleotides (nt) at the 5' end of the mRNA; it is essenti al for RNA stability and plays a role in translation. This element appears to form a small stem-loop that may interact with a previously described nuc leus-encoded factor to block 5'-->3' exoribonucleolytic degradation. Elemen ts II and III, located in the center and near the 3' end of the 5' untransl ated region, respectively, are essential for translation, but mutations in these elements do not affect mRNA stability. Element II is a maximum of 16 nt in length, does not form an obvious secondary structure, and appears to bind proteins that protect it from dimethyl sulfate modification. Element I II spans a maximum of 14 nt and appears to form a stem-loop in vivo, based on dimethyl sulfate modification and the sequences of intragenic suppressor s of element III mutations. Furthermore, mutations in element II result in changes in the RNA structure near element III, consistent with a long-range interaction that may promote translation.