In chloroplasts, the 3 untranslated regions of most mRNAs contain a stem-loop-forming inverted repeat (IR) sequence that is required for mRNA stability and correct 3-end formation. The IR regions of several mRNAs are also known to bind chloroplast proteins, as judged from in vitro gel mobility shift and UV cross-linking assays, and these RNA-protein interactions may be involved in the regulation of chloroplast mRNA processing and/or stability. Here we describe in detail the RNA and protein components that are involved in 3 IR-containing RNA (3 IR-RNA)-protein complex formation for the spinach chloroplast petD gene, which encodes subunit IV of the cytochrome b 6 /f complex. We show that the complex contains 55-, 41-, and 29-kDa RNA-binding proteins (ribonucleoproteins [RNPs]). These proteins together protect a 90-nucleotide segment of RNA from RNase T 1 digestion; this RNA contains the IR and downstream flanking sequences. Competition experiments using 3 IR-RNAs from the psbA or rbcL gene demonstrate that the RNPs have a strong specificity for the petD sequence. Site-directed mutagenesis was carried out to define the RNA sequence elements required for complex formation. These studies identified an 8-nucleotide AU-rich sequence downstream of the IR; mutations within this sequence had moderate to severe effects on RNA-protein complex formation. Although other similar sequences are present in the petD 3 untranslated region, only a single copy, which we have termed box II, appears to be essential for in vitro protein binding. In addition, the IR itself is necessary for optimal complex formation. These two sequence elements together with an RNP complex may direct correct 3-end processing and/or influence the stability of petD mRNA in chloroplasts.The roles of mRNA 3Ј untranslated regions (UTRs) in regulating gene expression have become prominent (1,18,30,44). In chloroplasts, the 3Ј UTRs of most mRNAs are flanked by stem-loop structure-forming inverted repeat (IR) sequences that participate in 3Ј-end formation and are also thought to impede the progress of processive exoribonucleases (32,36,39,40). Both the 3Ј IR and the 5Ј UTR have been shown by in vitro assays to associate with proteins which may regulate RNA processing, RNA stability, and/or translation initiation (10,28,32,38,46).The mechanism of chloroplast mRNA 3Ј-end formation has been studied by using both in vitro assays and chloroplast transformation. In vitro transcription experiments were used to show that although the chloroplast RNA polymerase is capable of recognizing rho-independent prokaryotic transcription termination signals (8, 36), most chloroplast 3Ј IRs have little or no transcription termination activity (36). Data obtained by comparing in vivo transcription rates upstream and downstream of the Chlamydomonas atpB 3Ј IR are consistent with the in vitro observations (39). This finding suggests that most chloroplast mRNAs are synthesized as precursors, which are subsequently processed to yield the uniform 3Ј termini found in vivo.Both RNases and R...