RNA-protein complexes that form in the spinach chloroplast atpI 5′ untranslated region can be divided into two subcomplexes, each comprised of unique cis-elements and trans-factors

Abstract

Control of gene expression in chloroplasts is critically dependent upon post-transcriptional mechanisms, most of which require formation of RNA-protein complexes. The 5' untranslated regions (5'UTRs) of chloroplast mRNAs have been shown to affect stability and/or translation of the message. These effects are mediated by the binding of specific protein(s) to the 5'UTR. We can detect such 5'UTR-protein complexes in vitro and have previously shown that the same polypeptide(s) bind many spinach chloroplast 5'UTRs (Robida et al. 2002). Here we report investigations on the RNA elements and protein factors involved in formation of these complexes. Comparison of the atpI 5'UTR, which serves as the representative 5'UTR for these experiments, among 12 angiosperms revealed two phylogenetically conserved regions upstream of a putative ribosome binding site. To determine whether the two conserved regions interact to form a single polypeptide-binding site, binding assays were performed with RNAs containing only one of the two. Those experiments revealed that the entire 5'UTR could be separated into two binding sites for chloroplast polypeptides, each containing one of the two conserved regions. Competition binding assays using the individual binding sites established that each was bound by different polypeptide(s). These data support the hypothesis that there are at least two unique polypeptides involved in these 5'UTR-protein complexes, each binding specifically to a different site within the 5'UTR.

Engineered chloroplast dsRNA silences cytochrome p450 monooxygenase, V‐ATPase and chitin synthase genes in the insect gut and disrupts Helicoverpa armigera larval development and pupation

“…Because the 3′ of PCR product of Pa and Pb primers have a 23~25 bp complementary sequence with the 5′ of PCR product from Pc and Pd primers, they should anneal into a dsRNA expression cassette (PpsbA:: ds RNA::TpsbA) (Table ). Formation of such dsRNA stem‐loop structures is very common in chloroplast 5′ UTR and 3′ UTR transcripts (Merhige et al ., ; Ruhlman et al ., ; Zou et al ., ). Two restriction enzyme sites ( Sal I and Pst I) were also introduced into this dsRNA expression cassettes by PCR (Table ).…”

The Role of Heterologous Chloroplast Sequence Elements in Transgene Integration and Expression

“…Detailed analyses have demonstrated that within these UTRs lie cis-elements, often forming secondary structures, which facilitate the interaction with nucleus-encoded RBPs (Yang et al, 1995;Hirose and Sugiura, 1996;Klaff et al, 1997;Alexander et al, 1998;Zou et al, 2003;Merhige et al, 2005). RBPs display an array of functions, including processing of polycistronic transcription units, RNA maturation and editing, transcript stability and turnover, and the recruitment of additional protein factors involved in the initiation of translation in response to the requirements of the cell (Nickelsen, 2003;Schmitz-Linneweber and Barkan, 2007).…”

Downstream promoter sequence of an Indian isolate of Rice tungro bacilliform virus alters tissue-specific expression in host rice and acts differentially in heterologous system

“…This probable inhibition of transcription by downstream elements seen in RTBV-WB promoter has been reported earlier for other plant promoters such as pollen-specific LAT59 (Curie and McCormick 1997), granule bound starch synthase (Kluth et al 2002) and PsbQ A gene (Gaur and Tyagi 2004). Moreover, the role of 5 0 -UTR in regulating transcription pausing or termination as well as mRNA stability and/or accumulation has also been well established for several plastid and nuclear genes where mRNA either interacts directly with proteins or forms stem loop structures (Selby et al 1989;Dickey et al 1992Dickey et al , 1998Caspar and Quail 1993;Bovy et al 1995;Higgs et al 1999;Nickelsen et al 1999;Vaistij et al 2000;Hua et al 2001;Bhat et al 2004;Lezhneva and Meurer 2004;Salvador et al 2004;Merhige et al 2005;Suay et al 2005). However, in-silico secondary structure prediction of the 5 0 -UTR for the C-, NE-and FL-type constructs using GeneQuest (DNASTAR Inc.) did not reveal any unique stem loop structures present solely in the NE-type (Fig.…”