The downstream atpE cistron is efficiently translated via its own cis-element in partially overlapping atpB–atpE dicistronic mRNAs in chloroplasts

The chloroplast atpB and atpE genes encode subunits β and ε of the ATP synthase, respectively. They are co-transcribed as dicistronic mRNAs in flowering plants. An unusual feature is an overlap (AUGA) of the atpB stop codon (UGA) with the atpE start codon (AUG). Hence, atpE translation has been believed to depend on atpB translation (i.e. translational coupling). Using an in vitro translation system from tobacco chloroplasts, we showed that both atpB and atpE cistrons are translated from the tobacco dicistronic mRNA, and that the efficiency of atpB translation is higher than that of atpE translation. When the atpB 5′-UTR was replaced with lower efficiency 5′-UTRs, atpE translation was higher than atpB translation. Removal of the entire atpB 5′-UTR arrested atpB translation but atpE translation still proceeded. Introduction of a premature stop codon in the atpB cistron did not abolish atpE translation. These results indicate that atpE translation is independent of atpB translation. Mutation analysis showed that the atpE cistron possesses its own cis-element(s) for translation, located ~25 nt upstream from the start codon

An SF1 affinity model to identify branch point sequences in human introns

Splicing factor 1 (SF1) binds to the branch point sequence (BPS) of mammalian introns and is believed to be important for the splicing of some, but not all, introns. To help identify BPSs, particularly those that depend on SF1, we generated a BPS profile model in which SF1 binding affinity data, validated by branch point mapping, were iteratively incorporated into computational models. We searched a data set of 117 499 human introns for best matches to the SF1 Affinity Model above a threshold, and counted the number of matches at each intronic position. After subtracting a background value, we found that 87.9% of remaining high-scoring matches identified were located in a region upstream of 3′-splice sites where BPSs are typically found. Since U2AF65 recognizes the polypyrimidine tract (PPT) and forms a cooperative RNA complex with SF1, we combined the SF1 model with a PPT model computed from high affinity binding sequences for U2AF65. The combined model, together with binding site location constraints, accurately identified introns bound by SF1 that are candidates for SF1-dependent splicing

Unexpected Diversity of Chloroplast Noncoding RNAs as Revealed by Deep Sequencing of the Arabidopsis Transcriptome

Noncoding RNAs (ncRNA) are widely expressed in both prokaryotes and eukaryotes. Eukaryotic ncRNAs are commonly micro- and small-interfering RNAs (18–25 nt) involved in posttranscriptional gene silencing, whereas prokaryotic ncRNAs vary in size and are involved in various aspects of gene regulation. Given the prokaryotic origin of organelles, the presence of ncRNAs might be expected; however, the full spectrum of organellar ncRNAs has not been determined systematically. Here, strand-specific RNA-Seq analysis was used to identify 107 candidate ncRNAs from Arabidopsis thaliana chloroplasts, primarily encoded opposite protein-coding and tRNA genes. Forty-eight ncRNAs were shown to accumulate by RNA gel blot as discrete transcripts in wild-type (WT) plants and/or the pnp1-1 mutant, which lacks the chloroplast ribonuclease polynucleotide phosphorylase (cpPNPase). Ninety-eight percent of the ncRNAs detected by RNA gel blot had different transcript patterns between WT and pnp1-1, suggesting cpPNPase has a significant role in chloroplast ncRNA biogenesis and accumulation. Analysis of materials deficient for other major chloroplast ribonucleases, RNase R, RNase E, and RNase J, showed differential effects on ncRNA accumulation and/or form, suggesting specificity in RNase-ncRNA interactions. 5′ end mapping demonstrates that some ncRNAs are transcribed from dedicated promoters, whereas others result from transcriptional read-through. Finally, correlations between accumulation of some ncRNAs and the symmetrically transcribed sense RNA are consistent with a role in RNA stability. Overall, our data suggest that this extensive population of ncRNAs has the potential to underpin a previously underappreciated regulatory mode in the chloroplast

Translation of partially overlapping psbD-psbC mRNAs in chloroplasts: the role of 5′-processing and translational coupling

The chloroplast psbD and psbC genes encode the D2 and CP43 proteins of the photosystem II complex, and they are generally cotranscribed. We report studies on the basic translation process of tobacco psbD-psbC mRNAs using an in vitro translation system from tobacco chloroplasts. The primary transcript has an unusually long 5′-UTR (905 nt). We show that it is translatable. Processing of the 5′-UTR greatly enhances the translation efficiency of the psbD cistron. A striking feature is that psbD and psbC cistrons overlap by 14 nt. Removal of the psbD 5′-UTR plus the start codon and introduction of a premature termination codon in the psbD cistron considerably reduce the translation efficiency of the downstream psbC cistron. These results indicate that translation of the psbC cistron depends largely on that of the upstream psbD cistron and thus shows translational coupling; however, a portion is independently translated. These observations, together with the presence of monocistronic psbC mRNAs, suggest that the psbD and psbC cistrons are translated via multiple processes to produce necessary amounts of D2 and CP43 proteins

Selection of Shine-Dalgarno sequences in plastids

Like bacterial genes, most plastid (chloroplast) genes are arranged in operons and transcribed as polycistronic mRNAs. Plastid protein biosynthesis occurs on bacterial-type 70S ribosomes and translation initiation of many (but not all) mRNAs is mediated by Shine-Dalgarno (SD) sequences. To study the mechanisms of SD sequence recognition, we have analyzed translation initiation from mRNAs containing multiple SD sequences. Comparing translational efficiencies of identical transgenic mRNAs in Escherichia coli and plastids, we find surprising differences between the two systems. Most importantly, while internal SD sequences are efficiently recognized in E. coli, plastids exhibit a bias toward utilizing predominantly the 5′-most SD sequence. We propose that inefficient recognition of internal SD sequences provides the raison d'être for most plastid polycistronic transcripts undergoing post-transcriptional cleavage into monocistronic mRNAs

Structure of the HERG K+ channel S5P extracellular linker - Role of an amphipathic alpha-helix in C-type inactivation

The HERG K+ channel has very unusual kinetic behavior that includes slow activation but rapid inactivation. These features are critical for normal cardiac repolarization as well as in preventing lethal ventricular arrhythmias. Mutagenesis studies have shown that the extracellular peptide linker joining the fifth transmembrane domain to the pore helix is critical for rapid inactivation of the HERG K+ channel. This peptide linker is also considerably longer in HERG K+ channels, 40 amino acids, than in most other voltage-gated K+ channels. In this study we show that a synthetic 42-residue peptide corresponding to this linker region of the HERG K+ channel does not have defined structural elements in aqueous solution; however, it displays two well defined helical regions when in the presence of SDS micelles. The helices correspond to Trp(585)-Ile(593) and Gly(604)-Tyr(611) of the channel. The Trp(585)-Ile(593) helix has distinct hydrophilic and hydrophobic surfaces. The Gly(604)-Tyr(611) helix corresponds to an N-terminal extension of the pore helix. Electrophysiological studies of HERG currents following application of exogenous S5P peptides show that the amphipathic helix in the S5P linker interacts with the pore region of the channel in a voltage-dependent manner

Complex chloroplast RNA metabolism: just debugging the genetic programme?

<p>Abstract</p> <p>Background</p> <p>The gene expression system of chloroplasts is far more complex than that of their cyanobacterial progenitor. This gain in complexity affects in particular RNA metabolism, specifically the transcription and maturation of RNA. Mature chloroplast RNA is generated by a plethora of nuclear-encoded proteins acquired or recruited during plant evolution, comprising additional RNA polymerases and sigma factors, and sequence-specific RNA maturation factors promoting RNA splicing, editing, end formation and translatability. Despite years of intensive research, we still lack a comprehensive explanation for this complexity.</p> <p>Results</p> <p>We inspected the available literature and genome databases for information on components of RNA metabolism in land plant chloroplasts. In particular, new inventions of chloroplast-specific mechanisms and the expansion of some gene/protein families detected in land plants lead us to suggest that the primary function of the additional nuclear-encoded components found in chloroplasts is the transgenomic suppression of point mutations, fixation of which occurred due to an enhanced genetic drift exhibited by chloroplast genomes. We further speculate that a fast evolution of transgenomic suppressors occurred after the water-to-land transition of plants.</p> <p>Conclusion</p> <p>Our inspections indicate that several chloroplast-specific mechanisms evolved in land plants to remedy point mutations that occurred after the water-to-land transition. Thus, the complexity of chloroplast gene expression evolved to guarantee the functionality of chloroplast genetic information and may not, with some exceptions, be involved in regulatory functions.</p

Metabolic Engineering of the Phenylpropanoid and Its Primary, Precursor Pathway to Enhance the Flavor of Fruits and the Aroma of Flowers

Plants produce a diverse repertoire of specialized metabolites that have multiple roles throughout their life cycle. Some of these metabolites are essential components of the aroma and flavor of flowers and fruits. Unfortunately, attempts to increase the yield and prolong the shelf life of crops have generally been associated with reduced levels of volatile specialized metabolites and hence with decreased aroma and flavor. Thus, there is a need for the development of new varieties that will retain their desired traits while gaining enhanced scent and flavor. Metabolic engineering holds great promise as a tool for improving the profile of emitted volatiles of domesticated crops. This mini review discusses recent attempts to utilize metabolic engineering of the phenylpropanoid and its primary precursor pathway to enhance the aroma and flavor of flowers and fruits