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

Suzuki, Haruka; Kuroda, Hiroshi; Yukawa, Yasushi; Sugiura, Masahiro

doi:10.1093/nar/gkr644

Cited by 23 publications

(24 citation statements)

References 53 publications

(63 reference statements)

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“…For example, our results definitively show that translation of the maize atpB and atpE ORFs is not coupled in vivo, corroborating conclusions based on in vitro data (Suzuki et al, 2011) but differing from results obtained when these genes were expressed heterologously in bacteria (Gatenby et al, 1989). In addition, we were able to address for the first time whether splicing needs to precede translation initiation in chloroplasts.…”

Section: Insights Into Translation Mechanisms In Chloroplastssupporting

confidence: 79%

“…The prominent peaks at positions 55,593 and 55,514 in the mutant profiles are also noteworthy: Each of these peaks span two array elements whose overlap contains the next strongest matches to the ideal Shine-Dalgarno sequence in the atpB ORF (Figure 6; see Supplemental Table 1 online). A peak at 54,645 in both the wild-type and mutant data maps to the known Shine-Dalgarno element for atpE (Suzuki et al, 2011). In fact, every appearance of five contiguous nucleotides matching the ideal Shine-Dalgarno element in atpB corresponds with a peak in ribosome footprints (Figure 6; see Supplemental Table 1 online).…”

Section: Evidence For Ribosome Pausing At Shine-dalgarno-like Sequencesmentioning

confidence: 95%

“…The translation of overlapping ORFs in bacteria is typically coupled, in that translation of the second ORF is dependent on the translation of the first (reviewed in Jackson et al, 2007). However, there is conflicting data concerning the coupled translation of overlapping ORFs in chloroplasts: Analysis of the overlapping chloroplast atpB/atpE ORFs in heterologous expression systems pointed to coupled translation (Gatenby et al, 1989), whereas in vitro studies with a tobacco (Nicotiana tabacum) chloroplast translation extract suggested independent translation (Suzuki et al, 2011). (A) Follow-up to atp4 ribosome profile data suggesting a defect in rpl14 expression ( Figure 3C; see Supplemental Figure 2C online).…”

Section: Translation Of Overlapping Chloroplast Reading Frames: Atpe mentioning

confidence: 99%

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A Rapid Ribosome Profiling Method Elucidates Chloroplast Ribosome Behavior in Vivo

2013

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The profiling of ribosome footprints by deep sequencing has revolutionized the analysis of translation by mapping ribosomes with high resolution on a genome-wide scale. We present a variation on this approach that offers a rapid and cost-effective alternative for the genome-wide profiling of chloroplast ribosomes. Ribosome footprints from leaf tissue are hybridized to oligonucleotide tiling microarrays of the plastid ORFeome and report the abundance and translational status of every chloroplast mRNA. Each assay replaces several time-consuming traditional methods while also providing information that was previously inaccessible. To illustrate the utility of the approach, we show that it detects known defects in chloroplast gene expression in several nuclear mutants of maize (Zea mays) and that it reveals previously unsuspected defects. Furthermore, it provided firm answers to several lingering questions in chloroplast gene expression: (1) the overlapping atpB/atpE open reading frames, whose translation had been proposed to be coupled, are translated independently in vivo; (2) splicing is not a prerequisite for translation initiation on an intron-containing chloroplast RNA; and (3) a feedback control mechanism that links the synthesis of ATP synthase subunits in Chlamydomonas reinhardtii does not exist in maize. An analogous approach is likely to be useful for studies of mitochondrial gene expression.

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Section: Insights Into Translation Mechanisms In Chloroplastssupporting

confidence: 79%

Section: Evidence For Ribosome Pausing At Shine-dalgarno-like Sequencesmentioning

confidence: 95%

Section: Translation Of Overlapping Chloroplast Reading Frames: Atpe mentioning

confidence: 99%

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A Rapid Ribosome Profiling Method Elucidates Chloroplast Ribosome Behavior in Vivo

2013

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“…Moreover, the 5=-UTR (AGGAG) of phaR is a Shine-Dalgarno (SD)-like sequence, while SD-like sequences (GAGGAAGGAGA) were also found 50 bp upstream of the ATG codon of the phaP gene. As reported in chloroplasts, a downstream cistron (atpE) of dicistronic mRNAs possesses its own cis-element for efficient translation (42); it is likely that phaP might also be translated independently, even though there is an overlap (ATGAGTGA) of the phaR ORF and the phaP ORF. Further research would also be dedicated to the exploration of this multilevel regulation between phaR and phaP in haloarchaea.…”

Section: Discussionmentioning

confidence: 94%

A Novel DNA-Binding Protein, PhaR, Plays a Central Role in the Regulation of Polyhydroxyalkanoate Accumulation and Granule Formation in the Haloarchaeon Haloferax mediterranei

Cai

Zhao

et al. 2015

Appl Environ Microbiol

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c Polyhydroxyalkanoates (PHAs) are synthesized and assembled as PHA granules that undergo well-regulated formation in many microorganisms. However, this regulation remains unclear in haloarchaea. In this study, we identified a PHA granule-associated regulator (PhaR) that negatively regulates the expression of both its own gene and the granule structural gene phaP in the same operon (phaRP) in Haloferax mediterranei. Chromatin immunoprecipitation-quantitative PCR (ChIP-qPCR) assays demonstrated a significant interaction between PhaR and the phaRP promoter in vivo. Scanning mutagenesis of the phaRP promoter revealed a specific cis-element as the possible binding position of the PhaR. The haloarchaeal homologs of the PhaR contain a novel conserved domain that belongs to a swapped-hairpin barrel fold family found in AbrB-like proteins. Amino acid substitution indicated that this AbrB-like domain is critical for the repression activity of PhaR. In addition, the phaRP promoter had a weaker activity in the PHA-negative strains, implying a function of the PHA granules in titration of the PhaR. Moreover, the H. mediterranei strain lacking phaR was deficient in PHA accumulation and produced granules with irregular shapes. Interestingly, the PhaR itself can promote PHA synthesis and granule formation in a PhaP-independent manner. Collectively, our results demonstrated that the haloarchaeal PhaR is a novel bifunctional protein that plays the central role in the regulation of PHA accumulation and granule formation in H. mediterranei. P olyhydroxyalkanoates (PHAs) are biodegradable polyesters synthesized by most genera of bacteria (1, 2) and some archaea (3-5). PHAs are accumulated as storage compounds of energy and carbon under imbalanced growth conditions (i.e., when nutrients such as nitrogen, phosphorus, or oxygen are limited but the carbon sources are in excess) (6).PHAs are often deposited in the cytoplasm as water-insoluble inclusions that are called PHA granules (6). Native PHA granules are found to be composed of 97.5% PHA, 2% proteins, and likely some amount of lipids (7). At least four types of proteins were found to be the PHA granule-associated proteins (PGAPs) in bacteria: PHA synthases, PHA depolymerases, regulators, and structural proteins (phasins [PhaPs]) (8, 9). In recent years, increasing new roles have been found for the PGAPs. Besides the classical phasin role of preventing PHA granules from coalescing, two distinct phasin-like proteins, PhaM and PhaF, have also been characterized as being crucial for granule distribution during cell division (10, 11).The PGAPs play important roles in PHA synthesis, PHA utilization, and granule formation and distribution (8, 9, 12), among which the regulatory proteins are responsible for ensuring the proper formation of PHA granules by influencing the expression of both phasins and themselves (13)(14)(15)(16)(17). A classic regulation model was presented in a poly(3-hydroxybutyrate) (PHB [a type of PHA])-accumulating bacterium, Ralstonia eutropha H16 (9). Briefly, the cy...

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“…These downstream cistrons are fully or mostly independently translated. In addition, they have additional promoters within the upstream genes and produce monocistronic atpE and psbC mRNAs, which are actively translated in vitro (42,44).…”

Section: Discussionmentioning

confidence: 99%

Additional pathway to translate the downstream ndhK cistron in partially overlapping ndhC-ndhK mRNAs in chloroplasts

Yukawa

Sugiura

2013

Proc. Natl. Acad. Sci. U.S.A.

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The chloroplast NAD(P)H dehydrogenase ( NDH ) C ( ndhC ) and ndhK genes partially overlap and are cotranscribed in many plants. We previously reported that the tobacco ndhC/K genes are translationally coupled but produce NdhC and NdhK, subunits of the NDH complex, in similar amounts. Generally, translation of the downstream cistron in overlapping mRNAs is very low. Hence, these findings suggested that the ndhK cistron is translated not only from the ndhC 5′UTR but also by an additional pathway. Using an in vitro translation system from tobacco chloroplasts, we report here that free ribosomes enter, with formylmethionyl-tRNA fMet , at an internal AUG start codon that is located in frame in the middle of the upstream ndhC cistron, translate the 3′ half of the ndhC cistron, reach the ndhK start codon, and that, at that point, some ribosomes resume ndhK translation. We detected a peptide corresponding to a 57-amino-acid product encoded by the sequence from the internal AUG to the ndhC stop codon. We propose a model in which the internal initiation site AUG is not designed for synthesizing a functional isoform but for delivering additional ribosomes to the ndhK cistron to produce NdhK in the amount required for the assembly of the NDH complex. This pathway is a unique type of translation to produce protein in the needed amount with the cost of peptide synthesis.

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

Cited by 23 publications

References 53 publications

A Rapid Ribosome Profiling Method Elucidates Chloroplast Ribosome Behavior in Vivo

A Rapid Ribosome Profiling Method Elucidates Chloroplast Ribosome Behavior in Vivo

A Novel DNA-Binding Protein, PhaR, Plays a Central Role in the Regulation of Polyhydroxyalkanoate Accumulation and Granule Formation in the Haloarchaeon Haloferax mediterranei

Additional pathway to translate the downstream ndhK cistron in partially overlapping ndhC-ndhK mRNAs in chloroplasts

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