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Masahiro Sugiura

Publications and source records attributed to Masahiro Sugiura.

At least 19 recordsLinked to original sources
PubMed2007

Complete nucleotide sequence of the freshwater unicellular cyanobacterium Synechococcus elongatus PCC 6301 chromosome: gene content and organization.

The entire genome of the unicellular cyanobacterium Synechococcus elongatus PCC 6301 (formerly Anacystis nidulans Berkeley strain 6301) was sequenced. The genome consisted of a circular chromosome 2,696,255 bp long. A total of 2,525 potential protein-coding genes, two sets of rRNA genes, 45 tRNA genes representing 42 tRNA species, and several genes for small stable RNAs were assigned to the chromosome by similarity searches and computer predictions. The translated products of 56% of the potential protein-coding genes showed sequence similarities to experimentally identified and predicted proteins of known function, and the products of 35% of the genes showed sequence similarities to the translated products of hypothetical genes. The remaining 9% of genes lacked significant similarities to genes for predicted proteins in the public DNA databases. Some 139 genes coding for photosynthesis-related components were identified. Thirty-seven genes for two-component signal transduction systems were also identified. This is the smallest number of such genes identified in cyanobacteria, except for marine cyanobacteria, suggesting that only simple signal transduction systems are found in this strain. The gene arrangement and nucleotide sequence of Synechococcus elongatus PCC 6301 were nearly identical to those of a closely related strain Synechococcus elongatus PCC 7942, except for the presence of a 188.6 kb inversion. The sequences as well as the gene information shown in this paper are available in the Web database, CYORF (http://www.cyano.genome.jp/).

Base Sequence↗
PubMed2006

A new in vitro translation system for non-radioactive assay from tobacco chloroplasts: effect of pre-mRNA processing on translation in vitro.

We previously developed an in vitro translation system derived from tobacco chloroplasts. Here, we report a significantly improved in vitro translation system. By modifying preparation procedures for chloroplast extracts and reaction conditions, we achieved 100-fold higher translation activity than the previous system. The new system does not require the supplement of Escherichia coli tRNAs due to the omission of micrococcal nuclease treatment, thus the tRNA population reflects the intrinsic tRNA population in tobacco chloroplasts. The rate of translation initiation from a variety of chloroplast mRNAs may be measured by monitoring the fluorescence intensity of synthesized green fluorescent protein, which is a non-radioactive detection method. Incorporation of an amino acid linked to a fluorescent dye also allows detection of the translation products in vitro. Using our new system, we found that mRNAs carrying unprocessed or processed atpH and rbcL 5'-UTRs were efficiently translated at similar rates, whereas translation of mRNAs with processed atpB and psbB 5'-UTRs was more efficient than those with unprocessed 5'-UTRs. These results suggest that the role of 5'-UTR processing in the regulation of chloroplast gene expression differs between mRNAs. The new in vitro translation system will be a powerful tool to investigate the mechanism of chloroplast mRNA translation.

5' Untranslated Regions↗
PubMed2006

Translation efficiencies of synonymous codons are not always correlated with codon usage in tobacco chloroplasts.

Codon usage in chloroplasts is different from that in prokaryotic and eukaryotic nuclear genomes. However, no experimental approach has been made to analyse the translation efficiency of individual codons in chloroplasts. We devised an in vitro assay for translation efficiencies using synthetic mRNAs, and measured the translation efficiencies of five synonymous codon groups in tobacco chloroplasts. Among four alanine codons (GCN, where N is U, C, A or G), GCU was the most efficient for translation, whereas the chloroplast genome lacks tRNA genes corresponding to GCU. Phenylalanine and tyrosine are each encoded by two codons (UUU/C and UAU/C, respectively). Phenylalanine UUC and tyrosine UAC were translated more than twice as efficiently than UUU and UAU, respectively, contrary to their codon usage, whereas translation efficiencies of synonymous codons for alanine, aspartic acid and asparagine were parallel to their codon usage. These observations indicate that translation efficiencies of individual codons are not always correlated with codon usage in vitro in chloroplasts. This raises an important issue for foreign gene expression in chloroplasts.

Amino Acid Sequence↗
PubMed2006

A survey of expressed tRNA genes in the chromosome I of Arabidopsis using an RNA polymerase III-dependent in vitro transcription system.

Eukaryotic tRNA genes are transcribed by RNA polymerase III. These tRNA genes are generally predicted using computer programs, and 620 tRNA genes in the Arabidopsis thaliana genome are currently annotated. However, no effort has been made to assay whether these predicted tRNA genes are all expressed, because it has been difficult to assay by routine in vivo methods. We report here a large-scale tRNA expression assay of predicted Arabidopsis tRNA genes using an RNA polymerase III-dependent in vitro transcription system developed by our group. DNA fragments including an annotated tRNA gene each were amplified by PCR and the resulting linear DNA was subjected to in vitro transcription. The addition of poly(dA-dT).poly(dA-dT) enhanced activity significantly and reduced background. The 124 predicted tRNA genes present in the Arabidopsis chromosome I were examined, and transcription activity and transcript stability from individual genes were determined. These results indicated that eight annotated genes are not expressed. Based on previous reports on pseudo-tRNA genes (e.g., Beier and Beier, Mol. Gen. Genet. 1992; 233: 201-208) and the present results, we estimated that 16% or more of the annotated tRNA genes in the chromosome I are not functional.

5' Flanking Region↗
PubMed2006

Translation initiation of cyanobacterial rbcS mRNAs requires the 38-kDa ribosomal protein S1 but not the Shine-Dalgarno sequence: development of a cyanobacterial in vitro translation system.

Little is known about the biochemical mechanism of translation in cyanobacteria though substantial studies have been made on photosynthesis, nitrogen fixation, circadian rhythm, and genome structure. To analyze the mechanism of cyanobacterial translation, we have developed an in vitro translation system from Synechococcus cells using a psbAI-lacZ fusion mRNA as a model template. This in vitro system supports accurate translation from the authentic initiation site of a variety of Synechococcus mRNAs. In Synechococcus cells, rbcL and rbcS encoding the large and small subunits, respectively, of ribulose-1,5-bisphosphate carboxylase/oxygenase are co-transcribed as a dicistronic mRNA, and the downstream rbcS mRNA possesses two possible initiation codons separated by three nucleotides. Using this in vitro system and mutated mRNAs, we demonstrated that translation starts exclusively from the upstream AUG codon. Although there are Shine-Dalgarno-like sequences in positions similar to those of the functional Shine-Dalgarno elements in Escherichia coli, mutation analysis indicated that these sequences are not required for translation. Assays with deletions within the 5'-untranslated region showed that a pyrimidine-rich sequence in the -46 to -15 region is necessary for efficient translation. Synechococcus cells contain two ribosomal protein S1 homologues of 38 and 33 kDa in size. UV cross-linking and immunoprecipitation experiments suggested that the 38-kDa S1 is involved in efficient translation via associating with the pyrimidine-rich sequence. The present in vitro translation system will be a powerful tool to analyze the basic mechanism of translation in cyanobacteria.

Base Sequence↗
PubMed2006

A simple in vitro RNA editing assay for chloroplast transcripts using fluorescent dideoxynucleotides: distinct types of sequence elements required for editing of ndh transcripts.

RNA editing is found in various transcripts from land plant chloroplasts. In tobacco chloroplasts, C-to-U conversion occurs at 36 specific sites including two sites identified in this work. Our RNA editing assay system using chloroplast extracts facilitated biochemical analyses of editing reactions but required mRNAs labeled with (32)P at specific sites. Here, we have improved the in vitro system using fluorescence-labeled chain terminators, ddGTP and ddATP, and have measured the editing activity at 19 sites in ndh transcripts. Editing activities varied from site to site. It has been reported that one editing site in ndhA mRNAs is present in spinach but absent in tobacco, but a corresponding editing capacity had been found in vivo in tobacco using biolistic transformation. We confirmed biochemically the existence of this activity in tobacco extracts. Using the non-radioactive assay, we examined sequences essential for editing within a 50-nt mRNA region encompassing an editing site. Editing of the ndhB-2 site requires a short sequence in front of the editing site, while that of the ndhF mRNA requires two separate regions, a sequence surrounding the editing site and a 5' distal sequence. These results suggest that distinct editing mechanisms are present in chloroplasts.

Chloroplasts↗
PubMed2006

Distinct modes of TATA box utilization by the RNA polymerase III transcription machineries from budding yeast and higher plants.

The TATA box is a key upstream control element for basal tRNA gene transcription by RNA polymerase III in some eukaryotes, such as the fission yeast (Schizosaccharomyces pombe) and higher plants, but not in others such as the budding yeast (Saccharomyces cerevisiae). To gain information on this differential TATA box requirement, we examined side-by-side the in vitro transcription properties of TATA-containing and TATA-mutated plant and S. cerevisiae tDNAs in homologous in vitro transcription systems from both organisms and in a hybrid system in which yeast TBP was replaced by its plant homologue. The data support the general conclusion that specific features of the plant transcription machinery, rather than upstream region architecture per se, are responsible for the much stronger TATA box dependence of the plant system. In both systems, however, a strong influence of the TATA box on transcription start site selection was observed. This was particularly striking in the case of plant tDNAs, where TATA-rich upstream regions were found to favour the use of alternative initiation sites. Replacement of yeast TBP with its plant counterpart did not confer any general TATA box responsiveness to the yeast transcription machinery. Interactions involving components other than TBP are thus responsible for the strong TATA box requirement of plant tDNA transcription.

Arabidopsis↗
PubMed2005

Plant 7SL RNA genes belong to type 4 of RNA polymerase III- dependent genes that are composed of mixed promoters.

The genes transcribed by RNA polymerase III (pol III) display a great diversity in terms of promoter structure and are placed in four groups accordingly. Type 3 subset of pol III genes has promoter elements which reside entirely upstream of the coding region of the gene whereas type 4 consists of genes with mixed promoters that enclose intra- and extragenic regulatory sequences. Plant 7SL RNA genes have been previously classified as type 3 of pol III genes requiring an upstream sequence element and a canonical TATA box for transcriptional activity in transfected plant protoplasts. We have identified two novel functional control regions within the coding region of an Arabidopsis 7SL RNA gene (At7SL-1) that resemble tRNA gene-specific A and B boxes with respect to sequence and position. Single and multiple nucleotide substitutions in either of these regions resulted in a pronounced reduction of transcription activity in tobacco nuclear extract that was not caused by a decreased stability as shown by decay kinetics of wild type and mutant RNA transcripts. These findings suggest that plant 7SL RNA genes should be actually placed in type 4 of pol III-transcribed genes. As a consequence of substantially different upstream promoters utilized by plant and human pol III, in vitro transcription of 7SL RNA genes in heterologous systems is severely impaired. A chimeric human 7SL RNA gene that contains the 5' flanking region up to position -300 of At7SL-1 is yet transcribed with a reduced efficiency in tobacco extract when compared with the plant wild-type gene, supporting the notion that internal regulatory elements contribute to full activity.

Arabidopsis↗
PubMed2004

Multiple elements required for translation of plastid atpB mRNA lacking the Shine-Dalgarno sequence.

The mechanism of translational initiation differs between prokaryotes and eukaryotes. Prokaryotic mRNAs generally contain within their 5'-untranslated region (5'-UTR) a Shine-Dalgarno (SD) sequence that serves as a ribosome-binding site. Chloroplasts possess prokaryotic-like translation machinery, and many chloroplast mRNAs have an SD-like sequence, but its position is variable. Tobacco chloroplast atpB mRNAs contain no SD-like sequence and are U-rich in the 5'-UTR (-20 to -1 with respect to the start codon). In vitro translation assays with mutated mRNAs revealed that an unstructured sequence encompassing the start codon, the AUG codon and its context are required for translation. UV crosslinking experiments showed that a 50 kDa protein (p50) binds to the 5'-UTR. Insertion of an additional initiation region (SD-sequence and AUG) in the 5'-UTR, but not downstream, arrested translation from the authentic site; however, no inhibition was observed by inserting only an AUG triplet. We hypothesize for translational initiation of the atpB mRNA that the ribosome enters an upstream region, slides to the start codon and forms an initiation complex with p50 and other components.

5' Untranslated Regions↗
PubMed2004

Functional Shine-Dalgarno-like sequences for translational initiation of chloroplast mRNAs.

Many of the chloroplast mRNAs possess Shine-Dalgarno (SD)-like sequences (typically GGAGG) in the 5'-untranslated regions, but the position is highly variable. Using a homologous in vitro translation system, we assessed the role for translation of SD-like sequences in four tobacco chloroplast mRNAs. The rbcL mRNA has a typical SD-like sequence at a position similar to the conserved position (-12 to -4 with respect to the start codon) observed in E. coli, and this sequence was found to be essential for translation. This was also the case for the atpE mRNA. However, SD-like sequences in the rps12 mRNA and in the petB mRNA is located far from (-44 to -42) and too close to (-5 to -2) the initiation codon, respectively, and these sequences were not essential for translation. These results indicate that functional SD-like sequences are located around 10 nucleotides upstream from the translational start codon. Competition assays confirmed that a functional SD-like sequence interacts with the 3' terminus of chloroplast 16S rRNA.

Base Sequence↗
PubMed2004

A systematic search for RNA editing sites in pea chloroplasts: an editing event causes diversification from the evolutionarily conserved amino acid sequence.

RNA editing in higher plant chloroplasts involves C-to-U conversion at specific sites in the transcripts. To examine whether pea shares editing sites with other angiosperms, a systematic search for editing sites in pea chloroplast transcripts was performed. Based on amino acid sequence alignment, 451 RNA editing sites were predicted from 60 transcripts. Sequence analysis of amplified cDNAs for these potential editing sites revealed 19 true editing sites from 13 transcripts. Together with those reported previously, the total number of editing sites is 27 from 16 transcripts in pea chloroplasts. Twenty-two sites are conserved among other plant species, whereas five sites are unique to pea. Among the 27 editing sites, seven are partially edited. The most interesting is the ndhG site 1, which has led to the diversification of the evolutionarily conserved amino acid sequence. This observation suggests that some of the editing events cause the diversity of amino acid sequences, and hence, that prediction of editing sites based on amino acid sequence alignment has its own limitations.

Amino Acid Sequence↗
PubMed2004

In vitro RNA editing systems from higher plant chloroplasts.

RNA editing in higher plant chloroplasts involves C-to-U conversion at ~30 specific sites. In vitro systems supporting accurate editing have been developed from tobacco and pea chloroplasts. mRNA substrates labeled with 32P at C residues to be edited provide sensitive detection of editing activity in vitro. The present systems allow the rapid identification of cis-elements using mutated mRNA substrates and trans-acting factors by ultraviolet crosslinking.

Base Sequence↗
PubMed2003

A site-specific factor interacts directly with its cognate RNA editing site in chloroplast transcripts.

RNA editing involves a variety of genetic systems and occurs by different mechanisms. In higher plant chloroplasts, specific sites of some transcripts are subject to C-to-U conversion. We have previously shown that site-specific trans-acting factors for psbE and petB mRNA editing bind corresponding cis-elements, which are located 5 nucleotides upstream from the editing site. Here we report that, by using mRNAs labeled either at the center of the upstream cis-element or at the editing site, the site-specific factors can be cross-linked with nucleotides at both positions. Mutations of nucleotides in the proximal region of the editing site revealed a correlation between editing activity and cross-linking efficiency of factors with the editing site, even though cross-linking with the upstream cis-element was unaffected. These observations suggest that the site-specific factor binds stably to the upstream ciselement, whereas it interacts weakly with the editing site. This finding raises the intriguing possibility that the site-specific factor is involved in both site-determination and C-to-U conversion in chloroplast RNA editing.

Base Sequence↗
PubMed2003

Molecular cloning, expression and characterization of three distinctive genes encoding methionine aminopeptidases in cyanobacterium Synechocystis sp. strain PCC6803.

Methionine aminopeptidase, known to be encoded by single genes in prokaryotes, is a cobalt-dependent enzyme that catalyzes the removal of N-terminal methionine residues from nascent polypeptides. Three ORFs encoding putative methionine aminopeptidases from the genome of cyanobacterium Synechocystis sp. strain PCC6803, designated as slr0786 ( map-1), slr0918 ( map-2) and sll0555 ( map-3) were cloned and expressed in Escherichia coli. The purified recombinant proteins encoded by map-1 and map-3 had much higher methionine aminopeptidase activity than the recombinant protein encoded by map-2. Comparative analysis revealed that the three recombinant enzymes differed in their substrate specificity, divalent ion requirement, pH, and temperature optima. The broad activities of the iso-enzymes are discussed in light of the structural similarities with other peptidase families and their levels of specificity in the cell. Potential application of cyanobacterial MetAPs in the production of recombinant proteins used in medicine is proposed. This is the first report of a prokaryote harboring multiple methionine aminopeptidases.

Amino Acid Sequence↗
PubMed2003

Identification and characterization of transcription factor IIIA and ribosomal protein L5 from Arabidopsis thaliana.

Thus far, no transcription factor IIIA (TFIIIA) from higher plants has been cloned and characterized. We have cloned and characterized TFIIIA and ribosomal protein L5 from Arabidopsis thaliana. Primary sequence comparison revealed a high divergence of AtTFIIIA and a relatively high conservation of AtL5 when compared with other organisms. The AtTFIIIA cDNA encodes a protein with nine Cys(2)-His(2)-type zinc fingers, a 23 amino acid spacer between fingers 1 and 2, a 66 amino acid spacer between fingers 4 and 5, and a 50 amino acid non-finger C-terminal tail. Aside from the amino acids required for proper zinc finger folding, AtTFIIIA is highly divergent from other known TFIIIAs. AtTFIIIA can bind 5S rDNA, as well as 5S rRNA, and efficiently stimulates the transcription of an Arabidopsis 5S rRNA gene in vitro. AtL5 identity was confirmed by demonstrating that this protein binds to 5S rRNA but not to 5S rDNA. Protoplast transient expression assays with green fluorescent protein fusion proteins revealed that AtTFIIIA is absent from the cytoplasm and concentrated at several nuclear foci including the nucleolus. AtL5 protein accumulates in the nucleus, especially in the nucleolus, and is also present in the cytoplasm.

Amino Acid Sequence↗
PubMed2003

Identification of RNA editing sites in chloroplast transcripts from the maternal and paternal progenitors of tobacco (Nicotiana tabacum): comparative analysis shows the involvement of distinct trans-factors for ndhB editing.

RNA editing alters genomic nucleotide sequences at the transcript level. In higher plant chloroplasts, C-to-U conversion is known to occur at around 30 specific sites. The tobacco cultivar Nicotiana tabacum is an amphidiploid derived from ancestors of N. sylvestris (maternal) and N. tomentosiformis (paternal). The chloroplast genome of N. tabacum is believed to originate from an ancestor of N. sylvestris. To study the evolution of RNA editing in higher plant chloroplasts, editing sites in the two likely progenitors have first been identified based on those found in N. tabacum. Altogether 34, 33, and 32 editing sites have been found in the chloroplast transcripts from N. tabacum, N. sylvestris, and N. tomentosiformis, respectively. Thirty-one sites are conserved among the three species, whereas remarkable differences are observed in the editing of ndhB and ndhD transcripts. Sites 7 and 8 in ndhB mRNAs are separated only by five nt, and both are edited in N. tabacum and N. sylvestris. However, site 8 is not edited in N. tomentosiformis, indicating that distinct trans-factors are involved in the two editing events. The first site in ndhD mRNAs is edited to produce an AUG start codon in N. sylvestris as well as in N. tabacum but not in N. tomentosiformis, suggesting that a distinct mechanism operates for the translational initiation of N. tomentosiformis ndhD mRNAs. Four to six sites are edited partially in green leaves. Some of these sites may represent evolutionary intermediates in the process of losing editing events.

Cell Lineage↗