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Selection of Shine-Dalgarno sequences in plastids.

Drechsel O, Bock R - Nucleic Acids Res. (2010)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institut für Molekulare Pflanzenphysiologie, Am Mühlenberg 1, D-14476 Potsdam-Golm, Germany.

ABSTRACT
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.

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Analysis of gfp expression in tobacco plastids. (A) Western blot analyses to determine GFP accumulation levels in transplastomic tobacco plants generated with pOD vectors. Loaded amounts of total protein are indicated below the blots. Dashes denote empty lanes. As a control for loading, the high-molecular weight region of the gel (which was not blotted) was stained with Coomassie and is shown below each blot. WT, wild-type (1 µg protein loaded); S5, transplastomic control (carrying the aadA but no gfp; ref. 71; 1 µg protein loaded); GFP, purified recombinant GFP used as standard for quantitation. (B) Analysis of gfp mRNA accumulation in pOD transplastomic plants. The lower band represents monocistronic gfp mRNA, the upper band is the result of read through transcription and has been observed before in studies using the same plastid transformation vector backbone (pRB95; 28). To control for equal loading, the blot was stripped and re-hybridized to a 16S rRNA-specific probe.
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Figure 3: Analysis of gfp expression in tobacco plastids. (A) Western blot analyses to determine GFP accumulation levels in transplastomic tobacco plants generated with pOD vectors. Loaded amounts of total protein are indicated below the blots. Dashes denote empty lanes. As a control for loading, the high-molecular weight region of the gel (which was not blotted) was stained with Coomassie and is shown below each blot. WT, wild-type (1 µg protein loaded); S5, transplastomic control (carrying the aadA but no gfp; ref. 71; 1 µg protein loaded); GFP, purified recombinant GFP used as standard for quantitation. (B) Analysis of gfp mRNA accumulation in pOD transplastomic plants. The lower band represents monocistronic gfp mRNA, the upper band is the result of read through transcription and has been observed before in studies using the same plastid transformation vector backbone (pRB95; 28). To control for equal loading, the blot was stripped and re-hybridized to a 16S rRNA-specific probe.

Mentions: Construction of vectors to analyze various combinations of plastid translation initiation signals. (A) Physical map of the targeting region in the plastid genome after integration of transformation constructs of the pOD series. The BglII restriction sites used for RFLP analysis are marked. The transgenes are targeted to the intergenic region between the trnfM and trnG genes (42). The GFP expression cassette consists of the ribosomal RNA operon promoter (Prrn) fused to a Shine-Dalgarno (SD) sequence element (see panel B) and the 3′-UTR from the plastid rps16 gene (Trps16). The expected sizes of gfp transcripts are indicated (cf. Figure 3B). The location of the RFLP probe is shown as black bar. The selectable marker gene aadA is driven by a chimeric ribosomal RNA operon promoter (Prrn) and fused to the 3′-UTR from the psbA gene (TpsbA; ref. 45) (B) Schematic maps of the different translation initiation signals tested in this study (pOD vector series). SD sequences are shown in orange, start codons (ATG) and mini-ORFs are indicated in green with the sequence given below the map. TEV: tobacco etch virus peptidase cleavage site; GFP: gene for the green fluorescent protein. The difference between the two basic vectors pOD1 and pOD19 lies in the mutational elimination of an in-frame stop codon upstream of the SD to facilitate translation of GFP from the first SD in constructs pOD20 and pOD21.


Selection of Shine-Dalgarno sequences in plastids.

Drechsel O, Bock R - Nucleic Acids Res. (2010)

Analysis of gfp expression in tobacco plastids. (A) Western blot analyses to determine GFP accumulation levels in transplastomic tobacco plants generated with pOD vectors. Loaded amounts of total protein are indicated below the blots. Dashes denote empty lanes. As a control for loading, the high-molecular weight region of the gel (which was not blotted) was stained with Coomassie and is shown below each blot. WT, wild-type (1 µg protein loaded); S5, transplastomic control (carrying the aadA but no gfp; ref. 71; 1 µg protein loaded); GFP, purified recombinant GFP used as standard for quantitation. (B) Analysis of gfp mRNA accumulation in pOD transplastomic plants. The lower band represents monocistronic gfp mRNA, the upper band is the result of read through transcription and has been observed before in studies using the same plastid transformation vector backbone (pRB95; 28). To control for equal loading, the blot was stripped and re-hybridized to a 16S rRNA-specific probe.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3045613&req=5

Figure 3: Analysis of gfp expression in tobacco plastids. (A) Western blot analyses to determine GFP accumulation levels in transplastomic tobacco plants generated with pOD vectors. Loaded amounts of total protein are indicated below the blots. Dashes denote empty lanes. As a control for loading, the high-molecular weight region of the gel (which was not blotted) was stained with Coomassie and is shown below each blot. WT, wild-type (1 µg protein loaded); S5, transplastomic control (carrying the aadA but no gfp; ref. 71; 1 µg protein loaded); GFP, purified recombinant GFP used as standard for quantitation. (B) Analysis of gfp mRNA accumulation in pOD transplastomic plants. The lower band represents monocistronic gfp mRNA, the upper band is the result of read through transcription and has been observed before in studies using the same plastid transformation vector backbone (pRB95; 28). To control for equal loading, the blot was stripped and re-hybridized to a 16S rRNA-specific probe.
Mentions: Construction of vectors to analyze various combinations of plastid translation initiation signals. (A) Physical map of the targeting region in the plastid genome after integration of transformation constructs of the pOD series. The BglII restriction sites used for RFLP analysis are marked. The transgenes are targeted to the intergenic region between the trnfM and trnG genes (42). The GFP expression cassette consists of the ribosomal RNA operon promoter (Prrn) fused to a Shine-Dalgarno (SD) sequence element (see panel B) and the 3′-UTR from the plastid rps16 gene (Trps16). The expected sizes of gfp transcripts are indicated (cf. Figure 3B). The location of the RFLP probe is shown as black bar. The selectable marker gene aadA is driven by a chimeric ribosomal RNA operon promoter (Prrn) and fused to the 3′-UTR from the psbA gene (TpsbA; ref. 45) (B) Schematic maps of the different translation initiation signals tested in this study (pOD vector series). SD sequences are shown in orange, start codons (ATG) and mini-ORFs are indicated in green with the sequence given below the map. TEV: tobacco etch virus peptidase cleavage site; GFP: gene for the green fluorescent protein. The difference between the two basic vectors pOD1 and pOD19 lies in the mutational elimination of an in-frame stop codon upstream of the SD to facilitate translation of GFP from the first SD in constructs pOD20 and pOD21.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Max-Planck-Institut für Molekulare Pflanzenphysiologie, Am Mühlenberg 1, D-14476 Potsdam-Golm, Germany.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus