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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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Related in: MedlinePlus

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.
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Figure 1: 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.

Mentions: Tobacco plastids and E. coli have identical anti-SD sequences in the 3′-end of their 16S ribosomal RNAs (5′-TGGATCACCTCCTT-3′; anti-SD motif underlined) and, therefore, plastid SD sequences can be recognized in E. coli and vice versa. The SD consensus sequence is GGAGG for both systems. In order to comparatively analyze the principles governing the recognition of SD sequences in plastids and in bacteria, we designed a strategy that allowed us to study translation of identical synthetic transcripts containing multiple SD sequences in E. coli and tobacco plastids. To this end, we constructed a GFP expression cassette driven by the plastid ribosomal operon promoter Prrn, a σ70-type promoter known to be active also in E. coli (27,50,43). A minilinker inserted between the transcriptional start site and the SD sequence upstream of the gfp reading frame allowed the convenient insertion of additional SD sequences, start codons, stop codons and mini-ORFs (open reading frames; Figure 1).Figure 1.


Selection of Shine-Dalgarno sequences in plastids.

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

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.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
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getmorefigures.php?uid=PMC3045613&req=5

Figure 1: 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.
Mentions: Tobacco plastids and E. coli have identical anti-SD sequences in the 3′-end of their 16S ribosomal RNAs (5′-TGGATCACCTCCTT-3′; anti-SD motif underlined) and, therefore, plastid SD sequences can be recognized in E. coli and vice versa. The SD consensus sequence is GGAGG for both systems. In order to comparatively analyze the principles governing the recognition of SD sequences in plastids and in bacteria, we designed a strategy that allowed us to study translation of identical synthetic transcripts containing multiple SD sequences in E. coli and tobacco plastids. To this end, we constructed a GFP expression cassette driven by the plastid ribosomal operon promoter Prrn, a σ70-type promoter known to be active also in E. coli (27,50,43). A minilinker inserted between the transcriptional start site and the SD sequence upstream of the gfp reading frame allowed the convenient insertion of additional SD sequences, start codons, stop codons and mini-ORFs (open reading frames; Figure 1).Figure 1.

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