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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

Analysis of gfp expression in E. coli. All experiments were repeated at least three times and identical results were obtained. Representative blots are shown here. (A) Western blot analyses to determine GFP accumulation levels in E. coli strains harboring the different pOD plasmids. 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. pRB95: empty vector control. (B) Analysis of gfp mRNA accumulation in pOD strains. As a loading control, the blot was stripped and re-hybridized to a 16S rRNA-specific probe.
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Figure 2: Analysis of gfp expression in E. coli. All experiments were repeated at least three times and identical results were obtained. Representative blots are shown here. (A) Western blot analyses to determine GFP accumulation levels in E. coli strains harboring the different pOD plasmids. 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. pRB95: empty vector control. (B) Analysis of gfp mRNA accumulation in pOD strains. As a loading control, the blot was stripped and re-hybridized to a 16S rRNA-specific probe.

Mentions: To be able to compare SD sequence recognition in plastids and eubacteria, we first analyzed all 20 constructs in E. coli cells. Determination of GFP accumulation levels revealed strong differences between the different transgenic bacterial strains (Figure 2A). As protein accumulation is the result of both mRNA accumulation levels and translational efficiency, we also determined gfp mRNA accumulation (Figure 2B) and calculated a value for the relative translational efficiency from the RNA accumulation and protein accumulation values (Table 2). RNA accumulation levels varied substantially between the different constructs (Figure 2B) suggesting that the modifications in the 5′-UTR affect mRNA stability. This is unsurprising, because secondary structure, length and sequence of the 5′-UTR are known to represent key determinants of transcript stability in E. coli (53,54). No clear correlation between 5′-UTR sequence, number of SD sequences and/or number of initiation codons and RNA stability was discernable (Table 2; Figures 1B and 2B) confirming that the complex principles that govern mRNA stability in E. coli do not allow to reliably predict transcript stabilities. As the analysis of RNA stability in E. coli was beyond the scope of this study, protein synthesis data were corrected for differences in mRNA abundance by calculating relative translational efficiency values (Table 2) and only these values are considered here. When total translational efficiencies were compared between the 20 constructs, striking differences were seen. First, increasing the spacing between two SD sequences to allow for simultaneous accommodation of two ribosomes increased translational efficiency significantly in the constructs where all GFP translation initiated at the same start codon (cf. pOD2 and pOD3 with pOD6 and pOD7; Table 2). This suggests that additional SD sequences attract more ribosomes, if sufficient spacing permits their simultaneous occupation. Presence of a start and a stop codon or a mini-ORF between two SD sequences resulted in decreased translational efficiency (pOD4, pOD5, pOD8 and pOD9; Table 2), possibly indicating that completion of translation initiation at upstream SD sequences competes with initiation at the downstream SD sequence that controls GFP synthesis.Figure 2.


Selection of Shine-Dalgarno sequences in plastids.

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

Analysis of gfp expression in E. coli. All experiments were repeated at least three times and identical results were obtained. Representative blots are shown here. (A) Western blot analyses to determine GFP accumulation levels in E. coli strains harboring the different pOD plasmids. 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. pRB95: empty vector control. (B) Analysis of gfp mRNA accumulation in pOD strains. As a loading control, 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 2: Analysis of gfp expression in E. coli. All experiments were repeated at least three times and identical results were obtained. Representative blots are shown here. (A) Western blot analyses to determine GFP accumulation levels in E. coli strains harboring the different pOD plasmids. 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. pRB95: empty vector control. (B) Analysis of gfp mRNA accumulation in pOD strains. As a loading control, the blot was stripped and re-hybridized to a 16S rRNA-specific probe.
Mentions: To be able to compare SD sequence recognition in plastids and eubacteria, we first analyzed all 20 constructs in E. coli cells. Determination of GFP accumulation levels revealed strong differences between the different transgenic bacterial strains (Figure 2A). As protein accumulation is the result of both mRNA accumulation levels and translational efficiency, we also determined gfp mRNA accumulation (Figure 2B) and calculated a value for the relative translational efficiency from the RNA accumulation and protein accumulation values (Table 2). RNA accumulation levels varied substantially between the different constructs (Figure 2B) suggesting that the modifications in the 5′-UTR affect mRNA stability. This is unsurprising, because secondary structure, length and sequence of the 5′-UTR are known to represent key determinants of transcript stability in E. coli (53,54). No clear correlation between 5′-UTR sequence, number of SD sequences and/or number of initiation codons and RNA stability was discernable (Table 2; Figures 1B and 2B) confirming that the complex principles that govern mRNA stability in E. coli do not allow to reliably predict transcript stabilities. As the analysis of RNA stability in E. coli was beyond the scope of this study, protein synthesis data were corrected for differences in mRNA abundance by calculating relative translational efficiency values (Table 2) and only these values are considered here. When total translational efficiencies were compared between the 20 constructs, striking differences were seen. First, increasing the spacing between two SD sequences to allow for simultaneous accommodation of two ribosomes increased translational efficiency significantly in the constructs where all GFP translation initiated at the same start codon (cf. pOD2 and pOD3 with pOD6 and pOD7; Table 2). This suggests that additional SD sequences attract more ribosomes, if sufficient spacing permits their simultaneous occupation. Presence of a start and a stop codon or a mini-ORF between two SD sequences resulted in decreased translational efficiency (pOD4, pOD5, pOD8 and pOD9; Table 2), possibly indicating that completion of translation initiation at upstream SD sequences competes with initiation at the downstream SD sequence that controls GFP synthesis.Figure 2.

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