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Identification of a plastid intercistronic expression element (IEE) facilitating the expression of stable translatable monocistronic mRNAs from operons.

Zhou F, Karcher D, Bock R - Plant J. (2007)

Bottom Line: At least some polycistronic transcripts are not translatable, and endonucleolytic processing may therefore be a prerequisite for translation to occur.As the requirements for intercistronic mRNA processing into stable monocistronic transcript are not well understood, we have sought to define minimum sequence elements that trigger processing and thus are capable of generating stable translatable monocistronic mRNAs.We describe here the in vivo identification of a small intercistronic expression element that mediates intercistronic cleavage into stable monocistronic transcripts.

View Article: PubMed Central - PubMed

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

ABSTRACT
Most plastid genes are part of operons and expressed as polycistronic mRNAs. Many primary polycistronic transcripts undergo post-transcriptional processing in monocistronic or oligocistronic units. At least some polycistronic transcripts are not translatable, and endonucleolytic processing may therefore be a prerequisite for translation to occur. As the requirements for intercistronic mRNA processing into stable monocistronic transcript are not well understood, we have sought to define minimum sequence elements that trigger processing and thus are capable of generating stable translatable monocistronic mRNAs. We describe here the in vivo identification of a small intercistronic expression element that mediates intercistronic cleavage into stable monocistronic transcripts. Separation of foreign genes by this element facilitates transgene stacking in operons, and thus will help to expand the range of applications of transplastomic technology.

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Identification of intercistronic mRNA processing sites in the tobacco psbB operon(a) Structure of the psbB operon. Genes above the lines are transcribed from left to right; the gene below the line (psbN) is transcribed in the opposite direction. The group II introns within the petB and petD coding regions are shown as open boxes. Transcription from the psbB promoter produces a pentacistronic mRNA that undergoes a complex series of processing steps resulting in monocistronic and oligocistronic mRNA species (Westhoff and Herrmann, 1988).(b) Partial sequence alignment of the psbT–psbH spacer region from tobacco, spinach and Arabidopsis. Shown is the 3′ part of the spacer, between the antisense psbN sequence and psbH. The intercistronic RNA processing site mapped in tobacco is indicated by a closed triangle. The sequences chosen as putative processing sequences in plastid transformation experiments are indicated by the double-arrowed lines.(c) Alignment of the psbH–petB spacer regions from tobacco, spinach and Arabidopsis. The major intercistronic RNA processing site mapped in tobacco is marked by a closed triangle; additionally identified minor processing sites are indicated by open triangles.(d) Location of intercistronic processing sites within putative RNA stem–loop structures. The major endonucleolytic cleavage sites are indicated by arrowheads.
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fig01: Identification of intercistronic mRNA processing sites in the tobacco psbB operon(a) Structure of the psbB operon. Genes above the lines are transcribed from left to right; the gene below the line (psbN) is transcribed in the opposite direction. The group II introns within the petB and petD coding regions are shown as open boxes. Transcription from the psbB promoter produces a pentacistronic mRNA that undergoes a complex series of processing steps resulting in monocistronic and oligocistronic mRNA species (Westhoff and Herrmann, 1988).(b) Partial sequence alignment of the psbT–psbH spacer region from tobacco, spinach and Arabidopsis. Shown is the 3′ part of the spacer, between the antisense psbN sequence and psbH. The intercistronic RNA processing site mapped in tobacco is indicated by a closed triangle. The sequences chosen as putative processing sequences in plastid transformation experiments are indicated by the double-arrowed lines.(c) Alignment of the psbH–petB spacer regions from tobacco, spinach and Arabidopsis. The major intercistronic RNA processing site mapped in tobacco is marked by a closed triangle; additionally identified minor processing sites are indicated by open triangles.(d) Location of intercistronic processing sites within putative RNA stem–loop structures. The major endonucleolytic cleavage sites are indicated by arrowheads.

Mentions: To identify sequence elements suitable for triggering processing of polycistronic transcripts into stable and translatable monocistronic mRNAs, we analyzed processing in the tobacco psbB operon (Figure 1a), which is one of the best characterized multi-gene operons in plastids (Felder et al., 2001; Meierhoff et al., 2003; Westhoff and Herrmann, 1988). The psbB operon consists of five genes, three of which encode photosystem II components (psbB, psbT and psbH), with the remaining two encoding subunits of the cytochrome b6f complex (petB and petD) (Figure 1a). The five genes are co-transcribed, giving rise to a long pentacistronic precursor RNA, which is then cleaved into smaller units by a complex series of processing events (Westhoff and Herrmann, 1988). Not all final processing products are monocistronic: the small psbT cistron remains associated with the upstream psbB, forming a di-cistronic mature mRNA, and the two cytochrome b6f components, petB and petD, are only inefficiently processed into monocistronic mRNAs, leaving a large proportion of the transcripts di-cistronic (Felder et al., 2001; Westhoff and Herrmann, 1988). Stem–loop-type secondary structures are found upstream of most cleavage sites, suggesting that they stabilize the 3′ ends of the mature transcripts (Stern and Gruissem, 1987; Westhoff and Herrmann, 1988).


Identification of a plastid intercistronic expression element (IEE) facilitating the expression of stable translatable monocistronic mRNAs from operons.

Zhou F, Karcher D, Bock R - Plant J. (2007)

Identification of intercistronic mRNA processing sites in the tobacco psbB operon(a) Structure of the psbB operon. Genes above the lines are transcribed from left to right; the gene below the line (psbN) is transcribed in the opposite direction. The group II introns within the petB and petD coding regions are shown as open boxes. Transcription from the psbB promoter produces a pentacistronic mRNA that undergoes a complex series of processing steps resulting in monocistronic and oligocistronic mRNA species (Westhoff and Herrmann, 1988).(b) Partial sequence alignment of the psbT–psbH spacer region from tobacco, spinach and Arabidopsis. Shown is the 3′ part of the spacer, between the antisense psbN sequence and psbH. The intercistronic RNA processing site mapped in tobacco is indicated by a closed triangle. The sequences chosen as putative processing sequences in plastid transformation experiments are indicated by the double-arrowed lines.(c) Alignment of the psbH–petB spacer regions from tobacco, spinach and Arabidopsis. The major intercistronic RNA processing site mapped in tobacco is marked by a closed triangle; additionally identified minor processing sites are indicated by open triangles.(d) Location of intercistronic processing sites within putative RNA stem–loop structures. The major endonucleolytic cleavage sites are indicated by arrowheads.
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Related In: Results  -  Collection

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fig01: Identification of intercistronic mRNA processing sites in the tobacco psbB operon(a) Structure of the psbB operon. Genes above the lines are transcribed from left to right; the gene below the line (psbN) is transcribed in the opposite direction. The group II introns within the petB and petD coding regions are shown as open boxes. Transcription from the psbB promoter produces a pentacistronic mRNA that undergoes a complex series of processing steps resulting in monocistronic and oligocistronic mRNA species (Westhoff and Herrmann, 1988).(b) Partial sequence alignment of the psbT–psbH spacer region from tobacco, spinach and Arabidopsis. Shown is the 3′ part of the spacer, between the antisense psbN sequence and psbH. The intercistronic RNA processing site mapped in tobacco is indicated by a closed triangle. The sequences chosen as putative processing sequences in plastid transformation experiments are indicated by the double-arrowed lines.(c) Alignment of the psbH–petB spacer regions from tobacco, spinach and Arabidopsis. The major intercistronic RNA processing site mapped in tobacco is marked by a closed triangle; additionally identified minor processing sites are indicated by open triangles.(d) Location of intercistronic processing sites within putative RNA stem–loop structures. The major endonucleolytic cleavage sites are indicated by arrowheads.
Mentions: To identify sequence elements suitable for triggering processing of polycistronic transcripts into stable and translatable monocistronic mRNAs, we analyzed processing in the tobacco psbB operon (Figure 1a), which is one of the best characterized multi-gene operons in plastids (Felder et al., 2001; Meierhoff et al., 2003; Westhoff and Herrmann, 1988). The psbB operon consists of five genes, three of which encode photosystem II components (psbB, psbT and psbH), with the remaining two encoding subunits of the cytochrome b6f complex (petB and petD) (Figure 1a). The five genes are co-transcribed, giving rise to a long pentacistronic precursor RNA, which is then cleaved into smaller units by a complex series of processing events (Westhoff and Herrmann, 1988). Not all final processing products are monocistronic: the small psbT cistron remains associated with the upstream psbB, forming a di-cistronic mature mRNA, and the two cytochrome b6f components, petB and petD, are only inefficiently processed into monocistronic mRNAs, leaving a large proportion of the transcripts di-cistronic (Felder et al., 2001; Westhoff and Herrmann, 1988). Stem–loop-type secondary structures are found upstream of most cleavage sites, suggesting that they stabilize the 3′ ends of the mature transcripts (Stern and Gruissem, 1987; Westhoff and Herrmann, 1988).

Bottom Line: At least some polycistronic transcripts are not translatable, and endonucleolytic processing may therefore be a prerequisite for translation to occur.As the requirements for intercistronic mRNA processing into stable monocistronic transcript are not well understood, we have sought to define minimum sequence elements that trigger processing and thus are capable of generating stable translatable monocistronic mRNAs.We describe here the in vivo identification of a small intercistronic expression element that mediates intercistronic cleavage into stable monocistronic transcripts.

View Article: PubMed Central - PubMed

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

ABSTRACT
Most plastid genes are part of operons and expressed as polycistronic mRNAs. Many primary polycistronic transcripts undergo post-transcriptional processing in monocistronic or oligocistronic units. At least some polycistronic transcripts are not translatable, and endonucleolytic processing may therefore be a prerequisite for translation to occur. As the requirements for intercistronic mRNA processing into stable monocistronic transcript are not well understood, we have sought to define minimum sequence elements that trigger processing and thus are capable of generating stable translatable monocistronic mRNAs. We describe here the in vivo identification of a small intercistronic expression element that mediates intercistronic cleavage into stable monocistronic transcripts. Separation of foreign genes by this element facilitates transgene stacking in operons, and thus will help to expand the range of applications of transplastomic technology.

Show MeSH
Related in: MedlinePlus