Both the nuclear and chloroplast genomes encode components of the chloroplast transcription machinery. In vascular plants, two enzymes and several accessory proteins are involved. One enzyme resembles the bacterial RNA polymerase, its core is encoded by the chloroplast, thus it is called PEP for Plastid Encoded Polymerase. It is completed by accessory proteins encoded in the nucleus. NEP (Nucleus Encoded Polymerase) is the second polymerase which resembles bacteriophage polymerases, is encoded in the nucleus and subsequently imported into the chloroplast. Although present in land plants, there is no evidence of a NEP in Chlamydomonas. Only one mutant of transcription was reported so far, affecting rbcL (Hong, 1997) but the interpretation of the results is debatable due to the extended time of the RNA pulse-labeling experiment. It is generally admitted that RNA polymerases are associated with plastid membrane fractions via anchoring of their DNA template (Azevedo., 2006; Sato, 1993; Sato, 1998; Young, 2003).
3 – Linker scan analysis of psbB cis-acting elements involved in transcript accumulation and CP47 translation.
I - Introduction
Figure 11: Comparison of the psbB-psbT-psbN-psbH-petB-petD gene cluster among different organisms. Genes are indicated by names above the filled black boxes. Gene clusters are represented by boxes linked by lines, an interrupted line indicates location at a distant site on the genome, genes on the opposite strands are indicated above and below the lines (from Hong et al., 1995).
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Two types of promoters have been described in C. reinhardtii (Klein, 1992):
- One resembles the bacterial sigma 70 type with a typical sequence (TATAATAT) at position -10 from the start site and a second element located upstream at position -35. It was described in particular for the 16S rRNA and appears to be conserved in many other C. reinhardtii promoters (Klein, 1992).
- The second form possesses in a similar fashion the -10 motif.
However the extent of conservation of the -35 element is significantly lower or sometimes absent, which clearly distinguishes this type of promoter from the first one. In this case, it seems that other sequences or structures, not fully characterized, are required to initiate transcription. In the case of the Chlamydomonas atpB promoter, its full activation depends on two sequences, one localized 22 bases upstream and the other one 60 bases downstream from the transcription start site, which together are thought to replace the -35 element (Klein, 1992).
Concerning the mechanism of transcription initiation, genome sequence comparison recently led to the characterization of a nucleus-encoded sigma 70-like factor in Chlamydomonas reinhardtii. In vitro, together with an E.coli RNA core polymerase, this sigma 70-like factor forms a chimeric holoenzyme that was shown to bind to spinach psbA RNA and to Chlamydomonas rrn16 promoters and to initiate transcription. In addition immunoblotting revealed an enrichment of this sigma 70-like factor in the chloroplast fraction. Thus after import into the chloroplast, this factor was proposed to function in transcription as in bacteria (Bohne, 2006). Intergenic regions can also influence transcription.
Indeed, the insertion of polydeoxyadenosine-rich sequences was reported to enhance transcription rates of surrounding genes (Lisitsky, 2001).
Transcription of plastid genes can occur in different ways. Products of chloroplast transcription can be either monocistronic, or polycistronic under the control of a unique upstream promoter. In land plants, chloroplast genes are mainly organized in clusters. Most of the time they are conserved between species and
3 – Linker scan analysis of psbB cis-acting elements involved in transcript accumulation and CP47 translation.
I - Introduction
Figure 12: Northern blots reveal differences in transcript stability between spinach and
Chlamydomonas. Blots from A to H correspond to the name of the probe used to hybridization and its location is indicated at the top in each gene cluster (from Westhoff et al,. 1986 and Vaistij et al., 1999).
psbB psbT psbH psbN 5´ UTR
1 kb
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often grouped by related functions (see figure 11). These long RNA molecules are subsequently matured to be properly expressed, even though in maize or in tobacco, polycistronic RNA molecules have been shown to be translated (Barkan, 1988; Quesada-Vargas, 2005). In higher plants, the proportion of polycistronic transcription units is rather high as for Arabidopsis, with 48 of the 87 chloroplast genes found in co-transcribed units containing between two and five genes each. In tobacco, up to 60 chloroplast genes are part of polycistronic units (Sugita, 1996).
In Chlamydomonas, approximately 20 genes out of the 99 were found in polycistronic transcription units (Drapier, 1998). The fact that only 20 co-transcribed genes have been detected in Chlamydomonas does not necessarily imply fewer polycistronic transcription units in this alga compared to Arabidopsis.
Indeed, one has to take into account that in Chlamydomonas, precursors, i.e. long and very often, low abundant RNA molecules, are processed much faster to shorter forms which then accumulate to higher levels. Indeed, RNA metabolism is variable between organisms. These differences may concern the synthesis rate, the processing efficiency, the stability, the splicing, the degradation, etc…. The example of the psbB gene cluster illustrates these features.
Comparison by Northern blotting of the accumulation of transcripts from spinach and Chlamydomonas reinhardtii (see figure 12) (Vaistij et al., 2000b; Westhoff and Herrmann, 1988) reveals that in Chlamydomonas the psbB-psbT-psbH tri-cistron precursor is not detectable. The longest detectable intermediate is the di-cistron psbB-psbT RNA of 2300 bases. The mature mRNAs of psbB and psbH are also detectable. In contrast in spinach, all precursors and intermediates of this transcription unit can be detected, even a penta-cistronic psbB-psbT-psbH-petB-petD transcript of 5600 bases can accumulate to detectable levels. This makes the study of precursors less straightforward in C. reinhardtii.
Finally, transcription does not appear to be a limiting step in chloroplast gene expression. Indeed, a 10 fold decrease of chloroplast gene copy number mediated by treatment with 5’-fluoro-2’-deoxyuridine does not affect the
3 – Linker scan analysis of psbB cis-acting elements involved in transcript accumulation and CP47 translation.
I - Introduction
Figure 13: Description of the various events leading to the formation of the mature 5’ extremity of Chlamydomonas mRNAs (Rochaix J.-D., eds, 1998). The black hexagon represents putative trans-actingfactors involved in defining the transcripts 5’end.
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overall mRNA levels in the organelle, indicating that proper accumulation of the transcripts is controlled downstream of transcription (Choquet, 2002).