The chloroplast atpA gene cluster in Chlamydomonas reinhardtii. Functional analysis of a polycistronic transcription unit

Abstract

Most chloroplast genes in vascular plants are organized into polycistronic transcription units, which generate a complex pattern of mono-, di-, and polycistronic transcripts. In contrast, most Chlamydomonas reinhardtii chloroplast transcripts characterized to date have been monocistronic. This paper describes the atpA gene cluster in the C. reinhardtii chloroplast genome, which includes the atpA, psbI, cemA, and atpH genes, encoding the alpha-subunit of the coupling-factor-1 (CF1) ATP synthase, a small photosystem II polypeptide, a chloroplast envelope membrane protein, and subunit III of the CF0 ATP synthase, respectively. We show that promoters precede the atpA, psbI, and atpH genes, but not the cemA gene, and that cemA mRNA is present only as part of di-, tri-, or tetracistronic transcripts. Deletions introduced into the gene cluster reveal, first, that CF1-alpha can be translated from di- or polycistronic transcripts, and, second, that substantial reductions in mRNA quantity have minimal effects on protein synthesis rates. We suggest that posttranscriptional mRNA processing is common in C. reinhardtii chloroplasts, permitting the expression of multiple genes from a single promoter.

Figure 1

Transcription of the C. reinhardtii chloroplast atpA gene cluster. A, Map of the atpA region of the chloroplast genome. R15, R7, and R8 are EcoRI restriction fragments (Rochaix, 1980). Other restriction sites are: P, PacI; H, HpaI; Hd, HindIII; and E47, Eco47III. The three promoters are indicated by bent arrows. The extents of transcripts are shown as numbered arrows, with estimated sizes in kilobase pairs shown at the right. B, RNA accumulation in wild-type cells. Blots of total RNA were hybridized with the probes shown at the top of each lane as described in Methods. The atpA-psbI-cemA probe was the 513-bp ScaI-DraII fragment of R7. Not all blots are from the same gel, so the relative migration of some species varies slightly. The asterisk in the psbI lane indicates a transcript of unknown origin, which is not seen when a larger probe is used (third lane).

Figure 2

5′-end mapping of atpA gene-cluster transcripts. Lanes R show primer-extension experiments with total RNA from wild-type cells, with the gene indicated under the panel. Relative to the translation-initiation codon, the primer for atpA annealed from +92 to +75, that for psbI annealed from +96 to +80, and that for atpH annealed from +27 to +9.

Figure 3

Analysis of chimeric uidA promoter fusions. A, The site of insertions of chimeric genes into the chloroplast genome. RNA-filter hybridizations are shown for atpH test constructs (B) and psbI test constructs (C). Both blots were hybridized with a uidA-coding-region probe; a psbA-coding-region probe was used for normalization of the psbI blot. For atpH, + and − indicate strains carrying the fusions in opposite orientations (A). For psbI, 1 and 2 are independent transformants in the (−) orientation, and the petD-uidA lane contains total RNA isolated from the strain DG2, which is known to accumulate uidA mRNA in vivo (Sakamoto et al., 1993). D, Run-on transcription from psbI-uidA transformants are shown. Two micrograms of each of the three plasmids shown at the right was fixed to a nylon filter using a slot-blot apparatus. pKS, pBluescript. Four separate filters were hybridized with ³²P-labeled transcripts from freeze-thaw-permeabilized cells of the strains shown across the top. Nonspecific hybridization can be seen for psbI-uidA lane1 (pKS) and for the wild type (uidA).

Figure 4

3′-End mapping of atpA gene cluster transcripts. Mapping was carried out by RNase (atpA) or S1 nuclease protection with the indicated number of units per microgram of RNA (cemA and atpH). Locations of probes and deduced 3′ ends are shown at the top of the figure and are described in Methods; the cemA 3′ end is close to or coincident with the atpH 5′ end. For atpA, the tRNA lane contained 10 μg of yeast tRNA instead of C. reinhardtii total RNA. For cemA and atpH, total RNA from strain ΔatpA was used; this strain accumulates increased levels of transcripts ending at cemA. Note that the probe for atpA was a uniformly labeled RNA (the bent part indicates pBluescript vector sequences), whereas that for cemA and atpH was an end-labeled DNA fragment. Electrophoresis was in a denaturing polyacrylamide gel for atpA, and in an alkaline agarose gel for cemA and atpH. The atpH 3′ end is marked on the gel as cemA/atpH 3′ because with this probe only cemA-atpH co-transcripts will be visible as protected fragments. MW, Molecular weight.

Figure 5

Characterization of the ΔatpA deletion strain. A, The horizontal gray dashed line shows the extent of the deletion. B, atpA-hybridizing transcripts, numbered as in Figure 1, after hybridization of an RNA blot with a 480-bp DraII-EcoRI fragment of R15, the 5′ end of the atpA gene. For the wild type (WT), transcript 2 is not visible in this exposure and is therefore labeled in gray. C, Pulse-labeling with [¹⁴C]acetate for 5 min, as described in Methods; β and α indicate subunits of the ATP synthase, and P5 is a PSII subunit. Strain 222E does not synthesize P5 because of a nuclear mutation causing instability of psbB mRNA (Monod et al., 1992), and was used as a negative control for this protein. In strains 222E and ΔatpA, there is increased labeling of the Rubisco large subunit, which migrates just above the β-subunit. This is typically seen for nonphotosynthetic mutants of C. reinhardtii under these experimental conditions (see fig. 3 in Drapier et al., 1992). D, Immunoblot using the antisera indicated at the right. The diminished accumulation of the β-subunit in ΔatpA reflects a posttranslational instability of the protein.

Figure 6

RNA accumulation in the deletion strains Δ1, Δ2, and Δ3. A, Map of the atpA gene cluster as shown in Figure 1. The extents of the deletions in Δ1, Δ2, and Δ3 are shown. B, RNA accumulation, with strains shown at the top of each panel and probes shown at the bottom. Transcripts 1 and 2 accumulate to relatively low levels in wild-type (WT) cells, and are more easily visualized with the cemA probe. An rbcL probe was used as a loading control.

Figure 7

Protein synthesis and accumulation in strains Δ1, Δ2, and Δ3. The strains were pulse labeled for 5 min with [¹⁴C]acetate (A) or analyzed by immunoblotting (B). Labeling is as for Figure 5.

Figure 8

Analysis of transformants with an insertion of the rbcL 3′ UTR downstream of psbI. A, Map of the atpA gene cluster showing the site of the rbcL 3′ UTR insertion. B, RNA-filter hybridization analysis using an atpA coding-region probe. Transcripts are numbered as in Figure 1. C, Separation of proteins after 5 min of pulse labeling with [¹⁴C]acetate. WT, Wild type.