We report the complete DNA sequence of the Euglena gracilis, Pringsheim strain Z chloroplast genome. This circular DNA is 143,170 bp, counting only one copy of a 54 bp tandem repeat sequence that is present in variable copy number within a single culture. The overall organization of the genome involves a tandem array of three complete and one partial ribosomal RNA operons, and a large single copy region. There are genes for the 16S, 5S, and 23S rRNAs of the 70S chloroplast ribosomes, 27 different tRNA species, 21 ribosomal proteins plus the gene for elongation factor EF-Tu, three RNA polymerase subunits, and 27 known photosynthesis-related polypeptides. Several putative genes of unknown function have also been identified, including five within large introns, and five with amino acid sequence similarity to genes in other organisms. This genome contains at least 149 introns. There are 72 individual group II introns, 46 individual group III introns, 10 group II introns and 18 group III introns that are components of twintrons (introns-within-introns), and three additional introns suspected to be twintrons composed of multiple group II and/or group III introns, but not yet characterized. At least 54,804 bp, or 38.3% of the total DNA content is represented by introns.

SummaryThe acetate-requiring Chlamydomonas reinhardtii nuclear mutant F16 harbors the mutation mcd1-1 and fails to accumulate the cytochrome b6/f complex. The primary defect of mcd1-1 was determined to be the instability of petD mRNA, which encodes subunit IV of the complex. Chimeric reporter genes introduced by chloroplast transformation demonstrated that the determinant of petD mRNA instability in the mcd1-1 background is located in the 5Ј untranslated region (UTR). However, when this 5Ј UTR was present downstream of other sequences in dicistronic or chimeric transcripts, the RNAs were no longer destabilized in the mcd1-1 background. Together, these results suggest that the 5Ј end of the petD 5Ј UTR interacts with the MCD1 product. The insertion of a polyguanosine sequence into the petD 5Ј UTR fused to a reporter gene allowed accumulation of the reporter gene transcript in the mutant background. Since polyguanosine forms a structure that is known to impede exonucleases, these data provide in vivo evidence that petD mRNA can be degraded by 5Ј→3Ј exoribonuclease activity. Furthermore, the data support a model in which protein binding to the petD 5Ј UTR protects the mRNA from 5Ј→3Ј degradation in wild-type cells.

SummaryMolecular genetic studies have shown that determinants of chloroplast mRNA stability lie in both the 5¢ and 3¢ untranslated regions. While it is wellknown that chloroplast mRNAs are unstable in the absence of certain nucleus-encoded factors, little is known of the decay mechanisms for chloroplast mRNA in wild-type cells. Here we used a poly(G) 18 sequence, which impedes both 5¢®3¢ and 3¢®5¢ exoribonucleolytic RNA decay in vivo, to study the degradation pathway of petD mRNA in wild-type and mcd1 mutant chloroplasts of Chlamydomonas; the mcd1 mutant lacks a nucleus-encoded factor required for petD mRNA accumulation. Upon inserting poly(G) at positions ±20, +25, +165 or +25/+165 relative to the mature petD 5¢ end, mRNAs accumulate with 5¢ ends corresponding to the poly(G) sequence, in addition to the normal RNA with its 5¢ end at +1. We interpret these results as evidence for continuous degradation of petD mRNA in wild-type cells by a 5¢®3¢ exoribonucleolytic activity. In the case of the ±20 insertion, the accumulating RNA can be interpreted as a processing intermediate, suggesting that 5¢ end maturation may also involve this activity. When examined in the mcd1 mutant background, petD mRNAs with the poly(G) 5¢ ends, but not normal +1 ends, accumulated. However, no expression of SUIV, the petD gene product, was detected. Insertion of poly(G) at +165 in wild-type cells did not demonstrably affect SUIV accumulation, suggesting that ribosomal scanning does not occur upstream of this position. However, since neither poly(G) ±20 nor +165 RNA could be translated in mcd1 cells, this raises the possibility that the MCD1 product is essential for translation.

A general characteristic of the 3' untranslated regions of plastid mRNAs is an inverted repeat sequence that can fold into a stem-loop structure. These stem-loops are superficially similar to structures involved in prokaryotic transcription termination, but were found instead to serve as RNA 3' end processing signals in spinach chloroplasts, and in the atpB mRNA of Chlamydomonas reinhardtii chloroplasts. In order to carry out a broad study of the efficiency of the untranslated sequences at the 3' ends of chloroplast genes in Chlamydomonas to function as transcription terminators, we performed in vivo run-on transcription experiments using Chlamydomonas chloroplast transformants in which different 3' ends were inserted into the chloroplast genome between a petD promoter and a reporter gene. The results showed that none of the 3' ends that were tested, in either sense or antisense orientation, prevented readthrough transcription, and thus were not highly efficient transcription terminators. Therefore, we suggest that most or all of the 3' ends of mature mRNAs in Chlamydomonas chloroplasts are formed by 3' end processing of longer precursors.

3-end processing of nucleus-encoded mRNAs includes the addition of a poly(A) tail that is important for translation initiation. Since the vast majority of chloroplast mRNAs acquire their 3 termini by processing yet are not polyadenylated, we asked whether 3 end maturation plays a role in chloroplast translation. A general characteristic of the 3 untranslated regions of chloroplast mRNAs is an inverted repeat (IR) sequence that can fold into a stem-loop structure. These stem-loops and their flanking sequences serve as RNA 3-end formation signals. Deletion of the Chlamydomonas chloroplast atpB 3 IR in strain ⌬26 results in reduced accumulation of atpB transcripts and the chloroplast ATPase ␤-subunit, leading to weakly photosynthetic growth. Of the residual atpB mRNA in ⌬26, approximately 1% accumulates as a discrete RNA of wild-type size, while the remainder is heterogeneous in length due to the lack of normal 3 end maturation. In this work, we have analyzed whether these unprocessed atpB transcripts are actively translated in vivo. We found that only the minority population of discrete transcripts of wild-type size is associated with polysomes and thus accounts for the ATPase ␤-subunit which accumulates in ⌬26. Analysis of chloroplast rbcL mRNA revealed that transcripts extending beyond the mature 3 end were not polysome associated. These results suggest that 3-end processing of chloroplast mRNA is required for or strongly stimulates its translation.Chloroplast genes are often organized into operons and gene clusters, which are transcribed into precursor transcripts that undergo complex processing events including splicing and intercistronic cleavages (reviewed in references 35 and 49). While intercistronic cleavages form some mRNA 5Ј and 3Ј termini, these can also be formed by other types of events. For example, 5Ј ends are often formed by endonucleolytic processing of primary transcripts, and this may be the exclusive mode of 5Ј end formation in chloroplasts in the green alga Chlamydomonas reinhardtii (reviewed in reference 12). Most plastid mRNAs contain inverted-repeat (IR) sequences in their 3Ј untranslated regions, which are believed to fold into stem-loop structures. These IR sequences do not function as efficient transcription terminators but instead are thought to stabilize upstream sequences and mediate correct 3Ј-end processing (36,37,44,47,48). In most cases, the 3Ј termini of mature transcripts lie immediately downstream of the IR.Plastid 3Ј IR sequences act to stabilize upstream mRNA segments in vitro and in vivo. When RNA molecules containing the IR sequences were incubated in spinach chloroplast protein extracts, they were correctly processed at their 3Ј ends and the products were stable for several hours. However, when the IR sequences were deleted from the same RNA molecules and incubated in an identical protein extract, the RNA molecules were rapidly degraded (17,37,44,46). The ability to introduce altered genes into the chloroplast of the green alga C. reinhardtii presented the opportunity to ...

In Chlamydomonas chloroplasts, atpB pre-mRNA matures through a two-step process. Initially, endonuclease cleavage occurs 8 -10 nt downstream of the mature 3 end, which itself lies at the end of a stem-loop-forming inverted repeat (IR) sequence. This intermediate product is then trimmed by a 3 3 5 exonuclease activity. Although the initial endonucleolytic cleavage by definition generates two products, the downstream product of atpB pre-mRNA endonucleolytic processing cannot be detected, even transiently. This product thus appears to be highly unstable, and it can be hypothesized that specific mechanisms exist to prevent its accumulation. In experiments described here, the atpB 3 maturation site was placed upstream of reporter genes in vivo. Constructs containing both the IR and endonuclease cleavage site (ECS) did not accumulate the reporter gene mRNA, whereas constructs containing only the IR did accumulate the reporter mRNA. The ECS alone gave an intermediate result, suggesting that the IR and ECS act synergistically. Additional secondary structures were used to test whether 5 3 3 and/or 3 3 5 exonuclease activities mediated degradation. Because these structures did not prevent degradation, rapid endonucleolytic cleavages most likely trigger RNA destruction after ECS cleavage. On the other hand, fragments resulting from cleavage within the endogenous atpB mRNA could occasionally be detected as antisense transcripts of the adjacent reporter genes. Because endonuclease cleavages are also involved in the 5 maturation of chloroplast mRNAs, where only the downstream cleavage product accumulates, it appears that chloroplast endoribonuclease activities have evolved mechanisms to selectively stabilize different ECS products.Maturation of mRNA can involve multiple steps in both prokaryotic and eukaryotic systems, and results in functional transcripts with a stability and subcellular localization appropriate to their functions. RNA sequence and secondary structure, along with ribonucleases and a variety of accessory factors, are used to achieve these goals. Built into this process is the necessity to recognize nonfunctional RNAs and eliminate them; destruction of nonsense codon-containing mRNAs is a good example of the complexity of such surveillance mechanisms (reviewed in Ref. 1).Our laboratory has focused on 5Ј end and 3Ј end maturation of chloroplast mRNAs, using both vascular plants and the unicellular green alga Chlamydomonas reinhardtii as models. In this respect, chloroplast transcripts have mostly prokaryotic features such as the lack of a trimethylguanosine 5Ј cap, a 3Ј stem-loop-forming inverted repeat (IR) 1 structure, and they are destabilized by polyadenylation (reviewed in Refs. 2 and 3). Processing of chloroplast mRNAs, with rare exceptions, depends on nucleus-encoded proteins, several of which have been identified genetically through screens for plants or Chlamydomonas strains unable to carry out photosynthesis. The phenotypes suggest that defects in intercistronic processing of polycistronic transcripts...

Mutations in the Chlamydomonas reinhardtii nuclear gene MCD1 specifically destabilize the chloroplast petD mRNA, which encodes subunit IV of the cytochrome b6/f complex. The MCD1 gene product is thought to interact with the mRNA 5' end to protect it from degradation by a 5' --> 3' exoribonuclease and may also have a role in translation initiation. Here we report the isolation and characterization of a semidominant, allele-specific, nucleus-encoded suppressor of the mcd1-2 mutation. The suppressor mutation, which defines a new locus MCD2, allows accumulation of 10% of the wild-type level of petD mRNA and as much as 50% of the wild-type subunit IV level. Taken together, these results suggest the suppressor mutation restores photosynthetic growth by stabilizing petD mRNA. In addition, it may promote increased translational efficiency, an inference supported by direct measurements of the subunit IV synthesis rate. Thus, both MCD1 and MCD2 may participate in both chloroplast RNA stability and translation initiation.

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