Polyadenylation of nucleus-encoded transcripts has a well-defined role in gene expression. The extent and function of polyadenylation in organelles and prokaryotic systems, however, are less well documented. Recent reports of polyadenylation-mediated RNA destabilization in Escherichia coli and in vascular plant chloroplasts prompted us to look for polyadenylation in plant mitochondria. Here, we report the use of reverse transcription-polymerase chain reaction to map multiple polyadenylate addition sites in maize mitochondrial cox2 transcripts. The lack of sequence conservation surrounding these sites suggests that polyadenylation may occur at many 3' termini created by endoribonucleolytic and/or exoribonucleolytic activities, including those activities involved in 3' end maturation. Endogenous transcripts could be efficiently polyadenylated in vitro by using maize mitochondrial lysates with an activity that added AMP more efficiently than GMP. Polyadenylated substrates were tested for stability in maize mitochondrial S100 extracts, and we found that, compared with nonpolyadenylated RNAs, the polyadenylated substrates were less stable. Taken together with the low abundance of polyadenylated RNAs in maize mitochondria, our results are consistent with a degradation-related process. The fact that polyadenylation does not dramatically destabilize plant mitochondrial transcripts, at least in vitro, is in agreement with results obtained for animal mitochondria but differs from those obtained for chloroplasts and E. coli. Because fully edited, partially edited, and unedited transcripts were found among the cloned polyadenylated cox2 cDNAs, we conclude that RNA editing and polyadenylation are independent processes in maize mitochondria.

Plant mitochondrial genes are often transcribed into complex sets of RNAs, resulting from multiple initiation sites and processing steps. To elucidate the role of initiation in generating the more than 10 cox2 transcripts found in maize mitochondria, we surveyed sequences upstream of cox2 for active promoters. Because the cox2 coding region is immediately downstream of a 0.7-kb recombination repeat, cox2 is under the control of two different sets of potential expression signals. Using an in vitro transcription assay, we localized four promoters upstream of the coding region in the so-called master chromosome, and two promoters upstream of the coding region in the recombinant subgenome. Ribonuclease protection analysis of labeled primary transcripts confirmed that all but one of these promoters is active in vivo. Primer extension was used to identify the promoter sequences and initiation sites, which agree with the consensus established earlier for maize mitochondria. This study identified two unusual promoters, the core sequences of which were composed entirely of adenines and thymines, and one of which was a complex promoter consisting of seven overlapping units. Deletion mutagenesis of the complex promoter suggested that each of its units was recognized independently by RNA polymerase. While each active promoter fit the maize core consensus sequence YRTAT, not all such sequences surveyed supported initiation. We conclude that in vitro transcription is a powerful tool for locating mitochondrial promoters and that, in the case of cox2, promoter multiplicity contributes strongly to transcript complexity.The transcriptional strategies of vascular plant mitochondrial genomes differ from those of their fungal and metazoan counterparts, with a major region being genome structure. Plant mitochondrial genomes are relatively large and the genes are mostly dispersed (1, 2). In contrast, metazoan genomes are compact and have a single promoter for each DNA strand, and the Saccharomyces cerevisiae mitochondrial genome is 75 kb, 1 and contains about 20 promoters (reviewed in Ref.3). The maize mitochondrial genome, the expression of which we have studied, can be mapped as a 570-kb circle with numerous repeated elements mediating recombination, leading to a complex set of overlapping molecules (4). Based on recent microscopic studies (5), these molecules are likely to be linearly permuted.Promoter analysis in plant mitochondria has been accelerated in the last several years by the development of in vitro transcription systems from wheat (6), maize (7), and pea (8). Extensive mutational analysis of the maize atpA and cox3 promoters showed that the only universally-present sequence required for transcription initiation in vitro was YRTAT (Y ϭ T or C and R ϭ G or A), located at or immediately upstream of the start site (9, 10). Even this degenerate sequence, however, is not found near all 5Ј termini identified as transcription start sites based on their ability to be capped in vitro by guanylyl transferase (11). While some of the...

We have previously used a homologous in vitro transcription system to define functional elements of the maize mitochondrial atpA promoter. These elements comprise a central domain extending from -7 to +5, relative to the transcription start site, and an upstream domain of 1-3 bp that is purine rich and centered around positions -11 to -12. Within the central domain lies an essential 5 bp core element. These elements are conserved in many mitochondrial promoters, but their functionality has only been tested for atpA. In this study we have introduced mutations into the corresponding elements of two cox3 promoters and show that while the core element is essential for cox3 promoter activity, upstream element mutations have little or no effect. To define the minimal sequence required for in vitro promoter activity a series of short cloned oligonucleotides corresponding to the atpA promoter was used. While some activity was seen with a 14 bp sequence, full activity required 26 bp, suggesting that elements other than the core and upstream region can influence promoter strength. Another series of clones showed that altered spacing between the upstream and core elements of atpA had a significant effect on promoter activity. These results further define important features of the plant mitochondrial transcriptional machinery.

Polyadenylation of nucleus-encoded transcripts has a well-defined role in gene expression. The extent and function of polyadenylation in organelles and prokaryotic systems, however, are less well documented. Recent reports of polyadenylation-mediated RNA destabilization in Escherichia coli and in vascular plant chloroplasts prompted us to look for polyadenylation in plant mitochondria. Here, we report the use of reverse transcription-polymerase chain reaction to map multiple polyadenylate addition sites in maize mitochondrial cox2 transcripts. The lack of sequence conservation surrounding these sites suggests that polyadenylation may occur at many 3 Ј termini created by endoribonucleolytic and/or exoribonucleolytic activities, including those activities involved in 3 Ј end maturation. Endogenous transcripts could be efficiently polyadenylated in vitro by using maize mitochondrial lysates with an activity that added AMP more efficiently than GMP. Polyadenylated substrates were tested for stability in maize mitochondrial S100 extracts, and we found that, compared with nonpolyadenylated RNAs, the polyadenylated substrates were less stable. Taken together with the low abundance of polyadenylated RNAs in maize mitochondria, our results are consistent with a degradation-related process. The fact that polyadenylation does not dramatically destabilize plant mitochondrial transcripts, at least in vitro, is in agreement with results obtained for animal mitochondria but differs from those obtained for chloroplasts and E. coli . Because fully edited, partially edited, and unedited transcripts were found among the cloned polyadenylated cox2 cDNAs, we conclude that RNA editing and polyadenylation are independent processes in maize mitochondria. INTRODUCTIONPresent-day mitochondria have almost certainly evolved from a prokaryotic endosymbiont (reviewed in Gray, 1992). These organelles possess their own genomes and gene expression machinery; however, during evolution, most of the genetic information of the mitochondrial ancestor was transferred to the nuclear genome. Mitochondrial genes in Saccharomyces cerevisiae and metazoans are transcribed by a nucleus-encoded T7-like RNA polymerase and accessory factors (reviewed in Tracy and Stern, 1995), and candidate plant nuclear genes encoding mitochondrial RNA polymerase have been identified (Cermakian et al., 1996; Hedtke et al., 1997;Young et al., 1998; Chang et al., 1999). In plants, promoter strength may play a regulatory role in gene expression (Mulligan et al., 1991), but post-transcriptional regulation also can occur by differential RNA stability (Finnegan and Brown, 1990).The maize mitochondrial genome is typical of those found in vascular plants. It can be genetically mapped as a single circular molecule of 570 kb, with multiple repeated sequences giving rise to a variety of stably inherited subgenomic recombination products (Lonsdale et al., 1984). As in other species, maize mitochondrial primary transcripts are subject to both cis -splicing (e.g., Fox and Leaver, 1981) ...

Plant mitochondrial genomes are highly recombinogenic, with a variety of species-specific direct and inverted repeats leading to in vivo accumulation of multiple DNA forms. In maize, the cox2 gene, which encodes subunit II of cytochrome c oxidase, lies immediately downstream of a 0.7-kilobase direct repeat, which is present in two copies in the 570-kilobase master chromosome. Promoters for cox2 exist upstream of both of these copies, in regions we have termed A and B. Three region B promoters are active for cox2 transcription in the master chromosome, whereas two region A promoters are active for cox2 transcription after recombination across the direct repeats. We have measured the proportion of genomes carrying region A or B upstream of cox2 in maize seedlings and found a ratio of approximately 1:6. Promoter strength, based on run-on transcription assays, shows a ratio of 1:4 for region A to region B promoters. These data allowed us to predict the relative contributions of region A and B to mitochondrial transcript accumulation, based on a simple product of genome-form abundance and promoter strength. When promoter use was determined by using quantitative reverse transcriptase-PCR, however, we found that region A promoters were used at an unexpectedly high rate when upstream of cox2 and used less than expected when not upstream of cox2. Thus, the use of this set of promoters seems to respond to genomic context. These results suggest a role for intragenomic and intergenomic recombination in regulating plant mitochondrial gene expression.