Abstract
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.
Highlights
Studied, can be mapped as a 570-kb circle with numerous repeated elements mediating recombination, leading to a complex set of overlapping molecules [4]
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 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 these promoters may be recognized by specific transcription factors [12], the frequency of active promoters has not been surveyed over large regions of any plant mitochondrial genome
Each promoter conforms to a previously established consensus sequence for maize mitochondrial promoters, there are some with unusual features
Summary
Studied, can be mapped as a 570-kb circle with numerous repeated elements mediating recombination, leading to a complex set of overlapping molecules [4]. Extensive mutational analysis of the maize atpA and cox 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, 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 these promoters may be recognized by specific transcription factors [12], the frequency of active promoters has not been surveyed over large regions of any plant mitochondrial genome. The maize mitochondrial genome, the expression of which we have
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