Isolation and characterization of the maize mitochondrial RNA polymerase
--Young, DA; Allen, R; Lonsdale, DM
To date only the mitochondrial RNA polymerases (mtRNAP) of the fungi Saccharomyces cerevisiae and Neurospora crassa have been isolated (Greenleaf et al., Proc Natl Acad Sci USA 83:3391, 1986; Chen et al., J Biol Chem 271:6537, 1996). The identification of rice ESTs with homology to the fungal polymerases provided the means to isolate the gene (rpomt) from maize. Using the rice EST clones and RT- PCR, maize cDNAs were isolated which were homologous to the yeast and Neurospora mitochondrial RNA polymerases. By employing a variety of 5'-RACE PCR techniques we have so far isolated approximately 3000 nucleotides of the maize cDNA, which we expect to be approximately 4000 nucleotides in length. We have yet to identify the 5'-terminus of the cDNA.
The known cDNA encodes 787 amino acids, which would produce a peptide of 89.2 kDa. Almost all 22 codons are used; codon usage and low G-C content of the cDNA suggests that the transcript is translated inefficiently. The amino alignment of the maize rpomt with the fungal polymerases is shown in Figure 1. The regions marked with symbols are domains that are highly conserved in many polymerases. Residues defined with an asterisk are found in all single-subunit polymerases while the domain marked between circles (hDhRGRhY; h=hydrophobic) is found in only DNA-directed RNA polymerases (Delarue et al., Protein Eng 3:461, 1990).
Southern analysis predicts that the gene is present as a single copy (Fig. 2). Screening of a genomic library resulted in the isolation of a number of clones which when mapped using restriction endonucleases fell into a single group. This supported the Southern analysis result. Approximately 20 kb of the rpomt locus have been sequenced. The cDNA is spread over 17.2 kb and is divided into 19 exons. The presence of a poly-A tail on the transcript and the identification of a partial PREM-1 retro-element sequence within an intron of the gene confirms that it is nuclear and not mitochondrially encoded.
Further experiments are in progress to isolate the 5' region of the gene. This will be followed by the determination of the rpomt encoded proteins subcellular location. Although the protein is highly homologous to the fungal polymerases (see Fig. 1) and such a gene is found in achloroplastic organisms (see Cermakain et al., Nucleic Acids Res 24:648, 1996) it has been proposed that a T3/T7-like RNA polymerase is also involved in chloroplast transcription (Allison et al., EMBO J 15:2802, 1996 Lerbs- Marche, Proc Natl Acad Sci USA 90:5509, 1993).
Figure 1. Prettybox amino acid alignment of the mitochondrial DNA-dependent RNA polymerases of yeast RPO41 (Sc) and Neurospora crassa cyt-5 (Ne) genes, with the maize rpomt (Rp) gene. Amino acids which are conserved between the majority of genes are black inverted, while similar amino acids are grey inverted. The figure is numbered relative to the known rpomt polypeptide. The five amino acids marked with an asterisk are those invariant in all DNA-directed polymerases, except multi-subunit RNA polymerases and RNA-directed polymerases. The phenylalanine (F786) residue marked with an arrow is conserved in all single-subunit RNA polymerases. This residue is proposed to interact with the incoming rNTP. Marked between dot symbols are the residues that constitute the hDhRGRhY domain which is conserved in all single-subunit DNA-directed RNA polymerases.
Figure 2. Genomic Southern analysis of B73N maize DNA. Each lane contains 20 g of DNA digested with the restriction endonuclease labelled above the corresponding lane. The Southern transferred gel was hybridised with a 1.7 kb rpomt genomic probe, and washed to a final stringency of 1 x SSC, 0.1 % SDS at 65 C. The sizes of positively hybridising bands are indicated by arrows. The molecular weights were deduced by comparison to a lane containing 1 kb ladder (BRL). The arrows inside the body of the figure indicate the locations of a faint positively hybridising HindIII band at 1.8 kb.
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