Functional analysis of two maize cDNAs encoding T7-like RNA polymerases.
We have characterized two maize cDNAs, rpoTm and rpoTp, that encode putative T7-like RNA polymerases. In vivo cellular localization experiments using transient expression of the green fluorescent protein suggest that their encoded proteins are targeted exclusively to mitochondria and plastids, respectively. An antibody raised against the C terminus of the rpoTp gene product identified mitochondrial polypeptides of approximately 100 kD. Their presence was correlated with RNA polymerase activity, and the antibody inhibited mitochondrial in vitro transcription activity. Together, these results strongly suggest that the product of rpoTm is involved in maize mitochondrial transcription. By contrast, immunoblot analysis and an antibody-linked polymerase assay indicated that rpoTp specifies a plastid RNA polymerase component. A quantitative reverse transcription-polymerase chain reaction assay was used to study the transcription of rpoTp and rpoTm in different tissues and under different environmental conditions. Although both genes were constitutively expressed, rpoTm transcripts were generally more prevalent in nonphotosynthetic tissues, whereas an increase in rpoTp transcripts paralleled chloroplast development. We suggest that these two genes encode constitutive components of the organelle transcription machinery but that their expression is nonetheless subject to modulation during plant development.
D000595:Amino Acid Sequence; D002451:Cell Compartmentation; D002736:Chloroplasts / Q000201:enzymology*; D003001:Cloning, Molecular; D017124:Conserved Sequence; D018076:DNA, Complementary / Q000235:genetics; D012321:DNA-Directed RNA Polymerases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D018628:Gene Dosage; D049452:Green Fluorescent Proteins; D008164:Luminescent Proteins / Q000235:genetics / Q000302:isolation & purification; D008928:Mitochondria / Q000201:enzymology*; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D018749:RNA, Plant / Q000096:biosynthesis / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification; D017386:Sequence Homology, Amino Acid; D014018:Tissue Distribution; D014764:Viral Proteins; D003313:Zea mays / Q000201:enzymology* / Q000235:genetics