tailieunhanh - Báo cáo khoa học: DNA-binding and transcription characteristics of three cloned sigma factors from mustard (Sinapis alba L.) suggest overlapping and distinct roles in plastid gene expression

We have isolated and studied the cloned sigma factors SASIG1-3 from mustard (Sinapis alba). In functional ana-lyses using both promoter and factor mutants,the three recombinant proteins all had similar basic properties but also revealed differences in promoter preference and requirements for single nucleotide positions. Directedmuta-genesis of SASIG1 identified critical residues within the conserved regions and necessary for binding of the )10 and)35 promoter elements,respectively. | Eur. J. Biochem. 270 1288-1300 2003 FEBS 2003 doi DNA-binding and transcription characteristics of three cloned sigma factors from mustard Sinapis alba L. suggest overlapping and distinct roles in plastid gene expression Anke Homann and Gerhard Link Plant Cell Physiology and Molecular Biology University of Bochum Germany We have isolated and studied the cloned sigma factors SASIG1-3 from mustard Sinapis alba . In functional analyses using both promoter and factor mutants the theee recombinant proteins all had similar basic properties but also revealed differences in promoter preference and requirements for single nucleotide positions. Directed mutagenesis of SASIG1 identified critical residues within the conserved regions and necessary for binding of the -10 and -35 promoter elements respectively. SASIG1 and 2 but nol S ASIG3. each hvve a typica 1 regkm 25 for binding of the extended -10 promoter element. SASIG3 has a prosequence reminiscent of rK from Bacillus subtilis ng that proteolytic cleavage from an inactive precursor is involved in the regulation of plastid transcription. In addi-tion SASIG2 was found to be more abundant in light-grown as compared to dark-grown mustard seedlings while the converse was true for SASIG3. Keywords plastid promoter RNA polymerase sigma factor transcription. Chloroplasts contain the photosynthetic machinery which is built-up and maintained by gene-regulatory mechanisms both inside and outside the organelle. At the level of transcription this involves the participation of multiple RNA polymerases at least two of which are located within the plastid compartment a a single-subunit type enzyme related to those of T-odd phages and mitochondria and b a multisubunit form resembling those of bacteria and eukaryotic nuclei reviewed in 1 . The former is a product of nuclear gene s nuclear-encoded phage-type plastid RNA polymerase NEP the latter whcd is the primrry enzyme for transcription