Poster: Organelle Biogenesis

Abs # 1138: Composition of the tobacco multisubunit plastid RNA polymerase and in vitroreconstitution of the holoenzyme

Presenter:	Suzuki, Jon Y., suzuki@waksman.rutgers.edu
Authors	Suzuki, Jon Y. (A)   Ytterberg, Jimmy  (B)   Beardslee, Thomas  (C)   Allison, Lori A (C)   van Wijk, Klaas  (B)   Maliga, Pal  (A)
Affiliations:	(A): Waksman Institute of Microbiology, Rutgers, The State University of New Jersey (B): Department of Plant Biology, Cornell University (C): Department of Biochemistry, University of Nebraska

The plastid-encoded plastid RNA polymerase (PEP) is similar to the multisubunit bacterial RNA polymerases. The plastid PEP core is encoded by plastid genes, and has the composition alpha₂, beta, beta', and beta". PEP composition was determined by His-tagging the tobacco alpha subunit C-terminus in vivo, affinity purification of the PEP complex by the His-tag, and mass spectrometry of the resulting proteins which were separated by 2D-gel electrophoresis. As expected, we identified the alpha, beta, beta', beta" subunits in our purified PEP preparation. In addition, we have identified four major and six minor proteins associated with the PEP. Based on their protein motifs, some of these proteins may be involved DNA interaction, RNA processing and or mRNA translation. These proteins may be involved in regulating PEP activity in response to environmental and developmental signals. Interestingly, the specificity factor, sigma required for promoter recognition was absent in the purified PEP. Therefore, we had the opportunity to reconstitute a homologous plastid RNA polymerase from the affinity-purified PEP core and sigma factors expressed in E. coli. Heterologous enzyme reconstituted from the PEP core and the E. coli sigma⁷⁰ factor had very low activity highlighting structural differences between the E. coli and plastid PEP cores. The homologous transcription system utilizing the plant PEP core will allow us to determine promoter specificity of individual plant sigma factors and their role in regulating plastid transcription.