Analysis of novel plastid proteins targeting nucleic acids: Identification of native RNA/DNA ligands and interacting proteins
Final Report Abstract
Accurate gene expression is essential for chloroplast biogenesis. In our project on functional examination of proteins involved in plastid gene expression we focused on pTAC12. In our previous work, we identified pTAC12 to be part of a large DNA-protein complex in chloroplasts referred to as plastid transcriptionally active chromosome (pTAC). Genetic studies of the ptac12 mutant showed profound changes in expression of specific genes and strong chloroplast biogenesis defects, but the molecular basis for the phenotype remained unclear. During the funding period, we have discovered pTAC12 as an integral subunit of the PEP-complex (plastid encoded RNA polymerase), the main RNA polymerase in mature chloroplasts. The same approach discovered additional, previously unidentified PEP-associated proteins (PAPs). A specific knowledge of the subunit composition will be useful for a better understanding of the plastid transcription system. Using molecular and biochemical tools we could show that ptac12 mutants and other mutants lacking specific PAPs, fail to accumulate the PEP-complex, despite normal levels of other subunits. This result suggest that the loss of PEP activity in these mutants is a consequence of the assembly defects. We were able to identify a subset of plastid RNAs that are synthesized by PEP- dependent transcription. Furthermore, our in vitro analysis of nucleic acid binding activities revealed that ZmpTAC12 interacts with both ssRNA and ssDNA, but possesses only a weak binding preference for particular nucleotide sequence. As such pTAC12 could enable rapid and efficient transcriptional or posttranscriptional processes, possibly by proper positioning the tethered DNA and/or RNA. Future insights will likely be gained from studies determining the properties of the protein-DNA/RNA interaction(s) in planta.
Publications
- (2011). Identification of essential subunits in the plastid-encoded RNA polymerase complex reveals building blocks for proper plastid development. Plant Physiol. 157, 1043-1055
Steiner S, Schröter Y, Pfalz J, Pfannschmidt T
(See online at https://doi.org/10.1104/pp.111.184515) - Essential nucleoid proteins in early chloroplast development. (2013). Trends Plant Sci. 18, 186-194
Pfalz J and Pfannschmidt T
(See online at https://doi.org/10.1016/j.tplants.2012.11.003) - (2014). A major role for the plastid-encoded RNA polymerase complex in the expression of plastid tRNAs. Plant Physiol. 164, 239-48
Carrier R, Zoschke R, Belcher S, Pfalz J, Barkan A
(See online at https://doi.org/10.1104/pp.113.228726) - (2015). Plastid nucleoids: evolutionary reconstruction of a DNA/protein structure with prokaryotic ancestry. Front Plant Sci. 6, 220
Pfalz J and Pfannschmidt T
(See online at https://doi.org/10.3389/fpls.2015.00220) - (2015). ZmpTAC12 binds single-stranded nucleic acids and is essential for accumulation of the plastid-encoded polymerase complex in maize. New Phytol. 206, 1024-37
Pfalz J, Holtzegel U, Barkan A, Weisheit W, Mittag M, Pfannschmidt T
(See online at https://doi.org/10.1111/nph.13248)
