Structural and functional analyses of multi-protein complexes regulating genotype to phenotype translations in plants
EMSL Project ID
48378
Abstract
DNA-dependent RNA polymerases decode genetic information stored in DNA by converting the information into RNA. This enzymatic activity, known as transcription, is a critical step in the process by which an organism's genotype gives rise to its phenotype. Importantly, genes do not exist in the nucleus as naked DNA molecules but as protein-DNA complexes collectively known as chromatin. Chemical modifications of chromatin play important roles in determining whether a given gene can be transcribed. Chromatin modifications include cytosine methylation of the DNA as well as modifications of the histone proteins that help organize the DNA. Chromatin modifications allow for "epigenetic" control - so named because it operates at a level above that of the genetic (DNA sequence) information. Genes can be active or silenced depending on their epigenetic state, which can vary in response to environmental conditions and can even be inherited. Two DNA-dependent RNA polymerases, Pol IV and Pol V, are key to RNA-directed DNA methylation, an important transcriptional silencing process in plants. Pol IV acts in partnership with an associated RNA-dependent RNA polymerase, RDR2, generating double-stranded precursors of small RNAs that then target matching gene sequences for silencing. The pairing of DNA-dependent and RNA-dependent RNA polymerases is unprecedented in biology, presumably facilitating decoding of double-stranded chromosomal DNA sequences into double-stranded RNA molecules. By using chemical crosslinking and mass spectrometry, combined with de novo assembly and reconstitution of functional Pol IV - RDR2 complexes, the goal of this project is to understand how Pol IV and RDR2 interact and couple their activities to synthesize double-stranded RNAs. This knowledge will provide a mechanistic understanding of how RNAs are synthesized and channeled into pathways responsible for targeted gene silencing.
Project Details
Project type
Large-Scale EMSL Research
Start Date
2014-10-01
End Date
2016-09-30
Status
Closed
Released Data Link
Team
Principal Investigator
Team Members
Related Publications
Haag JR, B Brower-Toland, EK Krieger, L Sidorenko, CD Nicora, AD Norbeck, A Irsigler, H LaRue, J Brzeski, KA Mcginnis, S Ivashuta, L Pasa-Tolic, VL Chandler, and CS Pikaard. 2014. "Functional Diversification of Maize RNA Polymerase IV and V subtypes via Alternative Catalytic Subunits." Cell Reports 9(1):378-390. doi:10.1016/j.celrep.2014.08.067
Ream TS, JR Haag, F Pontvianne, CD Nicora, AD Norbeck, L Pasa-Tolic, and CS Pikaard. 2015. "Subunit compositions of Arabidopsis RNA polymerases I and III reveal Pol I- and Pol III- specific forms of the AC40 subunit, and alternative C53 subunits ." Nucleic Acids Research 43(8):4163-4178. doi:10.1093/nar/gkv247