The single-polypeptide RNA polymerase of the T7 bacteriophage has been a model system for studying fundamental mechanisms of transcription ever since it was first identified almost 4 decades ago. Work over the past 15 years has revealed that the functions of this RNA polymerase extend beyond transcription of the phage genes, and that homologues of T7 RNA polymerase are widespread, occurring not only in eukaryotic mitochondria but also in mammalian nuclei, where they form the 4th major class of nuclear RNAPs (spRNAPIV). However, when compared to our detailed understanding of the structure and transcriptional mechanisms of T7RNAP, our understanding of the extra-transcriptional functions of this enzyme or of the mechanisms and biology of its mitochondrial and nuclear homologues is limited. To address these gaps in understanding we will: (1) Define the role of T7RNAP and its regulator T7 lysozyme in recruitment and assembly of the T7 DNA packaging machinery and, using both ensemble and single molecule experiments, describe the molecular details of the T7 DNA packaging reaction , (2) Determine crystal structures of yeast mitochondrial RNAP elongation and initiation complexes, and characterize the mechanism of promoter recognition by this RNA polymerase and of its activation by the mitochondrial transcription factor, (3) Identify the genes regulated by nuclear spRNAPIV and the effects of activation of respiration or of catabolite repression on spRNAPIV activity. These studies will advance our understanding of fundamental mechanisms of macromolecular complex assembly and of transcription processes in the mitochondrial and nuclear compartments of eukaryotic cells.
RNA polymerases are the central players in the expression of genetic information. Aberrant activity of RNA polymerases leads to human disease. Our work will increase our understanding of how RNA polymerases control genes that are turned on in cancer cells and genes that are involved in cellular carbohydrate and energy metabolism, processes important in aging and diabetes.
|Liu, Yu; Holmstrom, Erik; Zhang, Jinwei et al. (2015) Synthesis and applications of RNAs with position-selective labelling and mosaic composition. Nature 522:368-72|
|Velazquez, Gilberto; Sousa, Rui; Brieba, Luis G (2015) The thumb subdomain of yeast mitochondrial RNA polymerase is involved in processivity, transcript fidelity and mitochondrial transcription factor binding. RNA Biol 12:514-24|
|Drakulic, Srdja; Wang, Liping; CuÃ©llar, Jorge et al. (2014) Yeast mitochondrial RNAP conformational changes are regulated by interactions with the mitochondrial transcription factor. Nucleic Acids Res 42:11246-60|
|Velazquez, Gilberto; Guo, Qing; Wang, Liping et al. (2012) Conservation of promoter melting mechanisms in divergent regions of the single-subunit RNA polymerases. Biochemistry 51:3901-10|
|Sousa, Rui (2012) A dancer caught midstep: the structure of ATP-bound Hsp70. Mol Cell 48:821-3|
|Li, Yifeng; Sousa, Rui (2012) Novel system for in vivo biotinylation and its application to crab antimicrobial protein scygonadin. Biotechnol Lett 34:1629-35|
|Li, Yifeng; Sousa, Rui (2012) Expression and purification of E. coli BirA biotin ligase for in vitro biotinylation. Protein Expr Purif 82:162-7|
|Woo, Hyung-June; Jiang, Jianwen; Lafer, Eileen M et al. (2009) ATP-induced conformational changes in Hsp70: molecular dynamics and experimental validation of an in silico predicted conformation. Biochemistry 48:11470-7|
|Nayak, Dhananjaya; Siller, Sylvester; Guo, Qing et al. (2008) Mechanism of T7 RNAP pausing and termination at the T7 concatemer junction: a local change in transcription bubble structure drives a large change in transcription complex architecture. J Mol Biol 376:541-53|
|Schuermann, Jonathan P; Jiang, Jianwen; Cuellar, Jorge et al. (2008) Structure of the Hsp110:Hsc70 nucleotide exchange machine. Mol Cell 31:232-43|
Showing the most recent 10 out of 13 publications