The proposed work is aimed at increasing our understanding of structure- function relationships in bacteriophage T7 RNA polymerase (RNAP). Our structural and structure-function studies of T7 RNAP have provided us with its 3-dimensional structure and have identified within this structure the template binding cleft, the active site, a region involved in binding the rNTP, and a domain which binds the nascent RNA. Comparison of the T7 RNAP structure to other polymerases and integration of published structure- function studies of polymerases into this comparison makes it possible for us to develop the concept of modularity in polymerase structure-function organization. We have identified subdomains or 'modules' in T7 RNAP which are not a part of the structurally conserved polymerase domain. The functions of these additional modules are poorly understood. We will mutagenize these modules in order to determine their functions and to test functional hypotheses which have emerged from modeling studies. Our structural and structure-function studies of the extended thumb subdomain of T7 RNAP have shown that it is a flexible structure and that it functions to hold the ternary complex together during transcription (probably by wrapping around bound template). These studies have identified a putative hinge or 'knuckle' within the thumb which may underlie its flexibility. We will mutagenize this 'knuckle' to test this functional hypothesis. We will characterize the properties of a mutation which causes the polymerase to indiscriminately utilize both rNTPs and dNTPs in order to learn more about the mechanisms of substrate discrimination and to characterize the properties of this potentially useful research reagent. Finally, we plan to mutagenize a region in T7 RNAP likely to be responsible for substrate discrimination with the aim of changing the substrate specificity of T7 RNAP so that it will selectively utilize dNTPs. This work has two goals: 1. identification of the substrate discrimination elements through modification of substrate specificity, and 2. the engineering of a polymerase which synthesizes DNA but has the unique properties of an RNA polymerase (promoter specificity, de novo initiation, template unwinding). Such a novel enzyme could find application in genetic research and gene therapy protocols. Generally, it should be recognized that polymerases are critical enzymes in many applications: in clinical virology they are therapeutic targets; they are research tools in molecular biology; and they synthesize the nucleic acids that are the targets/agents of gene therapy and transgenic research. The ability to engineer or target polymerases could result in development of new therapeutics and research tools. Most of these developments cannot be anticipated, though we frame one potential application. To be able to carry out such engineering/targeting we need a deeper understanding of polymerase structure-function relationships. Our work with T7 RNAP is aimed at contributing to such enhanced understanding.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM052522-01A1
Application #
2191580
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1995-09-30
Project End
2000-03-31
Budget Start
1995-09-30
Budget End
1996-03-31
Support Year
1
Fiscal Year
1995
Total Cost
Indirect Cost
Name
University of Texas Health Science Center San Antonio
Department
Biochemistry
Type
Schools of Medicine
DUNS #
800772162
City
San Antonio
State
TX
Country
United States
Zip Code
78229
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; Cuéllar, 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
Sousa, Rui (2009) Comment on ""Xeno's paradox"". EMBO Rep 10:800
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; Guo, Qing; Sousa, Rui (2009) A promoter recognition mechanism common to yeast mitochondrial and phage t7 RNA polymerases. J Biol Chem 284:13641-7

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