Structural Characterisation of an ANTAR Domain Anti-Terminator Protein Bound to RNA
Access status:
USyd Access
Author/s
Walshe, James LachlanAbstract
Regulated transcription termination provides an efficient and responsive means to control gene expression. In bacteria, intrinsic termination occurs through the formation of an RNA stem-loop (T-loop), which disrupts the RNA polymerase elongation complex, resulting in its dissociation ...
See moreRegulated transcription termination provides an efficient and responsive means to control gene expression. In bacteria, intrinsic termination occurs through the formation of an RNA stem-loop (T-loop), which disrupts the RNA polymerase elongation complex, resulting in its dissociation from the DNA template. Bacteria have a number of strategies for over-riding intrinsic T-loops, one of which is the formation of mutually exclusive RNA structures. Proteins that promote the formation of alternative RNA structures are known as anti-terminators. The ANTAR domains are a family of RNA binding anti-terminators that are most commonly associated with two components signalling (TCS) pathways. Unlike the canonical DNA binding output domains of TCS proteins, ANTAR domains facilitate their response by binding to RNA. EutV from E. faecalis is an ANTAR domain protein that controls gene expression of the eut operon. EutV is a model protein for studying ANTAR induced anti-termination. The ANTAR domain binding motif consists of dual RNA hexaloops, with the second hexaloop overlapping with the 5’ end of the intrinsic T-loop. Despite the well-defined binding motif, it is unknown at the molecular level how the ANTAR domains interact with RNA. In this work we have determined the 2.16 Å resolution crystal structure of the ANTAR domain protein EutV in its RNA-free form. The dimeric structure of EutV shows a high degree of similarity to the previously characterised AmiR, suggesting a common dimeric organisation for these domains. We went on to solve the structure of EutV bound to RNA, which represents the first reported structure of an ANTAR domain bound to its cognate RNA binding motif. Our studies highlight the key interactions between conserved EutV residues and the RNA, as well as protein conformational changes undergone upon RNA binding. These interactions were validated by alanine mutagenesis, which allowed us to propose an updated model for ANTAR domain anti-termination.
See less
See moreRegulated transcription termination provides an efficient and responsive means to control gene expression. In bacteria, intrinsic termination occurs through the formation of an RNA stem-loop (T-loop), which disrupts the RNA polymerase elongation complex, resulting in its dissociation from the DNA template. Bacteria have a number of strategies for over-riding intrinsic T-loops, one of which is the formation of mutually exclusive RNA structures. Proteins that promote the formation of alternative RNA structures are known as anti-terminators. The ANTAR domains are a family of RNA binding anti-terminators that are most commonly associated with two components signalling (TCS) pathways. Unlike the canonical DNA binding output domains of TCS proteins, ANTAR domains facilitate their response by binding to RNA. EutV from E. faecalis is an ANTAR domain protein that controls gene expression of the eut operon. EutV is a model protein for studying ANTAR induced anti-termination. The ANTAR domain binding motif consists of dual RNA hexaloops, with the second hexaloop overlapping with the 5’ end of the intrinsic T-loop. Despite the well-defined binding motif, it is unknown at the molecular level how the ANTAR domains interact with RNA. In this work we have determined the 2.16 Å resolution crystal structure of the ANTAR domain protein EutV in its RNA-free form. The dimeric structure of EutV shows a high degree of similarity to the previously characterised AmiR, suggesting a common dimeric organisation for these domains. We went on to solve the structure of EutV bound to RNA, which represents the first reported structure of an ANTAR domain bound to its cognate RNA binding motif. Our studies highlight the key interactions between conserved EutV residues and the RNA, as well as protein conformational changes undergone upon RNA binding. These interactions were validated by alanine mutagenesis, which allowed us to propose an updated model for ANTAR domain anti-termination.
See less
Publisher
University of SydneyFaculty of Science
School of Life and Environmental Sciences
Type
PhD DoctoratePublication year
2019-06-28Licence
The author retains copyright of this thesis. It may only be used for the purposes of research and study. It must not be used for any other purposes and may not be transmitted or shared with others without prior permission.Share