STRUCTURAL STUDY OF HUMAN CATENIN ΒETA-LIKE PROTEIN 1 AND DOUBLE RNA PSEUDOKNOTS IN -1 RIBOSOMAL FRAMESHIFTING

Author

Guan Wang, Southern Illinois University CarbondaleFollow

Date of Award

5-1-2015

Degree Name

Doctor of Philosophy

Department

Chemistry

First Advisor

Du, Zhihua

Abstract

Project 1-- structure determination of human catenin-β-like protein 1 by x-ray crystallography Catenin-β-like protein 1 (CTNNBL1) is a highly conserved protein with multiple functions, one of which is to act as an interaction partner of the antibody-diversification enzyme activation-induced cytidine deaminase (AID) for its nuclear import and subnuclear trafficking. In this dissertation, the crystal structure of full-length human CTNNBL1 is reported. The protein contains six armadillo (ARM) repeats that pack into a superhelical ARM domain. This ARM domain is unique within the ARM protein family owing to the presence of several unusual structural features. Moreover, CTNNBL1 contains significant and novel non-ARM structures flanking both ends of the central ARM domain. A strong continuous hydrophobic core runs through the whole structure, indicating that the ARM and non-ARM structures fold together to form an integral structure. This structure defines a highly restrictive and discriminatory protein-binding groove that is not observed in other ARM proteins. The presence of a cluster of histidine residues in the groove implies a pH-sensitive histidine-mediated mechanism that may regulate protein binding activity. The many unique structural features of CTNNBL1 establish it as a distinct member of the ARM protein family. The structure provides critical insights into the molecular interactions between CTNNBL1 and its protein partners, especially AID. Project 2 -- study of double pseudoknots in the regulation of -1 programmed ribosomal frameshifting in RNA viruses −1 programmed ribosomal frameshifting (PRF) is utilized by many viruses to synthesize their enzymatic and structural proteins at a defined ratio. For efficient −1 PRF, two cis-acting elements are required--a heptanucleotide frameshift site and a downstream stimulator such as a pseudoknot. By searching for all possible pseudoknots within the full-length viral genomic mRNAs, we detected potential double pseudoknots at the −1 PRF junction in several animal viruses, including human immunodeficiency virus type-1 (HIV-1), transmissible gastroenteritis virus (TGEV), Barmah Forest virus (BFV), Fort Morgan virus (FMV), and Equine arteritis virus (EAV). We built structural models of the HIV-1 and EAV double pseudoknots to show that both the tandem and embedded mode of double pseudoknots are feasible and reasonable. We hypothesize that the fundamental reason for the viruses to utilize coaxially stacked double pseudoknots is to increase the overall stability of the frameshift regulating structure and while avoiding the use of an ultra stable single pseudoknot which may become a ribosomal roadblock. Results from this study significantly expand the repertoire of RNA structures and dynamics that may involve in the regulation of −1 PRF in viruses