The Biology and Biochemistry of PhoH2 proteins

Abstract

PhoH2 proteins are found in a diverse range of organisms. To date little is known about these proteins and the role they play in the organisms in which they reside. PhoH2 is a PIN PhoH domain fusion, and these proteins are currently annotated as having unknown function and are described as PhoH like. PhoH domains are thought to be ATPases and all characterised PIN domain proteins are RNases. Most efforts have focussed on determining the role of PIN domain proteins that comprise the toxic component (VapC) of VapBC toxin antitoxin systems, in which the PIN domain is coexpressed as part of an operon with an inhibitor (VapB). In the remaining cases where PIN domain proteins can be found such as PIN PhoH domain fusions, these cases remain unexamined. This thesis describes the biological and biochemical characterisation of the PIN PhoH protein, PhoH2 from Mycobacterium tuberculosis and Mycobacterium smegmatis, along with a preliminary structural characterisation of a thermophilic PhoH2 protein homologue.

The phoH2 gene from both mycobacterial organisms was found to be expressed as part of a long mRNA transcript. Examination of these transcripts revealed possible alternative 5’ start sites out of frame with the phoH2 gene. For protein overexpression, and ‘normal’ growth and colony formation with conditional overexpression, phoH2 from M. tuberculosis required 152 bp of the 5’ sequence directly upstream of the annotated phoH2 gene (annotated here as phoH2alt). PhoH2 proteins: PhoH2alt MTB, PhoH2alt MSMEG and PhoH2MSMEG show ATP/Mg²⁺ dependent, sequence specific RNA unwinding and cleavage. The sequence (A C) (A/U) (A/U) (G/C) U was deduced as a substrate for PhoH2, and PhoH2alt MTB also demonstrated unwinding and cleavage activity on its upstream ~152 base RNA transcript, suggesting a potential autoregulatory mechanism. Structural analysis of a thermophilic PhoH2 protein homologue has provided preliminary crystallographic data which along with electron microscopy suggest a ring like hexameric PhoH2 oligomer.