Rational Elicitation of Cold Sensitive Phenotypes

Abstract

Conditional gene expression and conditional mutants provide a means to modulate the expression of specific genes and to control the activity of their protein products in vivo, so as to be able to study their effects on the cell. Conditional mutants are functional under one set of conditions, termed ‘permissive’, while under other conditions that are ‘restrictive’, they become non-functional. The wild-type (Wt) is functional under both these conditions. Conditional mutants are especially useful for studying essential or lethal genes in an organism. In the case of temperature-sensitive (ts) and cold-sensitive mutants (cs), by using temperature shift as a condition, the target gene function can be modulated easily, rapidly, reversibly and selectively, at any stage in the life cycle of an organism. Cs mutants are less common and molecular determinants of cs phenotypes are poorly understood.This thesis presents a method for rational elicitation of cold sensitive phenotypes which involves design of partial loss-of-function mutants based solely on amino acid sequence, and coupling of such mutants to a heat responsive promoter to result in cs phenotypes. This approach has been validated for different proteins (CcdB, Gal4, Ura3 and Trp1) in different organisms (E. coli, S. cerevisiae and D. melanogaster). This is a straightforward approach which does not involve complex temperature-dependent, mutational effects.Additional characterization of purified Gal4 mutants by measuring protein thermal stability and DNA binding affinity, as well as measurements of transcript levels by qPCR were carried out, to understand the molecular basis of the cs phenotype. The pBAD series of vectors, containing the PBAD promoter and the araC regulator-activator, are very convenient for cloning and expression purposes. They have been widely for cloning and for graded expression of cloned genes. However, there have been reports of non-uniform gene expression across cells at sub-saturating concentrations of arabinose. This thesis also covers studies on this issue of heterogeneity of expression from PBAD promoters at the single cell level, using stable and degradable GFPs as reporters, in a variety of conditions such as constitutive versus autocatalytic expression of arabinose transporter, presence and absence of arabinose metabolising araBAD genes in the host, and varying time periods of induction. Several single amino acid substitutions which cause folding defects in the protein are seen to give rise to cs or ts phenotypes. The key to understanding the basis of such phenotypes caused by folding defective mutants lies in the folding pathway of the protein. To this end, the folding kinetics of E. coli CcdB were studied, which is prerequisite for understanding the folding defects in various ts/cs mutants of CcdB. This study looks at the folding of a dimeric protein, which is of great interest as it involves conformational changes as well as association steps. The results from various experimental observations, ligand binding studies and simulations lead to the conclusion that CcdB folds via parallel pathways, each involving an unstructured dimeric intermediate, to arrive at its native state. In summary, the thesis covers the research carried out ontuning conditional gene expression, rationally designing conditional mutants and understanding their folding mechanisms.