Differential Expression of the Lux Operon of V Harveyi

Johnston, Timothy

Abstract

It has been shown that mRNA's transcribed from the lux operon of V. harveyi have 3' termini in two regions of the operon. The first hypothesis is that there are two terminators and that the promoter proximal terminator is sometimes read through by RNA polymerase allowing transcription of the entire operon. The second is that there is only on terminator and that a portion of the mRNA is degraded from the 3' end to some stabilizing factor. During the sequencing of the luxB gene a region of dyad symmetry that begins 17 nucleotides 3' to the stop condon of the luxB gene was discovered which is in the region of the promoter proximal terminator and may function either as a transcriptional terminator as in hypothesis on or as a stabilizing factor as in hypothesis two. The purpose of this investigation is to first, sequence the remainder of the 3' end of the V. harveyi lux operon and identify the 3' termini of lux mRNA by S1 mapping and to second, determine whether those termini are the result of transcription or mRNA processing. A fragment containing the promoter proximal terminus will be cloned first, into a vector capable of quantitating transcription termination and second, 3' to where it already exists in a plasmid containing the lux operon. In the first construction termination will be detected and quantitated. In the second construction mRNA processing by exonuclease or endonuclease activity will be differentiated since exonuclease activity would result in a 3' end at the 3' location and endonuclease activity would result in a 3' end at the 5' location. These data will increase the understanding of the evolution and regulation of gene expression of bioluminescence in marine bacteria. This study will also lay the ground-work for the study of factors that stabilize or destabilize lux mRNA an contribute to the loss of luciferase activity in early stationary phase cultures.