Discovery and Characterization of Antiterminator Proteins in Bacteria

Abstract

Transcription is a discontinuous process, where each nucleotide incorporation cycle offers a decision between elongation, pausing, halting, or termination. In bacteria, many regulators—including protein antiterminators or cis-acting regulatory RNAs, such as riboswitches—exert their influence over transcription elongation. Through such mechanisms, these regulators can couple physiological or environmental signals to transcription attenuation, a process where RNA structure directly influences formation of transcription termination signals. However, through another regulatory mechanism called processive antitermination (PA), RNA polymerase can become induced to bypass termination sites over much greater distances than transcription attenuation can offer. These mechanisms are widespread in bacteria, although only a few mechanistic classes have been discovered overall. The aim of the research in this dissertation is two-fold: to identify novel genetic regulatory mechanisms targeting transcription termination and to systematically study the diversity and breadth distribution of these mechanisms among bacteria. This research focuses on two distinct mechanisms, each representing one of these mechanisms of antitermination. First, I detail discovery of LoaP, a specialized paralog of the universally conserved NusG transcription elongation factor. Our data demonstrate that Bacillus velezensis LoaP controls gene expression of antibiotic biosynthesis gene clusters by promoting readthrough of transcription termination sites. Additionally, we show that, unlike other bacterial NusG proteins, LoaP binds RNA with high affinity, and with apparent specificity for a sequence in the 5′ leader regions of its target operons. Second, we describe the interaction between a family of antitermination proteins containing the ANTAR RNA-binding domain with its target RNA. We show that ANTAR-containing proteins bind a tandem stem-loop RNA motif to prevent formation of terminator structures. Using a combination of mutagenesis strategies, we elucidate some of the RNA-binding requirements of a representative ANTAR protein. Finally, employed bioinformatic and phylogenetic approaches to place these regulators in the context of their entire protein families, learning about the distribution of these mechanisms, their association with particular potential regulons, and sequence composition of different protein subfamilies.