title: In vivo and in vitro analysis of RNases in Bacillus subtilis
title: In vivo und in vitro Analysen von RNases in Bacillus subtilis
titleType: AlternativeTitle
Creators: name: Hinrichs, Rebecca
Publisher: name: Philipps-Universität Marburg
Year: 2022
Subjects: Y-Komplex
Y-complex
Einzelmolekülverfolgung
RNasen
Chemistry &amp; allied sciences
Chemie
RNA degradation
RNase
single-molecule tracking
riboswitch
Bacillus subtilis

description: RNA degradation is a key process in the control of gene expression in bacteria and is essential for the cell’s homeostasis of nucleotide pools. A key player is the so-called RNA degradosome, proposed to be a membrane-associated complex containing endo- and exoribunucleases, as well as glycolytic enzymes and a DEAD-box RNA helicase. It is believed that endonuclease RNase Y is central to the formation of the RNA degradosome in Bacillus subtilis, leading to recruitment of RNases PnpA, RNase J1, and RNase J2, RNA helicase CshA, as well as glycolytic enzymes enolase and phosphofructokinase occurs. RNase Y has a transmembrane helix, and is "quasi“ essential; it is also required for mRNA processing following transcription. RNase Y also interacts with the so-called Y‑complex, consisting of YaaT, YlbF, and, YmcA (RicT, RicF, RicA), which is important for RNase Y-mediated processing of mRNA. How RNase Y can operate in two different protein complexes and acts in RNA decay as well as transcription-associated processes, is unclear. In this work, I show that the RNA degradosome is quite dynamic, having RNase Y, the glycolytic enzyme enolase, and the RNA helicase CshA and PnpA as central parts strongly reacting to a block in transcription, and thus to loss of mRNA substrate, while RNase J1 and J2 as well as the glycolytic enzyme phosphofructokinase show a much weaker response and are thus likely more peripheral components. The Y-complex clearly shows diffusion within the cytosol, but also the formation of membrane-associated accumulations, dissociating when transcription is blocked. Single molecule tracking (SMT) shows that the loss of one component of the Y-complex does not strongly affect the dynamics of the other proteins, suggesting that the complex forms a flexible association rather than a 1:1:1 stoichiometry. Biochemical analyses suggest that YaaT forms a membrane-anchor for the Y-complex, although it also has a cytosolic, freely diffusing fraction. A model will be presented that the Y-complex could function as an adaptor between nucleoid-associated mRNA synthesis and membrane-associated processing and degradation. II My thesis also presents a protocol for the successful purification of membrane-associated RNase Y, as a basis for further biochemical characterization of the protein, and interaction studies. Another essential process in which RNases play a crucial role is DNA replication. In addition to the RNA primers, which are necessary for the placement of Okazaki fragments, DNA/RNA hybrids must be processed and RNA must be removed to ensure the stability of the DNA. A part of the thesis work shows that B. subtilis replication forks intimately employ two RNases of the „H“ family, DNA polymerase A and exonuclease ExoR in vivo. Recruitment appears to be based on substrate availability rather than on specific protein/protein interactions, involving redundant enzymatic activities.