Publication Date

2014-09-17

Availability

Open access

Embargo Period

2014-09-16

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PHD)

Department

Biochemistry and Molecular Biology (Medicine)

Date of Defense

2014-06-03

First Committee Member

Murray P. Deutscher

Second Committee Member

Feng Gong

Third Committee Member

Kurt Schesser

Fourth Committee Member

Zhongwei Li

Fifth Committee Member

Arun Malhotra

Abstract

There are many ribonucleases in Escherichia coli and, presumably, each one has an important role in RNA metabolism. After an individual RNA has been fully transcribed, the fate of that RNA is determined by its sequence, structure, and the availability of RNases which may be determined by conditions inside and outside of the cell. During periods of exponential growth, the majority of RNA degradation is carried out by RNase II, RNase R and polynucleotide phosphorylase, and the short RNA fragments generated by the degradation process are reduced to individual nucleotides by oligoribonuclease. Maturation of RNAs is performed primarily by RNase III, RNase P, RNase E, RNase G, RNase T and RNase PH. These processes of degradation and maturation represent most of RNA metabolism in cells, and can be accounted for by the aforementioned RNases. However, there are two additional RNases that have no known primary role in RNA metabolism – RNase D and RNase BN. Here, I present in vivo and in vitro data showing that RNase D and, to a lesser extent, RNase BN are responsible for fine tuning regulatory elements in cells by removing at least one RNA that is deleterious for growth and survival when present in excess. This is the mRNA for CsrA, an RNA binding protein that acts in concert with the sigma factor RpoS when cells are growing slowly or not at all. I will also show that cells lacking RNase D take more time to exit lag phase than do cells in which RNase D is present and that this defect is not due to aberrant maturation of ribosomal RNAs. In order to identify RNAs that are substrates for RNase D and RNase BN in vivo, Northern analyses of many stable and messenger RNAs, electrophoresis of radiolabeled RNA and tRNA nucleotidyltransferase assays were carried out, among others. These ruled out tRNA and rRNA as the primary substrates for RNase D and RNase BN during stationary phase and recovery from prolonged starvation. They also revealed that the mRNA for CsrA is upregulated 3- to 4- fold in cells lacking RNase D during early stationary phase and that RNase D and RNase BN are expressed most abundantly during rapid growth. Assays of growth, cell morphology and ability to synthesize critical cellular components like flagella and extracellular matrix were utilized concurrently with the previously noted molecular techniques. These showed that cells lacking RNase D have a reduced ability to synthesize extracellular matrix, show reduced motility, took longer to recover from prolonged starvation, and synthesize more of the messenger molecule cyclic-di-GMP than wild type cells. Cells lacking RNase D and RNase BN were at a competitive disadvantage when grown in low pH conditions. Together, these studies have revealed that these two RNases do indeed have a role in E. coli allowing cells to more efficiently enter and exit rapid growth by fine-tuning the availability of at least one specific RNA that has a deleterious effect when overexpressed. The studies presented here indicate that the roles of of RNase R and RNase BN are subtle and while many substrates have been ruled out, undoubtedly additional substrates remain to be discovered.

Keywords

RNA metabolism, ribonuclease, RNA processing, exoribonuclease, RNA decay

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