Title

Cloning, purification and characterization, and biological function of the Escherichia coli exoribonuclease RNase R

Date of Award

2002

Availability

Article

Degree Name

Doctor of Philosophy (Ph.D.)

Department

Biochemistry and Molecular Biology

First Committee Member

Murray P. Deutscher, Committee Chair

Abstract

The gene that encodes the Escherichia coli exoribonuclease RNase R has been identified to be the vacB gene, which was previously shown to be required for expression of virulence in Shigella and enteroinvasive E. coli. Cloning of E. coli vacB led to overexpression of RNase R activity, and partial deletion or interruption of the cloned gene abolished this overexpression. Interruption of the chromosomal copy of vacB eliminated endogenous RNase R activity; however, the absence of RNase R by itself had no effect on cell growth. In contrast, cells lacking both RNase R and polynucleotide phosphorylase were found to be inviable.By placing vacB under the control of a T7 promoter, RNase R was overexpressed ∼100 fold and subsequently purified to near homogeneity in its native untagged form by a rapid procedure. The enzyme shares many catalytic properties with RNase II. Both enzymes are nonspecific processive ribonucleases that release 5'-nucleoside monophosphates and leave a short undigested oligonucleotide core. However, whereas RNase R shortens RNA processively to di- and trinucleotides, RNase II becomes more distributive when the length of the substrate reaches approximately 10 nucleotides, and it leaves an undigested core of 3--5 nucleotides. Both enzymes work on substrates with a 3'-phosphate group. RNase R and RNase II are most active on synthetic homopolymers such as poly(A), but their substrate specificities differ.To examine the molecular basis for the synthetic lethality of PNPase and RNase R, a temperature-sensitive (ts) mutant strain was constructed that carried pnp-200, a ts allele of the gene encoding PNPase, and rnr::kan, a deletion-interruption of the gene encoding RNase R. Upon shifting to the non-permissive temperature of 42°C, this strain ultimately ceases growth and loses viability. Microscopic examination revealed that the cells are elongated and contain multiple nucleoids, suggesting inhibition of cell division. Northern analysis of RNA isolated from cells after cessation of growth showed the accumulation of fragments of 16S and 23S rRNA, but not of 5S rRNA. Essentially no rRNA fragments were detected when the double mutant strain was grown at 31°C or when the PNP ts single mutant was grown at 42°C. These data indicate that PNPase and RNase R are required for removal of nonfunctional rRNA fragments, and they suggest that accumulation of these fragments disrupts ribosome maturation and ultimately leads to cell death. (Abstract shortened by UMI.)

Keywords

Biology, Molecular; Biology, Cell

Link to Full Text

http://access.library.miami.edu/login?url=http://gateway.proquest.com/openurl?url_ver=Z39.88-2004&rft_val_fmt=info:ofi/fmt:kev:mtx:dissertation&res_dat=xri:pqdiss&rft_dat=xri:pqdiss:3071255