Additional functional roles of {\it Escherichia coli\/} polynucleotide phosphorylase

Date of Completion

January 1997


Biology, Molecular|Biology, Genetics|Biology, Microbiology




Escherichia coli polynucleotide phosphorylase (PNPase) is generally believed to be involved in mRNA degradation via its 3$\sp\prime$ to 5$\sp\prime$ exoribonuclease activity. However, there are many unexplained observations related to the physiological role of this enzyme. The projects conducted in this thesis are aimed at understanding more about the in vivo function of PNPase. Two approaches were employed to try to achieve this goal. First, a biochemical approach involving the purification of PNPase associated proteins was used. Specifically, the previously documented $\beta$ subunit present in the pentameric form of PNPase was examined as an initial step toward understanding the mechanism and possibly the regulation of PNPase catalysis. Second, a genetic approach was undertaken which focused on characterization of mutant strains lacking PNPase in combination with other known enzymes involved in RNA metabolism, in particular, tRNA nucleotidyltransferase, poly(A) polymerase and RNase PH.^ From the purification of the $\beta$ subunit and the comparisons of the high and low molecular weight forms of PNPase in some of their catalytic properties, it was concluded that the $\beta$ subunit does not alter the biochemical activity of the $\alpha$ subunit. This result is consistent with the recent finding that the $\beta$ subunit is actually enolase.^ From analyses of mutant strains lacking PNPase, poly(A) polymerase and tRNA nucleotidyltransferase in various combinations, it was concluded that these three enzymes are involved in some common pathway in vivo, most likely the 3$\sp\prime$ end repair of tRNAs. Exactly how PNPase contributes to end repair is not completely understood.^ The detailed characterization of RNase PH$\sp-,$ PNPase$\sp-$ double mutant strain revealed an important functional aspect of these two Pi-dependent exoribonucleases in E. coli, i.e., they are required for normal ribosome biogenesis. tRNA processing is apparently normal in these cells despite the fact that the function of suppressor tRNA su3 is greatly reduced. The severe cold-sensitive growth and the abnormal ribosomal subunit profiles suggest a defect in ribosome assembly. The role of PNPase and RNase PH may not lie in the synthesis of a ribosome component, but rather in the expression of a chaperonin factor important for ribosome assembly. ^