Escherichia coli exoribonuclease T: Structure, function and mechanism of action

Date of Award




Degree Name

Doctor of Philosophy (Ph.D.)


Biochemistry and Molecular Biology

First Committee Member

Murray P. Deutscher, Committee Chair


Exoribonuclease T (RNase T) is one of eight distinct 3 ' to 5' exoribonucleases present in Escherichia coli. It plays an important role in stable RNA metabolism, including tRNA end turnover and 3' maturation of many stable RNAs. Although the substrates of RNase T in vivo share a common sequence feature consisting of a stable, double-stranded (ds) stem followed by a few unpaired 3' nucleotides, the enzyme was found to be a single strand-specific exoribonuclease that acts in the 3' to 5' direction in a non-processive manner. While other E. coli exoribonucleases stop several nucleotides downstream of an RNA duplex, RNase T can digest RNA up to the first base pair. The presence of a free 3'-hydroxyl group is required for the enzyme to initiate digestion. Studies with a variety of oligoribonucleotides revealed that RNase T discriminates against the removal of C residues. Although RNase T appears to bind as many as 10 nt of single-stranded RNA, its specificity is defined largely by the last four nucleotides. These findings explain many of the properties of RNase T during maturation of stable RNAs.RNase T was also found to have single strand-specific DNase activity. In contrast to its action on RNA, RNase T binds single-stranded (ss) DNA much more tightly and shows less sequence specificity. As with RNA, DNA secondary structure strongly affects its degradation by RNase T. Thus, RNase T action on a dsDNA with a single-stranded 3' extension efficiently generates blunt-ended DNA. This property of RNase T was applied to blunt-ended DNA cloning and provided much higher cloning efficiency than the currently used mung bean nuclease.As part of this study, we analyzed the structure and phylogenetic distribution of the known exoribonucleases. All of the exoribonucleases and their homologs have been grouped into six superfamilies. RNase T belongs to the DEDD exonuclease superfamily, characterized by four invariant acidic residues embedded within three highly conserved sequence motifs, which in DNA polymerases were shown to form the exonuclease active site. Mutagenesis of these motifs revealed that they are essential for RNase T activity, indicating that they likely form the RNase T catalytic center in a manner similar to that found in other DEDD exonucleases. We also identified by sequence analysis three short, but highly conserved, sequence segments (NBS) rich in positively-charged residues, which cluster to form a positively-charged surface patch based on structure modeling. Site-directed mutagenesis of these regions indicates that they are involved in substrate-binding. (Abstract shortened by UMI.)


Biology, Molecular; Chemistry, Biochemistry

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