Mechanism of Action of RNase T

Zuo, Y.; Deutscher, Murray P.

doi:10.1074/jbc.m207706200

Cited by 32 publications

(23 citation statements)

References 20 publications

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“…In E. coli RNase T, as well as all other DEDDh members, such as DP3E, DNA exonuclease I and oligoribonuclease, a conserved His (His 181 ) is present at the adjacent position in the sequence. This residue also is required for RNase T catalysis (8). In the tertiary structure modeled here, His 181 sits at a position similar to that of Tyr 497 in the Klenow fragment ( Fig.…”

Section: Homologymentioning

confidence: 99%

“…The bacterial strains and plasmids used here are as described in the companion study (8). Likewise, all the other reagents used in the experiments reported here have been described elsewhere (8).…”

Section: Materials-purified Escherichia Colimentioning

confidence: 99%

“…Purified mutant C168S RNase T was kindly provided by Dr. Zhongwei Li and was overexpressed and purified from E. coli cells as described previously (10). The bacterial strains and plasmids used here are as described in the companion study (8). Likewise, all the other reagents used in the experiments reported here have been described elsewhere (8).…”

Section: Materials-purified Escherichia Colimentioning

confidence: 99%

“…Methods-Assays for RNase T activity and recombinant DNA procedures were all carried out as described in the accompanying article (8).…”

Section: Materials-purified Escherichia Colimentioning

confidence: 99%

“…The enzyme forms a homodimer in vivo, and formation of the homodimer is required for it to function (7). In the companion article (8), RNase T was examined by sequence analysis and site-directed mutagenesis to identify regions of the protein important for catalysis, substrate binding, and dimerization. We showed first that the DEDD signature motifs probably form the RNase T catalytic center.…”

mentioning

confidence: 99%

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Mechanism of Action of RNase T

Zuo

Deutscher

2002

Journal of Biological Chemistry

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A detailed structural and functional model of E. coli RNase T was generated based on sequence analysis, homology modeling, and experimental observation. In the accompanying article, three short sequence segments (nucleic acid binding sequences (NBS)) important for RNase T substrate binding were identified. In the model, these segments cluster to form a positively charged surface patch. However, this patch is on the face of the RNase T monomer opposite the DEDD catalytic center. We propose that by dimerization, the NBS patch from one subunit is brought to the vicinity of the DEDD center of the second monomer to form a fully functional RNase T active site. In support of this model, mutagenetic studies show that one NBS1 residue, Arg 13 , sits at the catalytic center despite being on the opposite side of the monomer. Second, the complementarity of the RNase T subunits through the formation of homodimers was demonstrated by reconstitution of partial RNase T activity from monomers derived from two inactive mutant proteins, one defective in catalysis and one in substrate binding. These data explain why RNase T must dimerize to function. The model provides a detailed framework on which to explain the mechanism of action of RNase T.RNase T, a DEDD exonuclease, plays an important role in many aspects of stable RNA metabolism (1-6). The enzyme forms a homodimer in vivo, and formation of the homodimer is required for it to function (7). In the companion article (8), RNase T was examined by sequence analysis and site-directed mutagenesis to identify regions of the protein important for catalysis, substrate binding, and dimerization. We showed first that the DEDD signature motifs probably form the RNase T catalytic center. Second, sequence analysis identified several highly conserved, positively charged sequence segments, termed NBS1-3, 1 that are present in all RNase T orthologs, but absent from the closely related proofreading domains/subunits of DNA polymerases, that seem to be involved in the binding of nucleic acid substrates. Finally, we showed that residues at the C terminus of each monomer of RNase T are important for dimerization.Based on this information, additional experiments, and homology modeling, we present here a structural and functional model for RNase T. Based on this model, we show that the NBS segments cluster in the tertiary structure to form a positively charged surface patch suitable for nucleic acid binding. We also propose that the DEDD catalytic center and the NBS are on opposite sides of an RNase T monomer and are brought together to create a complete active site through dimerization. Direct evidence for the complementarity of the two RNase T subunits in the homodimer was obtained by reconstituting partial RNase T activity from two inactive mutants, one an NBS mutant defective in substrate binding and the other a DEDD mutant defective in catalysis. These results explain the requirement for dimerization to generate functional RNase T.

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Section: Homologymentioning

confidence: 99%

Section: Materials-purified Escherichia Colimentioning

confidence: 99%

Section: Materials-purified Escherichia Colimentioning

confidence: 99%

“…Methods-Assays for RNase T activity and recombinant DNA procedures were all carried out as described in the accompanying article (8).…”

Section: Materials-purified Escherichia Colimentioning

confidence: 99%

mentioning

confidence: 99%

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Mechanism of Action of RNase T

Zuo

Deutscher

2002

Journal of Biological Chemistry

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Structure and Degradation Mechanisms of 3′ to 5′ Exoribonucleases

Matos

Pobre

Reis

et al. 2011

Nucleic Acids and Molecular Biology

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Achieving Specific RNA Cleavage Activity by an Inactive Splicing Endonuclease Subunit Through Engineered Oligomerization

Calvin

2007

Journal of Molecular Biology

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Protein-protein interaction is a common strategy exploited by enzymes to control substrate specificity and catalytic activities. RNA endonucleases, which are involved in many RNA processing and regulation processes, are prime examples of this. How the activities of RNA endonucleases are tightly controlled such that they act on specific RNA is of general interest. We demonstrate in this study that an inactive RNA splicing endonuclease subunit can be switched "on" solely by oligomerization. Furthermore, we show that the mode of assembly correlates with different RNA specificities. The recently identified splicing endonuclease homolog from Sulfolobus solfataricus, despite possessing all of the putatively catalytic residues, has no detectable RNA cleavage activity on its own but is active upon mixing with its structural subunit. Guided by the previously determined three dimensional structure of the catalytic subunit, we altered its sequence such that it could potentially self-assemble thereby enabling its catalytic activity. We present the evidence for the specific RNA cleavage activity of the engineered catalytic subunit and for its formation of a functional tetramer. We also identify a higher order oligomer species that possesses distinct RNA cleavage specificity from that of previously characterized RNA splicing endonucleases.

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Mechanism of Action of RNase T

Cited by 32 publications

References 20 publications

Mechanism of Action of RNase T

Mechanism of Action of RNase T

Structure and Degradation Mechanisms of 3′ to 5′ Exoribonucleases

Achieving Specific RNA Cleavage Activity by an Inactive Splicing Endonuclease Subunit Through Engineered Oligomerization

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