Characterization of the Functional Domains of Escherichia coli RNase II

Amblar, Mónica; Barbas, Ana; Fialho, Arsénio M.; Arraiano, Cecília M.

doi:10.1016/j.jmb.2006.05.043

Cited by 68 publications

(97 citation statements)

References 52 publications

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“…This shows that the enzyme stalls 7-9 nt before reaching the double-stranded region (30,42) (Fig. 3).…”

Section: Comparing the Exoribonucleolytic Activity Of The Different Mmentioning

confidence: 86%

“…Purification of all proteins was performed by histidine affinity chromatography using HiTrap Chelating HP columns (GE Healthcare) and the AKTA fast protein liquid chromatography system (GE Healthcare) following the protocol previously described (30,31). Briefly, cell suspensions were lysed using a French press at 9000 p.s.i.…”

Section: Methodsmentioning

confidence: 99%

“…Activity Assays-Exoribonucleolytic activity was assayed using different RNA oligoribonucleotides as substrates (30): a poly(A) chain of 35 nt as a single-stranded substrate, a 30-mer oligoribonucleotide (5Ј-CCCGACACCAACCACUAAAAAA-AAAAAAAA-3Ј) annealed to the complementary 16-mer oligodeoxyribonucleotide (5Ј-AGTGGTTGGTGTCGGG-3Ј) as a double-stranded substrate with a 3Ј-single-stranded extension, and the DNA-RNA chimeric substrates Chi1 (5Ј-dTdTdTdTdTdTdTdTdTdTdTrCrCdTdT-3Ј) and Chi2 (5Ј-dTdTdT dTdTdTdTdTdTdTdTrCdTrCdT-3Ј). All the oligoribonucleotides were labeled at the 5Ј-end with [␥- 32 ATP] and T4 polynucleotide kinase and further purified with Microcon YM-3 Centrifugal Filter Devices (Millipore) to remove the non-incorporated nucleotides.…”

Section: Methodsmentioning

confidence: 99%

“…When the substrate tested was a double-stranded molecule with a 3Ј-single-stranded extension (16 -30ds), the wild-type enzyme degraded the single-stranded portion, generating 23-25-nt oligomers (30). This shows that the enzyme stalls 7-9 nt before reaching the double-stranded region (30,42) (Fig.…”

Section: Comparing the Exoribonucleolytic Activity Of The Different Mmentioning

confidence: 97%

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Determination of Key Residues for Catalysis and RNA Cleavage Specificity

Barbas

Matos

Amblar

et al. 2009

Journal of Biological Chemistry

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RNase II is the prototype of a ubiquitous family of enzymes that are crucial for RNA metabolism. In Escherichia coli this protein is a single-stranded-specific 3-exoribonuclease with a modular organization of four functional domains. In eukaryotes, the RNase II homologue Rrp44 (also known as Dis3) is the catalytic subunit of the exosome, an exoribonuclease complex essential for RNA processing and decay. In this work we have performed a functional characterization of several highly conserved residues located in the RNase II catalytic domain to address their precise role in the RNase II activity. We have constructed a number of RNase II mutants and compared their activity and RNA binding to the wild type using different singleor double-stranded substrates. The results presented in this study substantially improve the RNase II model for RNA degradation. We have identified the residues that are responsible for the discrimination of cleavage of RNA versus DNA. We also show that the Arg-500 residue present in the RNase II active site is crucial for activity but not for RNA binding. The most prominent finding presented is the extraordinary catalysis observed in the E542A mutant that turns RNase II into a "super-enzyme."Exoribonuclease II (RNase II) 5 is one of the major exoribonucleases involved in the degradation of RNA (1). This protein degrades RNA hydrolytically in the 3Ј to 5Ј direction, releasing 5Ј-nucleotide monophosphates in a processive manner. Its activity is sequence independent but sensitive to secondary structures, and poly(A) is the preferential substrate for this enzyme (2-6). In Escherichia coli RNase II is responsible for 90% of the hydrolytic activity in crude extracts (7). Moreover, its expression is differentially regulated at the transcriptional and post-transcriptional levels (8 -10), and the protein can be regulated by environmental conditions (10). The E. coli enzyme is the prototype of a widespread family of exoribonucleases, and RNase II homologues can be found in prokaryotes and eukaryotes (11). In the nucleus and in the cytoplasm of eukaryotic cells, RNase II homologues (Rrp44/Dis3) are part of the exosome, an essential multiprotein complex of exoribonucleases involved in processing, turnover, and quality control of different types of RNAs (12). Rrp44 is the only catalytically active nuclease in the human and yeast core exosome (13,14). Moreover, it was recently demonstrated that apart from the RNB catalytic domain, Rrp44p has a second nuclease domain, the PINc domain, that confers endonucleolytic activity to the protein (15, 16). In prokaryotic cells, apart from RNase II, there is an additional RNase II-like enzyme, RNase R, that is involved in RNA degradation and RNA and protein quality control (17)(18)(19)(20). RNase R has also been shown to be required for virulence (21).The determination of E. coli RNase II structure (22-24) showed that, contrary to the sequence predictions, RNase II consisted of four domains; they are two N-terminal cold shock domains (CSD1 and CSD2) involved in RNA b...

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“…This shows that the enzyme stalls 7-9 nt before reaching the double-stranded region (30,42) (Fig. 3).…”

Section: Comparing the Exoribonucleolytic Activity Of The Different Mmentioning

confidence: 86%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Comparing the Exoribonucleolytic Activity Of The Different Mmentioning

confidence: 97%

See 2 more Smart Citations

Determination of Key Residues for Catalysis and RNA Cleavage Specificity

Barbas

Matos

Amblar

et al. 2009

Journal of Biological Chemistry

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“…RNase II is synthesized by both the sporozoite and merozoite stages of the parasite [15]. There are orthologs predicted among the sequences of Plasmodium species and Escherichia coli where the enzyme activity was originally described [35]. However, in silico analysis revealed a sequence similarity of the following amino acid residues, HFTSPIRRYPD at different positions on Plasmodium species and E. coli.…”

Section: Bioinformatic Analysis Of Pf3d7_0906000mentioning

confidence: 99%

In Silico Characterization of Plasmodium falciparum and P. yoelii Translocon and Exoribonuclease II (RNase II) Identified in the Merozoite Rhoptry Proteome

Timta¹,

Yadavalli²,

Sam‐Yellowe³

2015

J Proteomics Bioinform

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Identification of temperature‐sensitive mutations and characterization of thermolabile RNase II variants

et al. 2018

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The RNase II family of ribonucleases is ubiquitous and critical for RNA metabolism. The rnb500 allele has been widely used for over 30 years; however, the underlying genetic changes which result in RNase II thermolabile activity remain unknown. Here, we combine molecular and biophysical studies to carry out an in vivo and in vitro investigation of RNase II mutation(s) that confer the rnb500 phenotype. Our findings indicate that RNase II thermolability is due to the Cys284Tyr mutation within the RNB domain, which abolishes activity by increasing protein kinetic instability at the nonpermissive temperature. These findings have important implications for the design of temperature‐sensitive variants of other RNase II enzymes, namely those with yet unknown functions.

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Characterization of the Functional Domains of Escherichia coli RNase II

Cited by 68 publications

References 52 publications

Determination of Key Residues for Catalysis and RNA Cleavage Specificity

Determination of Key Residues for Catalysis and RNA Cleavage Specificity

In Silico Characterization of Plasmodium falciparum and P. yoelii Translocon and Exoribonuclease II (RNase II) Identified in the Merozoite Rhoptry Proteome

Identification of temperature‐sensitive mutations and characterization of thermolabile RNase II variants

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