Structural analysis and in vitro processing to p5 rRNA of a 9S RNA molecule isolated from an rne mutant of E. coli

Ghora, Basanta K.; Apirion, David

doi:10.1016/0092-8674(78)90289-1

Cited by 238 publications

(190 citation statements)

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“…Moreover, it has been shown in vitro that RNaseE preferentially cleaves RNAs that have a 5Ј-monophosphate group (24). As primary transcripts in E. coli have a triphosphate group at their 5Ј end and the RNaseE-catalyzed hydrolysis generates downstream intermediates that have a 5Ј-monophosphate group (9), the ability of RNaseE to assess the phosphorylation status of 5Ј ends may ensure that after this enzyme has made the first cut in a primary transcript, it will tend to cut any sites downstream before starting on another intact transcript. The N-terminal half of RNaseE is sufficient to discriminate substrates that have a monophosphate group at the 5Ј end (25,26).…”

Section: Panel A)mentioning

confidence: 99%

“…4 -6) and the correct processing of precursors of, for example, ribosomal and transfer RNAs (7,8). RNaseE generates products terminating in a 3Ј-hydroxyl and a 5Ј-phosphate (9) indicating that the reaction involves the attack of water on the susceptible phosphodiester bond followed by scission of 3Ј-oxygen-phosphorus bond. The RNaseE polypeptide consists of 1061 residues (Fig.…”

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confidence: 99%

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Determination of the Catalytic Parameters of the N-terminal Half of Escherichia coli Ribonuclease E and the Identification of Critical Functional Groups in RNA Substrates

Redko

Tock

Adams

et al. 2003

Journal of Biological Chemistry

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Ribonuclease E is required for the rapid decay and correct processing of RNA in Escherichia coli. A detailed understanding of the hydrolysis of RNA by this and related enzymes will require the integration of structural and molecular data with quantitative measurements of RNA hydrolysis. Therefore, an assay for RNaseE that can be set up to have relatively high throughput while being sensitive and quantitative will be advantageous. Here we describe such an assay, which is based on the automated high pressure liquid chromatography analysis of fluorescently labeled RNA samples. We have used this assay to optimize reaction conditions, to determine for the first time the catalytic parameters for a polypeptide of RNaseE, and to investigate the RNaseE-catalyzed reaction through the modification of functional groups within an RNA substrate. We find that catalysis is dependent on both protonated and unprotonated functional groups and that the recognition of a guanosine sequence determinant that is upstream of the scissile bond appears to consist of interactions with the exocyclic 2-amino group, the 7N of the nucleobase and the imino proton or 6-keto group. Additionally, we find that a ribose-like sugar conformation is preferred in the 5 -nucleotide of the scissile phosphodiester bond and that a 2 -hydroxyl group proton is not essential. Steric bulk at the 2 position in the 5 -nucleotide appears to be inhibitory to the reaction. Combined, these observations establish a foundation for the functional interpretation of a three-dimensional structure of the catalytic domain of RNaseE when solved.RNaseE is a single-stranded-specific endoribonuclease (1-3) that is required for the rapid decay of many if not most transcripts in Escherichia coli (for reviews, see Refs. 4 -6) and the correct processing of precursors of, for example, ribosomal and transfer RNAs (7,8). RNaseE generates products terminating in a 3Ј-hydroxyl and a 5Ј-phosphate (9) indicating that the reaction involves the attack of water on the susceptible phosphodiester bond followed by scission of 3Ј-oxygen-phosphorus bond. The RNaseE polypeptide consists of 1061 residues (Fig.

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Section: Panel A)mentioning

confidence: 99%

mentioning

confidence: 99%

Determination of the Catalytic Parameters of the N-terminal Half of Escherichia coli Ribonuclease E and the Identification of Critical Functional Groups in RNA Substrates

Redko

Tock

Adams

et al. 2003

Journal of Biological Chemistry

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“…While me is an essential gene (Gegenheimer eta/., 1977;Ghora and Apirion, 1978), its overexpression can interfere with cell growth and viability. Cloning of me on a highcopy-number plasmid increases cell doubling time and can lead to plasmid loss or the acquisition of mutations that reduce RNase E activity (Claverie-Martin et a/., 1991).…”

Section: Rne Proteinmentioning

confidence: 99%

“…(1977), and a temperature-sensitive (ts) mutant that failed to produce p5S RNA and accumulated the 9 s RNA precursor was identified soon afterwards (Apirion and Lassar, 1978;Ghora and Apirion, 1978). Extracts from these mutant bacteria contained a thermolabile enzyme that cleaved 9s RNA in vifro at two sites (Ghora and Apirion, 1978;Misra and Apirion, 1979); this endonuclease was named RNase E and the gene affecting the activity of the enzyme was designated me. Nevertheless, the observation that not all RNA molecules accumulating in me'' bacteria after a shift to elevated temperature are substrates for RNase E in vitro (Ray and Apirion, 1981) and the finding that mutations in the rne gene also alter the activities of other E. coli ribonucleases in vivo (Gurevitz eta/., 1982) led to speculation that rne may affect RNase E activity indirectly.…”

Section: Introductionmentioning

confidence: 99%

RNase E: still a wonderfully mysterious enzyme

Cohen

McDowall

1997

Molecular Microbiology

130

106

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SummaryRibonuclease E (RNase E), which is encoded by an essential Escherichia coli gene known variously as me, ams, and hmp, was discovered initially as an rRNA-processing enzyme but is now known to have a general role in RNA decay. Multiple functions, including the ability t o cleave RNA endonucleolytically in AU-rich single-strand regions, RNA-binding capabilities, and the ability to interact with polynucleotide phosphorylase and other proteins implicated in the processing and degradation of RNA, are encoded by its 1061 amino acid residues. The presence of homologues and functional analogues of the m e gene in a variety of prokaryotic and eukaryotic species suggests that its functions have been highly conserved during evolution. While much has been learned in recent years about the structure and functions of RNase E, there is continuing mystery about possible additional activities and molecular interactions of this enzyme.

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“…The RNA degradosome is required for the normal maturation of transfer and ribosomal RNA and for degradation of most messenger RNAs (16)(17)(18). In degradosome-dependent mRNA decay, RhlB facilitates the degradation of structured RNA, and RNaseE provides the endoribonuclease activity that cuts the RNA into fragments that are further degraded by the 3Ј35Ј exoribonuclease activity of PNPase (reviewed in ref.…”

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confidence: 99%

RNaseE and the other constituents of the RNA degradosome are components of the bacterial cytoskeleton

Taghbalout

Rothfield

2007

Proc. Natl. Acad. Sci. U.S.A.

114

115

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RNaseE is the main component of the RNA degradosome of Escherichia coli, which plays an essential role in RNA processing and decay. Localization studies showed that RNaseE and the other known degradosome components (RNA helicase B, polynucleotide phosphorylase, and enolase) are organized as helical filamentous structures that coil around the length of the cell. These resemble the helical structures formed by the MreB and MinD cytoskeletal proteins. Formation of the RNaseE cytoskeletal-like structure requires an internal domain of the protein that does not include the domains required for any of its known interactions or the minimal domain required for endonuclease activity. We conclude that the constituents of the RNA degradosome are components of the E. coli cytoskeleton, either assembled as a primary cytoskeletal structure or secondarily associated with another underlying cytoskeletal element. This suggests a previously unrecognized role for the bacterial cytoskeleton, providing a mechanism to compartmentalize proteins that act on cytoplasmic components, as exemplified by the RNA processing and degradative activities of the degradosome, to regulate their access to important cellular substrates.RNA processing ͉ PNPase ͉ enolase ͉ RNA helicase

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Structural analysis and in vitro processing to p5 rRNA of a 9S RNA molecule isolated from an rne mutant of E. coli

Cited by 238 publications

References 31 publications

Determination of the Catalytic Parameters of the N-terminal Half of Escherichia coli Ribonuclease E and the Identification of Critical Functional Groups in RNA Substrates

Determination of the Catalytic Parameters of the N-terminal Half of Escherichia coli Ribonuclease E and the Identification of Critical Functional Groups in RNA Substrates

RNase E: still a wonderfully mysterious enzyme

RNaseE and the other constituents of the RNA degradosome are components of the bacterial cytoskeleton

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