Poly(A)- and poly(U)-specific RNA 3′ tail shortening by E. coli ribonuclease E

Huang, Hong; Liao, Jian; Cohen, Stanley N.

doi:10.1038/34219

Cited by 81 publications

(78 citation statements)

References 27 publications

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“…The experimental design and substrates we used were intended to elucidate the inherent mode of cleavage-site selection by the RNase E catalytic domain in the absence of such modifying and͞or confounding factors. Consistent with the 3Ј to 5Ј directionality of cleavages we observed for short oligonucleotide substrates containing multiple identical cleavage sites is kinetic evidence showing that removal of poly(A) tails from RNAI precedes, rather than follows, cleavage of a site near the 5Ј end of this 108-nt-long natural substrate (11).…”

Section: Discussionsupporting

confidence: 59%

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The catalytic domain of RNase E shows inherent 3′ to 5′ directionality in cleavage site selection

Yin

Vickers

Cohen

2002

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

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RNase E, a multifunctional endoribonuclease of Escherichia coli, attacks substrates at highly specific sites. By using synthetic oligoribonucleotides containing repeats of identical target sequences protected from cleavage by 2 -O-methylated nucleotide substitutions at specific positions, we investigated how RNase E identifies its cleavage sites. We found that the RNase E catalytic domain (i.e., N-Rne) binds selectively to 5 -monophosphate RNA termini but has an inherent mode of cleavage in the 3 to 5 direction. Target sequences made uncleavable by the introduction of 2 -O-methylmodified nucleotides bind to RNase E and impede cleavages at normally susceptible sites located 5 to, but not 3 to, the protected target. Our results indicate that RNase E can identify cleavage sites by a 3 to 5 ''scanning'' mechanism and imply that anchoring of the enzyme to the 5 -monophosphorylated end of these substrates orients the enzyme for directional cleavages that occur in a processive or quasiprocessive mode. In contrast, we find that RNase G, which has extensive structural homology with and size similarity to N-Rne, and can functionally complement RNase E gene deletions when overexpressed, has a nondirectional and distributive mode of action.has a demonstrated role in the processing of ribosomal RNA (1, 2), the chemical degradation of bulk cellular RNA (3-7), the decay of specific regulatory, messenger, and structural RNAs (for recent reviews, see refs. 8 and 9), the control of plasmid DNA replication (10), and the removal of poly(A) tails from transcripts (11,12). RNase E cleaves preferentially at specific sites in single-strand RNA segments rich in AϩU nucleotides (13)(14)(15)(16)). An inherent mode of action involving entry of RNase E at the 5Ј end of substrates followed by a 5Ј to 3Ј wave of cleavages has been inferred from the greater in vivo stability of fragments at the 3Ј ends of certain RNA substrates (17-20) together with evidence that (i) the enzyme prefers a free 5Ј end for endonucleolytic cleavage (18,20), (ii) RNase E degradation efficiency is affected by 5Ј-phosphorylation in vivo (10) and in vitro (19,20), and (iii) 5Ј regions of secondary structure can impede cleavages in vivo (21). However, RNA degradation in vivo also can occur in the opposite direction (e.g., ref. 22).As RNase E cleavage of complex substrates potentially can be affected by differences in the affinity of the enzyme for target sites having different sequences (15, 16), by secondary structure (23), and in vivo also by the attachment of ribosomes (24-26) and͞or RNA-binding proteins (27), conclusions about the inherent mode of action of RNase E from studies of long natural substrates may be problematical. We wished to elucidate the mechanism of target-site selection by RNase E in the absence of these confounding factors; to do this, we designed, and chemically synthesized, oligoribonucleotide substrates that contain repeats of identical target sequences and are devoid of regions able to engage in base paring. We found that the RNase E catalytic...

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Section: Discussionsupporting

confidence: 59%

“…8 and 9), the control of plasmid DNA replication (10), and the removal of poly(A) tails from transcripts (11,12). RNase E cleaves preferentially at specific sites in single-strand RNA segments rich in AϩU nucleotides (13)(14)(15)(16)).…”

mentioning

confidence: 99%

The catalytic domain of RNase E shows inherent 3′ to 5′ directionality in cleavage site selection

Yin

Vickers

Cohen

2002

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

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“…The functional groups in additional sequence determinants that have recently been shown to affect the efficiency of cleavage by E. coli RNaseE (54) can be characterized using the approach described here. It is now clearly established experimentally that RNaseE is sensitive to the presence of specific nucleotides at multiple positions relative to the site of cleavage even though it can hydrolyze substrates with low sequence complexity such as oligomeric adenosine and uracil (36,55). In addition to sequence, our analysis of substitutions of the 2Ј-OH on the ribose sugar directly 5Ј to the phosphodiester bond that is normally cleaved by RNaseE suggests that a ribose-like sugar pucker is important for efficient hydrolysis.…”

Section: Fig 5 the Bases Introduced At Nucleotide Position 4 In Br10mentioning

confidence: 98%

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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“…1B). RNase E also exhibits an unusual cleavage activity on RNA substrates containing a 3 poly(A) or poly(U) tail (10), although the significance of this activity is not yet clear.…”

Section: Initiation Of Degradationmentioning

confidence: 99%

Degradation of mRNA in Escherichia coli

Jain

2002

IUBMB Life

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SummaryDegradation of messenger RNAs (mRNAs) is a universal process that occurs in every cell and has important implications for nucleotide metabolism and gene expression. One organism in which mRNA degradation has been thoroughly studied is the bacterium Escherichia coli (E. coli). In this review I describe what is presently known about the different processes involved in the conversion of mRNAs from high molecular weight species to mononucleotides in E. coli. The ribonucleases and accessory factors involved in mRNA degradation, and features on mRNAs that make them resistant or sensitive to degradation will also be described. At the conclusion of this review, some of the anticipated directions of future research on this topic will be discussed.

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Poly(A)- and poly(U)-specific RNA 3′ tail shortening by E. coli ribonuclease E

Cited by 81 publications

References 27 publications

The catalytic domain of RNase E shows inherent 3′ to 5′ directionality in cleavage site selection

The catalytic domain of RNase E shows inherent 3′ to 5′ directionality in cleavage site selection

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

Degradation of mRNA in Escherichia coli

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