The CafA Protein Required for the 5′-Maturation of 16 S rRNA Is a 5′-End-dependent Ribonuclease That Has Context-dependent Broad Sequence Specificity

Tock, Mark R.; Walsh, A. P.; Carroll, Gregory T.; McDowall, Kenneth J.

doi:10.1074/jbc.275.12.8726

Cited by 139 publications

(180 citation statements)

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“…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). There is evidence for "5Ј end dependence" in vivo and it has been suggested that this facet of RNaseE-catalyzed cleavage may contribute to the finding that RNA decay occurs in general without the production of significant levels of intermediates in E. coli (27).…”

Section: Panel A)mentioning

confidence: 96%

“…The mechanism by which the b site within the context of a 9 S transcript is cleaved by RNaseE despite the absence of an upstream G remains to be determined. It is possible that the recruitment of RNaseE is regulated by some secondary structural element(s) (17) as suggested relatively early in the study of RNaseE (1,30) or the distance between susceptible sequences and a monophosphate at the 5Ј end (25). From the above, it can be seen that an understanding of the recognition and hydrolysis of RNA by RNaseE will require a systematic analysis of the contribution of RNA sequence and structure, including the phosphorylation status at the 5Ј end, the multiple domains within the C-terminal half of RNaseE, and the effects of other degradosome component.…”

Section: Panel A)mentioning

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: 96%

Section: Panel A)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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“…In view of the homology of RNase G to the catalytic domain of RNase E, it is not surprising that they have a similar cleavage site specificity, with both preferring to cut RNA within singlestranded regions that are AU-rich (8,9). Another important property shared by these two endonucleases is their preference for RNA substrates that bear a single phosphate group at an unpaired 5Ј end.…”

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confidence: 99%

“…Another important property shared by these two endonucleases is their preference for RNA substrates that bear a single phosphate group at an unpaired 5Ј end. Remarkably, such substrates are cleaved by full-length RNase E, N-RNase E, or RNase G more than an order of magnitude faster than otherwise identical RNAs bearing a 5Ј-terminal triphosphate or hydroxyl group, even though cleavage may occur far from the 5Ј end (9)(10)(11). The greater susceptibility of 5Ј-monophosphorylated RNAs to cleavage by these enzymes may help to ensure the rapid degradation of the downstream products of initial endonucleolyic cleavage, which differ from their primary-transcript precursors in being 5Ј-monophosphorylated rather than 5Ј-triphosphorylated.…”

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Catalytic activation of multimeric RNase E and RNase G by 5′-monophosphorylated RNA

Jiang

Belasco

2004

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

112

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RNase E is an endonuclease that plays a central role in RNA processing and degradation in Escherichia coli. Like its E. coli homolog RNase G, RNase E shows a marked preference for cleaving RNAs that bear a monophosphate, rather than a triphosphate or hydroxyl, at the 5 end. To investigate the mechanism by which 5 -terminal phosphorylation can influence distant cleavage events, we have developed fluorogenic RNA substrates that allow the activity of RNase E and RNase G to be quantified much more accurately and easily than before. Kinetic analysis of the cleavage of these substrates by RNase E and RNase G has revealed that 5 monophosphorylation accelerates the reaction not by improving substrate binding, but rather by enhancing the catalytic potency of these ribonucleases. Furthermore, the presence of a 5 monophosphate can increase the specificity of cleavage site selection within an RNA. Although monomeric forms of RNase E and RNase G can cut RNA, the ability of these enzymes to discriminate between RNA substrates on the basis of their 5 phosphorylation state requires the formation of protein multimers. Among the molecular mechanisms that could account for these properties are those in which 5 -end binding by one enzyme subunit induces a protein structural change that accelerates RNA cleavage by another subunit.M essenger RNAs are labile molecules whose longevity directly influences the synthesis rates of the proteins they encode. Despite the importance of mRNA degradation for gene expression, little is understood about the molecular mechanisms that govern differences in mRNA stability, even in so well studied an organism as Escherichia coli. Elucidating these mechanisms is crucial for explaining how RNA sequence and structure control mRNA lifetimes.The degradation of most E. coli mRNAs is thought to begin with internal cleavage by the endonuclease RNase E (1), although in some cases decay begins instead with cleavage by RNase III or RNase G (an RNase E homolog) (2, 3). The resulting mRNA fragments are then degraded to mononucleotides by a combination of further endonucleolytic cleavage and 3Ј exonucleolytic digestion.RNase E is a 1,061-aa protein comprising functionally distinct domains. The catalytically active amino-terminal half of the protein (N-RNase E: residues 1-498) is alone sufficient for the enzyme's ribonuclease activity, whereas the carboxyl half of the protein (residues 499-1061) contains both an arginine-rich region and a carboxy-terminal domain that serves as a scaffold for the assembly of a multiprotein complex known as the RNA degradosome (4, 5). By contrast, RNase G, which comprises 489 amino acid residues, is similar in sequence to the amino half of RNase E but lacks an arginine-rich region and a scaffold domain (6, 7).In view of the homology of RNase G to the catalytic domain of RNase E, it is not surprising that they have a similar cleavage site specificity, with both preferring to cut RNA within singlestranded regions that are AU-rich (8, 9). Another important property shared by these two en...

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“…The 5′ end of substrate mRNA dramatically affects its ability to be cleaved by RNase E, with the rate constant for 5′ monophosphate RNA more than an order of magnitude greater than that for RNA with a 5′ triphosphate or 5′ hydroxyl 3,4 . The basis for this 5′ end selectivity became clear from the crystal structure of the homotetrameric catalytic domain 5 , which showed the 5′ monophosphate end is hydrogen bonded into a pocket at the base of an RNA-binding channel.…”

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confidence: 99%

The end defines the means in bacterial mRNA decay

Schoenberg

2007

Nat Chem Biol

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Bacterial mRNAs begin with a triphosphate on the first transcribed nucleotide, but the endonuclease long thought to initiate mRNA decay in E. coli (RNase E) only works well on RNA with a 5′ monophosphate. The first committed step in the degradation of mRNA in that organism now appears to be the conversion of the 5′ triphosphate to a monophosphate, a process that is functionally similar to mRNA decapping in eukaryotes.The 5′ end of newly synthesized RNAs bears a triphosphate derived from the first transcribed nucleotide. In eukaryotes the distal phosphate is replaced by an inverted, methylated GMP to form the m 7 GpppX-cap; however, no such modification occurs in prokaryotes. The hydrolysis of the cap to a 5′ monophosphate is the first committed step in eukaryotic mRNA decay, and a paper by Celesnik et al. 1 that appeared in the July 6, 2007 issue of Molecular Cell shows that a remarkably similar process occurs in E. coli.In eukaryotes the process of mRNA decay generally involves loss of the 3′ poly(A) tail and removal of the cap by a decapping enzyme (Dcp2). The body of the mRNA is then degraded from the 5′ end by the 5′-3′ exonuclease Xrn1 and from the 3′ end by the exosome 2 . Prokaryotes such as E. coli use a distinctly different process in which mRNA is cleaved by the endonuclease RNase E to generate an upstream product with a 3′ hydroxyl and a downstream product with a 5′ monophosphate. The upstream product is rapidly cleared by 3′-5′ exonucleases and the 5′ monophosphate end of each downstream cleavage product activates subsequent rounds of endonuclease cleavage and exonuclease clearance (see bottom of Fig. 1). Thus, on the surface bacterial mRNA decay appears to be distinctly different from the major decay process in eukaryotes.The 5′ end of substrate mRNA dramatically affects its ability to be cleaved by RNase E, with the rate constant for 5′ monophosphate RNA more than an order of magnitude greater than that for RNA with a 5′ triphosphate or 5′ hydroxyl 3,4 . The basis for this 5′ end selectivity became clear from the crystal structure of the homotetrameric catalytic domain 5 , which showed the 5′ monophosphate end is hydrogen bonded into a pocket at the base of an RNA-binding channel. This interaction is thought to cause an allosteric change in the protein that juxtaposes a magnesium-coordinated hydroxyl with the scissile phosphate at a downstream site in the RNA substrate.Given that the 5′ end of bacterial mRNA is a 5′ triphosphate Celesnik et al. 1 asked whether in E. coli there might be a previously unidentified step that triggers RNase E cleavage by converting the 5′ terminus to a monophosphate. To address this they developed a splinted ligation assay in which an antisense DNA oligonucleotide that extends past the RNA 5′ end is used to position another oligonucleotide (oligo X) adjacent to 5′ most nucleotide. DNA ligase will covalently join oligo X to the 5′ end of the RNA if it has a 5′ monophosphate, but not a 5′ hydroxl or 5′ triphosphate. By carefully optimizing reaction conditions and ...

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The CafA Protein Required for the 5′-Maturation of 16 S rRNA Is a 5′-End-dependent Ribonuclease That Has Context-dependent Broad Sequence Specificity

Cited by 139 publications

References 53 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

Catalytic activation of multimeric RNase E and RNase G by 5′-monophosphorylated RNA

The end defines the means in bacterial mRNA decay

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