In-Gel Analysis of Site-Specific RNases

Nilsson, Rune‐Pär; Gabain, Alexander von; Kaberdin, Vladimir R.

doi:10.2144/02332bm03

Cited by 4 publications

(3 citation statements)

References 1 publication

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“…Interestingly, RNase E mutants containing as few as 402 N-terminal amino acids retain RNA cleavage activity, albeit to a reduced degree as compared with longer forms of RNase E (Nilsson et al 2002). Three of the dominantnegative mutants (DNX21, DNX23, and DNX25) were Figure 6.-Model for the dominant-negative phenotype conferred by truncated RNase E variants.…”

Section: Discussionmentioning

confidence: 99%

Identification and Analysis of Escherichia coli Ribonuclease E Dominant-Negative Mutants

2006

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The Escherichia coli (E. coli) ribonuclease E protein (RNase E) is implicated in the degradation and processing of a large fraction of RNAs in the cell. To understand RNase E function in greater detail, we developed an efficient selection method for identifying nonfunctional RNase E mutants. A subset of the mutants was found to display a dominant-negative phenotype, interfering with wild-type RNase E function. Unexpectedly, each of these mutants contained a large truncation within the carboxy terminus of RNase E. In contrast, no point mutants that conferred a dominant-negative phenotype were found. We show that a representative dominant-negative mutant can form mixed multimers with RNase E and propose a model to explain how these mutants can block wild-type RNase E function in vivo.

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Section: Discussionmentioning

confidence: 99%

Identification and Analysis of Escherichia coli Ribonuclease E Dominant-Negative Mutants

2006

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“…SDS protein gels containing covalently attached oligonucleotides were prepared according to the standard protocol (Sambrook et al 1989) except that the solution used to generate the resolving gel(s) was supplemented with acrylamide-modified oligonucleotide (∼0.3 pmole/mL) prior to polymerization. After electrophoresis, the gels were washed extensively with 25% (vol/vol) isopropanol to remove SDS (Blank et al 1982) and then proteins were refolded using the procedure described by (Nilsson et al 2002). The activity staining was performed at 30-37°C for 1 h, followed by 2-h incubation at ambient temperature.…”

Section: Activity Stainingmentioning

confidence: 99%

Expanding the Use of Zymography by the Chemical Linkage of Small, Defined Substrates to the Gel Matrix

Kaberdin¹,

McDowall²

2003

Genome Res.

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In the postgenomic era, the comprehensive proteomic analysis of metabolic and signaling pathways is inevitably faced with the challenge of large-scale identification and characterization of polypeptides with a particular enzymatic activity. Previous work has shown that a wide variety of enzymatic activities of microbial, plant, and animal origin can be assigned to individual polypeptides using in-gel activity staining (zymography). However, a number of limitations, such as special substrate requirements, the lack of a standard procedure, and difficulties in distinguishing enzymes with overlapping activities have precluded the widespread use of zymography as a routine laboratory method. Here we demonstrate that, by employing small-defined substrates that are covalently attached to the gel matrix, we can largely overcome the aforementioned problems and assay readily a number of different classes of enzymatic activities within gels after standard SDS-polyacrylamide electrophoresis. Moreover, this development is compatible with the two-dimensional separation of proteins and thus has great potential in the high-throughput screening and characterization of complex biological and clinical samples.

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“…There are a number of lysine, tyrosine, cysteine, and histidine residues within the NTH of E. coli RNaseE. Of these, three lysines at positions 103, 109, and 149 and three tyrosines at positions 42, 123, and 256 are highly conserved across the RNaseE family 6 and located within the smallest RNaseE polypeptide that has been found to have hydrolytic activity (52). Hydrated metal ions such as Mg(H 2 0) 6 2ϩ could also be one of the ionizable groups that are required for the hydrolysis of RNA by RNaseE.…”

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

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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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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In-Gel Analysis of Site-Specific RNases

Cited by 4 publications

References 1 publication

Identification and Analysis of Escherichia coli Ribonuclease E Dominant-Negative Mutants

Identification and Analysis of Escherichia coli Ribonuclease E Dominant-Negative Mutants

Expanding the Use of Zymography by the Chemical Linkage of Small, Defined Substrates to the Gel Matrix

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

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