Studies of the RNA Degradosome-organizing Domain of the Escherichia coli Ribonuclease RNase E

Callaghan, Anastasia J.; Aurikko, Jukka P.; Ilag, Leopold L.; Grossmann, J. Günter; Chandran, Vidya; Kühnel, Karin; Poljak, Leonora; Carpousis, Agamemnon J.; Robinson, Carol V.; Symmons, Martyn F.; Luisi, Ben F.

doi:10.1016/j.jmb.2004.05.046

Cited by 152 publications

(241 citation statements)

References 54 publications

(64 reference statements)

Supporting

Mentioning

208

Contrasting

Unclassified

Order By: Relevance

“…4). This contrasts with the behavior of WT RhlB and all other helicases used in the present work, which elute later and are likely to be monomers based on our current and earlier mass spectrometry data (22).…”

Section: Analysis Of Interactions Of Rhlb Surface Mutants With Rnase E-contrasting

confidence: 92%

“…However, the effects of only a very few cofactors or accessory proteins on the enzymatic functions of RNA helicases have been characterized in vitro. As part of the present work we have extended previous studies that have reported on the E. coli DEAD box RNA helicase, RhlB, whose ATPase activity is stimulated by interaction with its partner protein, the endoribonuclease RNase E (19,22,23). Evidence indicates that the boost of ATPase activity is not caused by increased recruitment of RNA (18).…”

Section: Discussionsupporting

confidence: 75%

“…Recombinant Expression and Purification of RNA Helicases and Variants-Wild-type (WT) 5 and site-directed variants of RhlB, RhlE, and SrmB were expressed in E. coli strain BL21(DE3) and isolated and purified as previously described (22,23). A final purification step was introduced to the published procedure involving S200 size exclusion chromatography.…”

Section: Methodsmentioning

confidence: 99%

“…The final expression constructs produced proteins that were highly overexpressed in E. coli, were entirely soluble, and could be purified to Ͼ95% as judged from SDS-PAGE. From previous studies it was found that RhlB forms a complex with RNase E (19,(21)(22)(23). To aid in investigating the properties of this complex, a di-cistronic expression vector was constructed encoding WT RhlB or its site-directed variants and an N-terminal His 6 -tagged 67-amino acid peptide of RNase E corresponding to residues 696 -762.…”

Section: Site-directed Mutagenesis Of Three E Coli Dead Box Proteinsmentioning

confidence: 99%

“…The C-terminal domain is predicted to be predominantly unstructured, with some small regions of propensity for secondary structure being restricted to specific sites of recognition (22). The site of interaction on RNase E for RhlB has been identified to lie between residues 698 and 762 (23).…”

mentioning

confidence: 99%

See 4 more Smart Citations

Allosteric Activation of the ATPase Activity of the Escherichia coli RhlB RNA Helicase

Worrall

Howe

McKay

et al. 2008

Journal of Biological Chemistry

View full text Add to dashboard Cite

Helicase B (RhlB) is one of the five DEAD box RNA-dependent ATPases found in Escherichia coli. Unique among these enzymes, RhlB requires an interaction with the partner protein RNase E for appreciable ATPase and RNA unwinding activities. To explore the basis for this activating effect, we have generated a di-cistronic vector that overexpresses a complex comprising RhlB and its recognition site within RNase E, corresponding to residues 696 -762. Complex formation has been characterized by isothermal titration calorimetry, revealing an avid, enthalpyfavored interaction between the helicase and RNase E-(696 -762) with an equilibrium binding constant (K a ) of at least 1 ؋ 10 8 M ؊1 . We studied ATPase activity of mutants with substitutions within the ATP binding pocket of RhlB and on the putative interaction surface that mediates recognition of RNase E. For comparisons, corresponding mutations were prepared in two other E. coli DEAD box ATPases, RhlE and SrmB. Strikingly, substitutions at a phenylalanine near the Q-motif found in DEAD box proteins boosts the ATPase activity of RhlB in the absence of RNA, but completely inhibits it in its presence. The data support the proposal that the protein-protein and RNA-binding surfaces both communicate allosterically with the ATPase catalytic center. We conjecture that this communication may govern the mechanical power and efficiency of the helicases, and is tuned in individual helicases in accordance with cellular function.RNA helicases are a diverse set of proteins found in all three kingdoms of life that possess the ability to unwind short stretches of RNA duplexes in reactions that require the hydrolysis of nucleoside triphosphates (1). They are the largest group of enzymes in eukaryotic RNA metabolism and are involved in virtually all aspects of cellular RNA manipulation including transcription, splicing, RNA nuclear export, and ribosome biogenesis (2, 3). The DEAD box helicases are grouped in helicase superfamily 2, whose members contain nine conserved motifs, including the Walker B motif DEAD from which they take their common name (Fig. 1). Escherichia coli contains five genes encoding for DEAD box proteins; csdA (formerly called deaD), dbpA, rhlB, rhlE, and srmB (4). The gene products have a common core of 350 -400 amino acids, consisting of two domains that have the same fold found in the DNA-recombination protein RecA. Flanking this two-domain core are variable regions that are thought to be responsible for the differing properties and functions of the five helicases (4).DEAD box helicases can unwind short RNA duplexes that are no more than two helical turns in length (ϳ22 bp), and some helicases can displace avidly bound proteins from RNA (1, 5). A role of DEAD box RNA helicases as nonspecific RNA unfolding enzymes has been described (6). Yang and Jankowsky (7) have elegantly shown that DEAD box helicases use a single strand RNA region to facilitate loading onto duplex RNA where only a few base pairs are disrupted in an ATP-dependent manner, leading to destabiliza...

show abstract

Section: Analysis Of Interactions Of Rhlb Surface Mutants With Rnase E-contrasting

confidence: 92%

Section: Discussionsupporting

confidence: 75%

Section: Methodsmentioning

confidence: 99%

Section: Site-directed Mutagenesis Of Three E Coli Dead Box Proteinsmentioning

confidence: 99%

mentioning

confidence: 99%

See 3 more Smart Citations

Allosteric Activation of the ATPase Activity of the Escherichia coli RhlB RNA Helicase

Worrall

Howe

McKay

et al. 2008

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

Water‐Soluble Nile Blue Derivatives: Syntheses and Photophysical Properties

Jose

Ueno

Burgess

2008

Chemistry A European J

View full text Add to dashboard Cite

Four water-soluble 2-hydroxy-Nile Blue derivatives, 1 a, 1 b, 2 a, and 2 b, were prepared by condensation reactions performed under relatively mild conditions (90 degrees C, N,N-dimethylformamide with no added acid). These fluorescent probes had more favorable fluorescence characteristics than two known water-soluble Nile Blue derivatives. Specifically, they were superior to the known dyes with respect to their quantum yields in aqueous media and the sharpness of their fluorescence emissions. Concentration-dependant UV absorption and fluorescence emission studies indicated that the dyes did not aggregate in aqueous solution at concentrations of less than 1-4 microM. The new water-soluble materials 1 a, 1 b, 2 a, and 2 b emit in a desirable region of the fluorescence spectrum (lambda=670-675 nm). Overall they are potentially interesting for labeling biomolecules in aqueous environments.

show abstract

Showing your ID: intrinsic disorder as an ID for recognition, regulation and cell signaling

2005

View full text Add to dashboard Cite

Regulation, recognition and cell signaling involve the coordinated actions of many players. To achieve this coordination, each participant must have a valid identification (ID) that is easily recognized by the others. For proteins, these IDs are often within intrinsically disordered (also ID) regions. The functions of a set of well-characterized ID regions from a diversity of proteins are presented herein to support this view. These examples include both more recently described signaling proteins, such as p53, alpha-synuclein, HMGA, the Rieske protein, estrogen receptor alpha, chaperones, GCN4, Arf, Hdm2, FlgM, measles virus nucleoprotein, RNase E, glycogen synthase kinase 3beta, p21(Waf1/Cip1/Sdi1), caldesmon, calmodulin, BRCA1 and several other intriguing proteins, as well as historical prototypes for signaling, regulation, control and molecular recognition, such as the lac repressor, the voltage gated potassium channel, RNA polymerase and the S15 peptide associating with the RNA polymerase S-protein. The frequent occurrence and the common use of ID regions in important protein functions raise the possibility that the relationship between amino acid sequence, disordered ensemble and function might be the dominant paradigm for the molecular recognition that serves as the basis for signaling and regulation by protein molecules.

show abstract

Studies of the RNA Degradosome-organizing Domain of the Escherichia coli Ribonuclease RNase E

Cited by 152 publications

References 54 publications

Allosteric Activation of the ATPase Activity of the Escherichia coli RhlB RNA Helicase

Allosteric Activation of the ATPase Activity of the Escherichia coli RhlB RNA Helicase

Water‐Soluble Nile Blue Derivatives: Syntheses and Photophysical Properties

Showing your ID: intrinsic disorder as an ID for recognition, regulation and cell signaling

Contact Info

Product

Resources

About