An RNA degradosome assembly in Caulobacter crescentus.
Nucleic Acids Res. 2010 Oct 15. [Epub ahead of print]
Authors: Hardwick SW, Chan VS, Broadhurst RW, Luisi BF.
In many bacterial species, the multi-enzyme RNA degradosome assembly makes key contributions to RNA metabolism. Powering the turnover of RNA and the processing of structural precursors, the RNA degradosome has differential activities on a spectrum of transcripts and contributes to gene regulation at a global level. Here, we report the isolation and characterization of an RNA degradosome assembly from the α-proteobacterium Caulobacter crescentus, which is a model organism for studying morphological development and cell-cycle progression.
Molecular recognition between Escherichia coli enolase and ribonuclease E.
Acta Crystallogr D Biol Crystallogr. 2010 Sep;66(Pt 9):1036-40. Epub 2010 Aug 13.
Authors: Nurmohamed S, McKay AR, Robinson CV, Luisi BF.
In Escherichia coli and many other bacterial species, the glycolytic enzyme enolase is a component of the multi-enzyme RNA degradosome, an assembly that is involved in RNA processing and degradation. Enolase is recruited into the degradosome through interactions with a small recognition motif located within the degradosome-scaffolding domain of RNase E. Here, the crystal structure of enolase bound to its cognate site from RNase E (residues 823-850) at 1.9 A resolution is presented. The structure suggests that enolase may help to organize an adjacent conserved RNA-binding motif in RNase E.
An ancient anion-binding structural module in RNA and DNA helicases.
Proteins. 2010 Jun;78(8):1900-8.
RNA and DNA helicases manipulate or translocate along single strands of nucleic acids by grasping them using a conserved structural motif. We have examined the available crystal structures of helicases of the two principal superfamilies, SF1 and SF2, and observed that the most conserved interactions with the nucleic acid occur between the phosphosugar backbone of a trinucleotide and the three strand-helix loops within a (beta-strand/alpha-helix)(3) structural module.
The use of novel organic gels and hydrogels in protein crystallization
J Appl Crystallogr. 2010 Feb;43(1):58-63. Epub 6 Jan 2010.
The use of an organic solvent-based gel prepared from polyethylene oxide and a polyvinyl alcohol hydrogel for protein crystallization was investigated. The preparation, properties and application of the gels for protein crystallization are described, and the advantages and limitations of the approach are discussed. The gels are compared with agar, which is a popular aqueous gel used for protein crystallization. The growth behaviour and diffraction quality of crystals prepared in these gel media were evaluated for two model soluble proteins, thaumatin and lysozyme, and for two bacterial membrane proteins, TolC and AcrB.
The regulatory protein RraA modulates RNA-binding and helicase activities of the E. coli RNA degradosome.
RNA 2010 Mar;16(3):553-62. Epub 2010 Jan 27.
The Escherichia coli endoribonuclease RNase E is an essential enzyme having key roles in mRNA turnover and the processing of several structured RNA precursors, and it provides the scaffold to assemble the multienzyme RNA degradosome. The activity of RNase E is inhibited by the protein RraA, which can interact with the ribonuclease's degradosome-scaffolding domain. Here, we report that RraA can bind to the RNA helicase component of the degradosome (RhlB) and the two RNA-binding sites in the degradosome-scaffolding domain of RNase E. In the presence of ATP, the helicase can facilitate the exchange of RraA for RNA stably bound to the degradosome. Our data suggest that RraA can affect multiple components of the RNA degradosome in a dynamic, energy-dependent equilibrium. The multidentate interactions of RraA impede the RNA-binding and ribonuclease activities of the degradosome and may result in complex modulation and rerouting of degradosome activity.
Prof. Ben Luisi
Department of Biochemistry
University of Cambridge
80 Tennis Court Road
CB2 1GA Cambridge
Drugs and certain proteins are transported across the membranes of Gram-negative bacteria by energy-activated pumps. The outer membrane component of these pumps is a channel that opens from a sealed resting state during the transport process. We describe two crystal structures of the Escherichia coli outer membrane protein TolC in its partially open state. Opening is accompanied by the exposure of three shallow intraprotomer grooves in the TolC trimer, where our mutagenesis data identify a contact point with the periplasmic component of a drug efflux pump, AcrA.
Polynucleotide phosphorylase (PNPase) is a processive exoribonuclease that contributes to messenger RNA turnover and quality control of ribosomal RNA precursors in many bacterial species. In Escherichia coli, a proportion of the PNPase is recruited into a multi-enzyme assembly, known as the RNA degradosome, through an interaction with the scaffolding domain of the endoribonuclease RNase E. Here, we report crystal structures of E. coli PNPase complexed with the recognition site from RNase E and with manganese in the presence or in the absence of modified RNA.
In Escherichia coli, the glycolytic enzyme enolase is a component of the RNA degradosome, which is an RNase E mediated assembly involved in RNA processing and transcript turnover. The recruitment of enolase by the RNA degradosome has been implicated in the turnover of certain transcripts, and it is mediated by a small segment of roughly a dozen residues that lie within a natively unstructured sub-domain of RNase E. Here, we present the crystal structure of enolase in complex with its recognition site from RNase E at 1.6Å resolution.
The coordinated regulation of gene expression is required for homeostasis, growth and development in all organisms. Such coordination may be partly achieved at the level of messenger RNA stability, in which the targeted destruction of subsets of transcripts generates the potential for cross-regulating metabolic pathways. In Escherichia coli, the balance and composition of the transcript population is affected by RNase E, an essential endoribonuclease that not only turns over RNA but also processes certain key RNA precursors.