The regulatory protein and component interactions of soluble methane monooxygenase

The purpose of this study was to investigate the regulatory protein (protein B) and component interactions of soluble methane monooxygenase (sMMO). sMMO is a multi-component enzyme which catalyses the oxidation of methane to methanol. It consists of three proteins, a hydroxylase, a reductase and protein B (Colby and Dalton, 1978). Protein B contains no metals, cofactors or prosthetic groups and has a molecular mass of 16 kDa. It has been shown that protein B is absolutely necessary for the hydroxylase activity of the sMMO complex and is a powerful regulator of the enzyme (Green and Dalton, 1985). It has also been found that 12 amino acids are cleaved from the N-terminus of protein B from Me. eapsulatus (Bath) to form an inactive truncate, known as protein B' and mutation of the Met12_Gly13 cleavage site to Met12 -GIn I3 to give the single mutant protein G13Q, improved the stability of the protein (Lloyd et al., 1997). Much of this work has concentrated on the study of the catalytic and regulatory significance of the 12 amino acids cleaved from protein B. Mc. eapsulatus (Bath) protein B appears to- cleave autocatalytically, generating the inactive protein B' truncate. The secondary structures of proteins B and B' were seen to be the same, although the overall structure was identified as differing slightly and protein B was shown to be capable of existing in a monomer-dimer equilibrium, whereas protein B' was identified as existing in a monomer form. An homologous protein B from Ms. trichosporium OB3b, identified as being more a-helical in character, has been shown to be more stable than Mc. eapsulatus (Bath) protein B but still undergoes the inactivating cleavage reaction to form truncates, although the cleavage sites differ between the two proteins. The construction, expression and purification of N-terminal truncates of Mc. capsulatus (Bath) protein B identified that the presence of the first 7 amino acids was essential for protein B activity within the sMMO system and a decrease in specific activity was observed as each amino acid from 1 to 7 was lost. Upon loss of the 7th amino acid, tyrosine, the truncate protein was observed to be totally inactive and also much more prone to cleavage, but unchanged in terms of secondary structure. Protein concentration was observed as having an effect on the stability of Mc. capsulatus (Bath) protein B and, the single mutant G13Q, with increased concentrations improving stability. This effect was not observed for the double mutant MI2A:G13Q, although it was shown to be more stable than the other proteins under more dilute conditions. The addition of a magnesium salt also improved the stability of protein B. Studies into the interactions of protein B with the other proteins within the sMMO complex have also been performed. Evidence that the hydroxylase undergoes a large conformational change upon the binding of the reductase and protein B has been obtained and modelled to suggest that one trimer of the hydroxylase dimer rotates by 1800 relative to the other upon complex formation. It also showed the sMMO complex to form in a stoichiometry of 1:2:2 hydroxylase:reductase:protein B. Other data suggest that -sMMO component binding occurs on only one trimer of the hydroxylase dimer under different conditions

Hfq binding changes the structure of Escherichia coli small noncoding RNAs OxyS and RprA, which are involved in the riboregulation of rpoS

OxyS and RprA are two small noncoding RNAs (sRNAs) that modulate the expression of rpoS, encoding an alternative sigma factor that activates transcription of multiple Escherichia coli stress-response genes. While RprA activates rpoS for translation, OxyS down-regulates the transcript. Crucially, the RNA binding protein Hfq is required for both sRNAs to function, although the specific role played by Hfq remains unclear. We have investigated RprA and OxyS interactions with Hfq using biochemical and biophysical approaches. In particular, we have obtained the molecular envelopes of the Hfq–sRNA complexes using small-angle scattering methods, which reveal key molecular details. These data indicate that Hfq does not substantially change shape upon complex formation, whereas the sRNAs do. We link the impact of Hfq binding, and the sRNA structural changes induced, to transcript stability with respect to RNase E degradation. In light of these findings, we discuss the role of Hfq in the opposing regulatory functions played by RprA and OxyS in rpoS regulation

An improved method for surface immobilisation of RNA: application to small Non-Coding RNA - mRNA pairing

Characterisation of RNA and its intermolecular interactions is increasing in importance as the inventory of known RNA functions continues to expand. RNA-RNA interactions are central to post-transcriptional gene regulation mechanisms in bacteria, and the interactions of bacterial small non-coding RNAs (sRNAs) with their mRNA targets are the subject of much current research. The technology of surface plasmon resonance (SPR) is an attractive approach to studying these interactions since it is highly sensitive, and allows interaction measurements to be recorded in real-time. Whilst a number of approaches exist to label RNAs for surface-immobilisation, the method documented here is simple, quick, efficient, and utilises the high-affinity streptavidin-biotin interaction. Specifically, we ligate a biotinylated nucleotide to the 3' end of RNA using T4 RNA ligase. Although this is a previously recognised approach, we have optimised the method by our discovery that the incorporation of four or more adenine nucleotides at the 3' end of the RNA (a poly-A-tail) is required in order to achieve high ligation efficiencies. We use this method within the context of investigating small non-coding RNA (sRNA)-mRNA interactions through the application of surface technologies, including quantitative SPR assays. We first focus on validating the method using the recently characterised Escherichia coli sRNA-mRNA pair, MicA-ompA, specifically demonstrating that the addition of the poly-A-tail to either RNA does not affect its subsequent binding interactions with partner molecules. We then apply this method to investigate the novel interactions of a Vibrio cholerae Qrr sRNA with partner mRNAs, hapR and vca0939; RNA-RNA pairings that are important in mediating pathogenic virulence. The calculated binding parameters allow insights to be drawn regarding sRNA-mRNA interaction mechanisms

South-Siberian mountain mires: Perspectives on a potentially vulnerable remote source of biodiversity

Changes in climate, land-use and pollution are having disproportionate impacts on ecosystems and biodiversity of arctic and mountain ecosystems. While these impacts are well-documented for many areas of the Arctic and alpine regions, some isolated and inaccessible mountain areas are poorly studied. Furthermore, even in well-studied regions, assessments of biodiversity and species to environmental change are biased towards vascular plants and cryptogams, particularly bryophytes are far less represented. This paper aims to document the environments of the remove and inaccessible Altai-Sayan mountain mires and particularly their bryofloras where threatened specias exist and species new to the regional flora are still being found. As these mountain mires are relatively inaccessible, changes in drivers of change ad their ecosystem and biodiversity impacts have not been monitored. However, the remoteness of the mires has so far protected them and their species. In this study, we describe the mires, their bryophyte species and the expected impacts of environmental stressors to bring attention to the urgency of documenting change and conserving these pristine ecosystems

High-density functional-RNA arrays as a versatile platform for studying RNA-based interactions

We are just beginning to unravel the myriad of interactions in which non-coding RNAs participate. The intricate RNA interactome is the foundation of many biological processes, including bacterial virulence and human disease, and represents unexploited resources for the development of potential therapeutic interventions. However, identifying specific associations of a given RNA from the multitude of possible binding partners within the cell requires robust high-throughput systems for their rapid screening. Here, we present the first demonstration of functional-RNA arrays as a novel platform technology designed for the study of such interactions using immobilized, active RNAs. We have generated high-density RNA arrays by an innovative method involving surface-capture of in vitro transcribed RNAs. This approach has significant advantages over existing technologies, particularly in its versatility in regards to binding partner character. Indeed, proof-of-principle application of RNA arrays to both RNA-small molecule and RNA-RNA pairings is demonstrated, highlighting their potential as a platform technology for mapping RNA-based networks and for pharmaceutical screening. Furthermore, the simplicity of the method supports greater user-accessibility over currently available technologies. We anticipate that functional-RNA arrays will find broad utility in the expanding field of RNA characterization

An Investigation into the Potential of Targeting Escherichia coli rne mRNA with Locked Nucleic Acid (LNA) Gapmers as an Antibacterial Strategy

The increase in antibacterial resistance is a serious challenge for both the health and defence sectors and there is a need for both novel antibacterial targets and antibacterial strategies. RNA degradation and ribonucleases, such as the essential endoribonuclease RNase E, encoded by the rne gene, are emerging as potential antibacterial targets while antisense oligonucleotides may provide alternative antibacterial strategies. As rne mRNA has not been previously targeted using an antisense approach, we decided to explore using antisense oligonucleotides to target the translation initiation region of the Escherichia coli rne mRNA. Antisense oligonucleotides were rationally designed and were synthesised as locked nucleic acid (LNA) gapmers to enable inhibition of rne mRNA translation through two mechanisms. Either LNA gapmer binding could sterically block translation and/or LNA gapmer binding could facilitate RNase H-mediated cleavage of the rne mRNA. This may prove to be an advantage over the majority of previous antibacterial antisense oligonucleotide approaches which used oligonucleotide chemistries that restrict the mode-of-action of the antisense oligonucleotide to steric blocking of translation. Using an electrophoretic mobility shift assay, we demonstrate that the LNA gapmers bind to the translation initiation region of E. coli rne mRNA. We then use a cell-free transcription translation reporter assay to show that this binding is capable of inhibiting translation. Finally, in an in vitro RNase H cleavage assay, the LNA gapmers facilitate RNase H-mediated mRNA cleavage. Although the challenges of antisense oligonucleotide delivery remain to be addressed, overall, this work lays the foundations for the development of a novel antibacterial strategy targeting rne mRNA with antisense oligonucleotides