Kenneth J. McDowall

Structure of Escherichia coli RNase E catalytic domain and implications for RNA turnover

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. RNase E cleaves RNA internally, but its catalytic power is determined by the 5′ terminus of the substrate, even if this lies at a distance from the cutting site. Here we report crystal structures of the catalytic domain of RNase E as trapped allosteric intermediates with RNA substrates. Four subunits of RNase E catalytic domain associate into an interwoven quaternary structure, explaining why the subunit organization is required for catalytic activity. The subdomain encompassing the active site is structurally congruent to a deoxyribonuclease, making an unexpected link in the evolutionary history of RNA and DNA nucleases. The structure explains how the recognition of the 5′ terminus of the substrate may trigger catalysis and also sheds light on the question of how RNase E might selectively process, rather than destroy, specific RNA precursors.

RNase E: still a wonderfully mysterious enzyme

Ribonuclease E (RNase E), which is encoded by an essential Escherichia coli gene known variously as rne, ams, and hmp, was discovered initially as an rRNA‐processing enzyme but is now known to have a general role in RNA decay. Multiple functions, including the ability to cleave RNA endonucleolyticaliy in AU‐rich single‐strand regions, RNA‐binding capabilities, and the ability to interact with polynucleotide phosphorylase and other proteins implicated in the processing and degradation of RNA, are encoded by its 1061 amino acid residues. The presence of homologues and functional analogues of the rne gene in a variety of prokaryotic and eukaryotic species suggests that its functions have been highly conserved during evolution. While much has been learned in recent years about the structure and functions of RNase E, there is continuing mystery about possible additional activities and molecular interactions of this enzyme.

Transcriptional activation of the pathway-specific regulator of the actinorhodin biosynthetic genes in Streptomyces coelicolor

The Streptomyces produce a plethora of secondary metabolites including antibiotics and undergo a complex developmental cycle. As a means of establishing the pathways that regulate secondary metabolite production by this important bacterial genus, the model species Streptomyces coelicolor and its relatives have been the subject of several genetic screens. However, despite the identification and characterization of numerous genes that affect antibiotic production, there is still no overall understanding of the network that integrates the various environmental and growth signals to bring about changes in the expression of biosynthetic genes. To establish new links, we are taking a biochemical approach to identify transcription factors that regulate antibiotic production in S. coelicolor. Here we describe the identification and characterization of a transcription factor, designated AtrA, that regulates transcription of actII‐ORF4, the pathway‐specific activator of the actinorhodin biosynthetic gene cluster in S. coelicolor. Disruption of the corresponding atrA gene, which is not associated with any antibiotic gene cluster, reduced the production of actinorhodin, but had no detectable effect on the production of undecylprodigiosin or the calcium‐dependent antibiotic. These results indicate that atrA has specificity with regard to the biosynthetic genes it influences. An orthologue of atrA is present in the genome of Streptomyces avermitilis, the only other streptomycete for which there is a publicly available complete sequence. We also show that S. coelicolor AtrA can bind in vitro to the promoter of strR, a transcriptional activator unrelated to actII‐ORF4 that is the final regulator of streptomycin production in Streptomyces griseus. These findings provide further evidence that the path leading to the expression of pathway‐specific activators of antibiotic biosynthesis genes in disparate Streptomyces may share evolutionarily conserved components in at least some cases, even though the final activators are not related, and suggests that the regulation of streptomycin production, which serves an important paradigm, may be more complex than represented by current models.

The permease gene nagE2 is the key to N‐acetylglucosamine sensing and utilization in Streptomyces coelicolor and is subject to multi‐level control

The availability of nutrients is a major determinant for the timing of morphogenesis and antibiotic production in the soil‐dwelling bacterium Streptomyces coelicolor. Here we show that N‐acetylglucosamine transport, the first step of an important nutrient signalling cascade, is mediated by the NagE2 permease of the phosphotransferase system, and that the activity of this permease is linked to nutritional control of development and antibiotic production. The permease serves as a high‐affinity transporter for N‐acetylglucosamine (Km of 2.6 µM). The permease complex was reconstituted with individually purified components. This showed that uptake of N‐acetylglucosamine requires a phosphoryl group transfer from phosphoenolpyruvate via the phosphotransferases EI, HPr and IIACrr to NagF, which in turn phosphorylates N‐acetylglucosamine during transport. Transcription of the nagF and nagE2 genes is induced by N‐acetylglucosamine. Nutrient signalling by N‐acetylglucosamine that triggers the onset of development was abolished in the nagE2 and nagF mutants. nagE2 is subject to multi‐level control by the global transcription factor DasR and the activator AtrA that also stimulates genes for antibiotic actinorhodin biosynthesis. Hence, it is apparent that streptomycetes tightly control the nutritional state in a complex manner to ensure the correct timing for the developmental programme.

Rapid cleavage of RNA by RNase E in the absence of 5′ monophosphate stimulation

The best characterized pathway for the initiation of mRNA degradation in Escherichia coli involves the removal of the 5′‐terminal pyrophosphate to generate a monophosphate group that stimulates endonucleolytic cleavage by RNase E. We show here however, using well‐characterized oligonucleotide substrates and mRNA transcripts, that RNase E can cleave certain RNAs rapidly without requiring a 5′‐monophosphorylated end. Moreover, the minimum substrate requirement for this mode of cleavage, which can be categorized as ‘direct’ or ‘internal’ entry, appears to be multiple single‐stranded segments in a conformational context that allows their simultaneous interaction with RNase E. While previous work has alluded to the existence of a 5′ end‐independent mechanism of mRNA degradation, the relative simplicity of the requirements identified here for direct entry suggests that it could represent a major means by which mRNA degradation is initiated in E. coli and other organisms that contain homologues of RNase E. Our results have implications for the interplay of translation and mRNA degradation and models of gene regulation by small non‐coding RNAs.

Characterization of an oxytetracycline-resistance gene, otrA, of Streptomyces rimosus.

The sequence of a 2657 bp DNA fragment containing the coding and regulatory regions of the oxytetracycline (OTC)‐resistance gene, otrA, from the OTC producer Streptomyces rimosus was determined. The predicted amino acid sequence of OtrA had extensive identity with tetracycline‐resistance genes from other bacteria which mediate resistance via non‐covalent ribosomal modification. The N‐terminal domain had extremely high identity with the GTP‐binding sites of elongation factors, such as EF‐G and EF‐Tu, suggesting that binding and hydrolysis of GTP is important to the function of the protein. Significant identity with EF‐G was present throughout the polypeptide. Transcriptional activity upstream of the otrA coding region was investigated. An Escherichia coli‐ type promoter, of otrA p1, was identified. Transcriptional read through of otrA from the upstream gene (otcZ) was also detected in S. rimosus cultures. A divergent promoter activity was identified with sub‐clones of the OtrA fragment in promoter probe vectors analysed in Streptomyces lividans. However, this activity was not identified in a subclone containing more than haif of the otrA coding sequence in S. lividans or at all in S. rimosus, indicating that OtrA negatively regulates the expression of the divergent transcript. The data are consistent with regulation of antibiotic production by OtrA to prevent ‘suicide’.

Translational activation by the noncoding RNA DsrA involves alternative RNase III processing in the rpoS 5′-leader

The intricate regulation of the Escherichia coli rpoS gene, which encodes the stationary phase sigma-factor sigmaS, includes translational activation by the noncoding RNA DsrA. We observed that the stability of rpoS mRNA, and concomitantly the concentration of sigmaS, were significantly higher in an RNase III-deficient mutant. As no decay intermediates corresponding to the in vitro mapped RNase III cleavage site in the rpoS leader could be detected in vivo, the initial RNase III cleavage appears to be decisive for the observed rapid inactivation of rpoS mRNA. In contrast, we show that base-pairing of DsrA with the rpoS leader creates an alternative RNase III cleavage site within the rpoS/DsrA duplex. This study provides new insights into regulation by small regulatory RNAs in that the molecular function of DsrA not only facilitates ribosome loading on rpoS mRNA, but additionally involves an alternative processing of the target.

Sensing of 5' monophosphate by Escherichia coli RNase G can significantly enhance association with RNA and stimulate the decay of functional mRNA transcripts in vivo.

The RNase E/G family of endoribonucleases has a central role in RNA degradation and processing. Previous work has shown that their cleavage of substrates in vitro can be stimulated by the presence of a 5′ monophosphate group. It has not however, established the importance of this activation for any natural RNA processing or decay pathway in vivo. Here we provide for Escherichia coli RNase G the first evidence that the sensing of a 5′ monophosphate is required in vivo for the normal rapid decay of functional mRNAs; moreover, we show in vitro that, in contrast to a previous study, the presence of a 5′ monophosphate can enhance the affinity of RNase G binding to RNA. The implications of these results along with our finding that the maturation of 16S rRNA is unaffected in cells containing an RNase G mutant impaired in 5′ end sensing are discussed with regard to current models of RNA processing and decay and the molecular mechanism that underlies RNA cleavage by the RNase E/G family.

Dietary zinc oxide affects the expression of genes associated with inflammation: transcriptome analysis in piglets challenged with ETEC K88.

The post-weaning growth check in commercial pig production systems is often associated with gastrointestinal infection, in particular that caused by enterotoxigenic Escherichia coli (ETEC) K88. Pharmacological doses of zinc oxide (ZnO) in the post-weaning diet reduce the incidence of diarrhoea and improve piglet performance. In the present study, piglets reared indoors or outdoors and weaned onto diets with or without pharmacological levels of ZnO were orally challenged with ETEC K88. Quantitative real-time PCR was performed on RNA extracted from jejunal lamina propria and Peyers patch samples, to compare expression of a variety of candidate genes between treatments. Candidate genes were selected from an initial microarray study using pooled RNA to identify differentially expressed genes. Dietary treatment with ZnO was associated with significant differences in the transcript abundance of several genes. Zinc supplementation was associated with a marked decrease in expression of immune response genes concerned with inflammation, and possibly related to the stage of infection. Interestingly, evidence was also obtained that a reduced level of MUC4 (a proposed ETEC K88 receptor) was associated with zinc supplementation suggesting a mechanism that might influence ETEC infection. These findings indicate that zinc oxide supplementation may reduce the level of inflammation caused by ETEC challenge.

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

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.