Binding of a biosynthetic intermediate to AtrA modulates the production of lidamycin by Streptomyces globisporus.

The control of secondary production in streptomycetes involves the funneling of environmental and physiological signals to the cluster-situated (transcriptional) regulators (CSRs) of the biosynthetic genes. For some systems, the binding of biosynthetic products to the CSR has been shown to provide negative feedback. Here we show for the production of lidamycin (C-1027), a clinically relevant antitumor agent, by Streptomyces globisporus that negative feedback can extend to a point higher in the regulatory cascade. We show that the DNA-binding activity of the S. globisporus orthologue of AtrA, which was initially described as a transcriptional activator of actinorhodin biosynthesis in S. coelicolor, is inhibited by the binding of heptaene, a biosynthetic intermediate of lidamycin. Additional experiments described here show that S. globisporus AtrA binds in vivo as well as in vitro to the promoter region of the gene encoding SgcR1, one of the CSRs of lidamycin production. The feedback to the pleiotropic regulator AtrA is likely to provide a mechanism for coordinating the production of lidamycin with that of other secondary metabolites. The activity of AtrA is also regulated by actinorhodin. As AtrA is evolutionarily conserved, negative feedback of the type described here may be widespread within the streptomycetes.

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.

Two-dimensional gel electrophoresis for identifying proteins that bind DNA or RNA.

Electrophoretic mobility shift assays (EMSAs) are commonly used to analyze nucleic acid-protein interactions. When nucleic acid is bound by protein, its mobility during gel electrophoresis is reduced. Similarly, the final position of protein within a complex is shifted when compared to its free state. Here we provide a protocol for a simple approach that uses these mobility differences to identify nucleic acid-binding proteins. Following EMSA, denaturing gel electrophoresis is implemented to provide a second dimension of separation. Protein that binds a specific nucleic acid is identified as a spot(s) whose presence at a particular position(s) is dependent on nucleic acid within the initial binding reaction. The polypeptide in a spot can be subsequently identified by mass spectrometry. As EMSAs can be performed using partially purified or cell extracts, this approach substantially reduces the need for protein purification. It should facilitate the identification of a nucleic acid-binding protein within approximately 4 d.

"Zn-link": a metal-sharing interface that organizes the quaternary structure and catalytic site of the endoribonuclease, RNase E.

Ribonuclease E is an essential hydrolytic endonuclease in Escherichia coli, and it plays a central role in maintaining the balance and composition of the messenger RNA population. The enzyme is also required for rRNA and tRNA processing. We have shown earlier that the highly conserved catalytic domain of E. coli RNase E is a homotetramer [Callaghan, A. J. et al. (2003) Biochemistry 42, 13848-13855]. Here, we report that this quaternary organization requires zinc. Two protomers share a single zinc ion, and quantitative analysis indicates that each protein contributes two cysteine thiols toward the coordination of the metal. The candidate cysteines are part of a motif that is conserved in the RNase E protein family, and mutation of these residues causes the partial loss of zinc, the complete disruption of the tetramer into dimers, and effective catalytic inactivation. However, these mutations do not affect RNA binding. The tetramer can be artificially maintained by disulfide bond formation, which fully displaces the zinc but largely preserves the catalytic activity. Thus, catalytic activity does not require zinc directly but does require the quaternary structure, for which the metal is essential. We propose that the RNase E tetramer has two nonequivalent subunit interfaces, one of which is mediated by a single, tetrathiol-zinc complex, which we refer to as a "Zn-link" motif. One or both interfaces organize the active site, which is distinct from the primary site of RNA binding.

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.

Expanding the use of zymography by the chemical linkage of small, defined substrates to the gel matrix.

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.