Unwinding of a DNA replication fork by a hexameric viral helicase.

Hexameric helicases are motor proteins that unwind double-stranded DNA (dsDNA) during DNA replication but how they are optimised for strand separation is unclear. Here we present the cryo-EM structure of the full-length E1 helicase from papillomavirus, revealing all arms of a bound DNA replication fork and their interactions with the helicase. The replication fork junction is located at the entrance to the helicase collar ring, that sits above the AAA + motor assembly. dsDNA is escorted to and the 5´ single-stranded DNA (ssDNA) away from the unwinding point by the E1 dsDNA origin binding domains. The 3´ ssDNA interacts with six spirally-arranged ß-hairpins and their cyclical top-to-bottom movement pulls the ssDNA through the helicase. Pulling of the RF against the collar ring separates the base-pairs, while modelling of the conformational cycle suggest an accompanying movement of the collar ring has an auxiliary role, helping to make efficient use of ATP in duplex unwinding.

Structural basis for DNA strand separation by a hexameric replicative helicase.

Hexameric helicases are processive DNA unwinding machines but how they engage with a replication fork during unwinding is unknown. Using electron microscopy and single particle analysis we determined structures of the intact hexameric helicase E1 from papillomavirus and two complexes of E1 bound to a DNA replication fork end-labelled with protein tags. By labelling a DNA replication fork with streptavidin (dsDNA end) and Fab (5' ssDNA) we located the positions of these labels on the helicase surface, showing that at least 10 bp of dsDNA enter the E1 helicase via a side tunnel. In the currently accepted 'steric exclusion' model for dsDNA unwinding, the active 3' ssDNA strand is pulled through a central tunnel of the helicase motor domain as the dsDNA strands are wedged apart outside the protein assembly. Our structural observations together with nuclease footprinting assays indicate otherwise: strand separation is taking place inside E1 in a chamber above the helicase domain and the 5' passive ssDNA strands exits the assembly through a separate tunnel opposite to the dsDNA entry point. Our data therefore suggest an alternative to the current general model for DNA unwinding by hexameric helicases.

A flexible brace maintains the assembly of a hexameric replicative helicase during DNA unwinding.

The mechanism of DNA translocation by papillomavirus E1 and polyomavirus LTag hexameric helicases involves consecutive remodelling of subunit-subunit interactions around the hexameric ring. Our biochemical analysis of E1 helicase demonstrates that a 26-residue C-terminal segment is critical for maintaining the hexameric assembly. As this segment was not resolved in previous crystallographic analysis of E1 and LTag hexameric helicases, we determined the solution structure of the intact hexameric E1 helicase by Small Angle X-ray Scattering. We find that the C-terminal segment is flexible and occupies a cleft between adjacent subunits in the ring. Electrostatic potential calculations indicate that the negatively charged C-terminus can bridge the positive electrostatic potentials of adjacent subunits. Our observations support a model in which the C-terminal peptide serves as a flexible 'brace' maintaining the oligomeric state during conformational changes associated with ATP hydrolysis. We argue that these interactions impart processivity to DNA unwinding. Sequence and disorder analysis suggest that this mechanism of hexamer stabilization would be conserved among papillomavirus E1 and polyomavirus LTag hexameric helicases.

The C-terminal region of the exosome-associated protein Rrp47 is specifically required for box C/D small nucleolar RNA 3'-maturation.

Cells lacking the exosome-associated protein Rrp47 show similar defects in stable RNA processing to those observed in the absence of the catalytic subunit Rrp6, but the precise mechanism(s) by which Rrp47 functions together with Rrp6 remains unclear. Deletion complementation analyses defined an N-terminal region of Rrp47, largely coincident with the bioinformatically defined Sas10/C1D domain, which was sufficient for protein function in vivo. In vitro protein interaction studies demonstrated that this domain of Rrp47 binds the PMC2NT domain of Rrp6. Expression of the N-terminal domain of Rrp47 in yeast complemented most RNA-processing defects associated with the rrp47Δ mutant but failed to complement the defect observed in 3'-end maturation of box C/D small nucleolar RNAs. Consistent with these results, protein capture assays revealed an interaction between the C-terminal region of Rrp47 and the small nucleolar ribonucleoproteins Nop56 and Nop58. Filter binding assays demonstrated that deletion of the lysine-rich sequence at the C terminus of Rrp47 blocked RNA binding in vitro. Furthermore, a protein mutated both at the C terminus and within the N-terminal domain showed a synergistic defect in RNA binding without impacting on its ability to interact with Rrp6. These studies provide evidence for a role of Rrp47 in registering a small nucleolar ribonucleoprotein particle assembly, functionally characterize the Sas10/C1D domain of Rrp47, and show that both the C terminus of Rrp47 and the N-terminal domain contribute to its RNA-binding activity.

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.

The PMC2NT domain of the catalytic exosome subunit Rrp6p provides the interface for binding with its cofactor Rrp47p, a nucleic acid-binding protein.

The exosome complex is a key component of the cellular RNA surveillance machinery and is required for normal 3' end processing of many stable RNAs. Exosome activity requires additional factors such as the Ski or TRAMP complexes to activate the complex or facilitate substrate binding. Rrp47p promotes the catalytic activity of the exosome component Rrp6p, but its precise function is unknown. Here we show that recombinant Rrp47p is expressed as an apparently hexameric complex that specifically binds structured nucleic acids. Furthermore, pull-down assays demonstrated that Rrp47p interacts directly with the N-terminal region of Rrp6p that contains the functionally uncharacterized PMC2NT domain. Strains expressing a mutant form of Rrp6p lacking the N-terminal region failed to accumulate Rrp47p at normal levels, exhibited a slow growth phenotype characteristic of rrp47-Delta mutants and showed RNA processing defects consistent with loss of Rrp47p function. These findings suggest Rrp47p promotes Rrp6p activity by facilitating binding via the PMC2NT domain to structural elements within RNA. Notably, characterized Rrp6p substrates such as the 5.8S+30 species are predicted to contain helices at their 3' termini, while others such as intergenic or antisense cryptic unstable transcripts could potentially form extensive double-stranded molecules with overlapping mRNAs.

The identification of nucleic acid-interacting proteins using a simple proteomics-based approach that directly incorporates the electrophoretic mobility shift assay.

Proteins that interact with nucleic acids are central to numerous cellular processes, and their continuing characterization represents one of the foremost challenges in the postgenomic era. Here we describe a simple proteomics-based approach for the identification by mass spectrometry of proteins in crude extracts that interact with nucleic acids. It incorporates the electrophoretic mobility shift assay and is based on the finding that when a protein forms a complex with nucleic acid its electrophoretic mobility is affected as well as that of the nucleic acid. Our method should greatly reduce and in some cases may even eliminate the need for extensive protein purification and as such should contribute significantly to the functional annotation of the proteome. Furthermore it requires no prior knowledge of the molecular mass, quaternary structure, or pI of the interacting protein. Proof of principle is demonstrated using a recently discovered transcription factor; however, the approach should also have application in the identification of proteins that interact with RNA.

Transcript analysis reveals an extended regulon and the importance of protein-protein co-operativity for the Escherichia coli methionine repressor.

We have used DNA arrays to investigate the effects of knocking out the methionine repressor gene, metJ, on the Escherichia coli transcriptome. We assayed the effects in the knockout strain of supplying wild-type or mutant MetJ repressors from an expression plasmid, thus establishing a rapid assay for in vivo effects of mutations characterized previously in vitro. Repression is largely restricted to known genes involved in the biosynthesis and uptake of methionine. However, we identified a number of additional genes that are significantly up-regulated in the absence of repressor. Sequence analysis of the 5' promoter regions of these genes identified plausible matches to met-box sequences for three of these, and subsequent electrophoretic mobility-shift assay analysis showed that for two such loci their repressor affinity is higher than or comparable with the known metB operator, suggesting that they are directly regulated. This can be rationalized for one of the loci, folE, by the metabolic role of its encoded enzyme; however, the links to the other regulated loci are unclear, suggesting both an extension to the known met regulon and additional complexity to the role of the repressor. The plasmid gene replacement system has been used to examine the importance of protein-protein co-operativity in operator saturation using the structurally characterized mutant repressor, Q44K. In vivo, there are detectable reductions in the levels of regulation observed, demonstrating the importance of balancing protein-protein and protein-DNA affinity.

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.

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.