Pcf11 and Clp1 are subunits of cleavage factor IA (CFIA), an essential polyadenylation factor in Saccahromyces cerevisiae. We have determined the structure of a ternary complex of Clp1 together with ATP and the Clp1-binding region of Pcf11. Clp1 contains three domains, a small N-terminal β sandwich domain, a C-terminal domain containing a novel α/β-fold and a central domain that binds ATP. The arrangement of the nucleotide binding site is similar to that observed in SIMIBI-class ATPase subunits found in other multisubunit macromolecular complexes. However, despite this similarity, nucleotide hydrolysis does not occur. The Pcf11 binding site is also located in the central domain where three highly conserved residues in Pcf11 mediate many of the protein–protein interactions. We propose that this conserved Clp1–Pcf11 interaction is responsible for maintaining a tight coupling between the Clp1 nucleotide binding subunit and the other components of the polyadenylation machinery. Moreover, we suggest that this complex represents a stabilized ATP bound form of Clp1 that requires the participation of other non-CFIA processing factors in order to initiate timely ATP hydrolysis during 3′ end processing.

NusA is a key regulator of bacterial transcriptional elongation, pausing, termination and antitermination, yet relatively little is known about the molecular basis of its activity in these fundamental processes. In Mycobacterium tuberculosis, NusA has been shown to bind with high affinity and specificity to BoxB-BoxA-BoxC antitermination sequences within the leader region of the single ribosomal RNA (rRNA) operon. We have determined high-resolution X-ray structures of a complex of NusA with two short oligo-ribonucleotides derived from the BoxC stem-loop motif and have characterised the interaction of NusA with a variety of RNAs derived from the antitermination region. These structures reveal the RNA bound in an extended conformation to a large interacting surface on both KH domains. Combining structural data with observed spectral and calorimetric changes, we now show that NusA binding destabilises secondary structure within rRNA antitermination sequences and propose a model where NusA functions as a chaperone for nascently forming RNA structures.

The Protein Data Bank in Europe (PDBe) (http://www.ebi.ac.uk/pdbe/) is actively working with its Worldwide Protein Data Bank partners to enhance the quality and consistency of the international archive of bio-macromolecular structure data, the Protein Data Bank (PDB). PDBe also works closely with its collaborators at the European Bioinformatics Institute and the scientific community around the world to enhance its databases and services by adding curated and actively maintained derived data to the existing structural data in the PDB. We have developed a new database infrastructure based on the remediated PDB archive data and a specially designed database for storing information on interactions between proteins and bound molecules. The group has developed new services that allow users to carry out simple textual queries or more complex 3D structure-based queries. The newly designed ‘PDBeView Atlas pages’ provide an overview of an individual PDB entry in a user-friendly layout and serve as a starting point to further explore the information available in the PDBe database. PDBe’s active involvement with the X-ray crystallography, Nuclear Magnetic Resonance spectroscopy and cryo-Electron Microscopy communities have resulted in improved tools for structure deposition and analysis.

We describe a method to analyze the sequence specificity of an RNA-binding domain. The method, which we have named scaffold-independent analysis, reports on the specificity for each nucleotide position within an RNA target, uncoupled from the surrounding structural and sequence context. We expect this information to improve our understanding of protein-RNA interfaces in ssRNA binding domains (e.g., KH or RRM domains) and to be useful to the design of novel protein-RNA recognition surfaces. Our NMR binding assays using the third KH domain of the Nova-1 protein provide a proof-of-principle for the method and novel information on the specificity of this domain for its RNA targets.

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Creatininase (CrnA) from Pseudomonas putida is a homohexameric heat-stable enzyme composed of 259 amino acids per subunit. The molecular weight of each monomer is 28.4 kDa. The enzyme hydrolyses creatinine to yield creatine. Crystals of this protein have been grown from ethanol/PEG 8000. They belong to the monoclinic space group P2(1), with unit-cell parameters a = 74.8, b = 95.7, c = 116.9 A, alpha = gamma = 90, beta = 103.8 degrees. The diffraction limit is 2.5 A. The self-rotation function of the native data set is consistent with a CrnA hexamer in the asymmetric unit and suggests D(3) point-group symmetry of the enzyme.

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