Kinetochores are proteinaceous assemblies that mediate the interaction of chromosomes with the mitotic spindle. The 180 kDa Ndc80 complex is a direct point of contact between kinetochores and microtubules. Its four subunits contain coiled coils and form an elongated rod structure with functional globular domains at either end. We crystallized an engineered "bonsai" Ndc80 complex containing a shortened rod domain but retaining the globular domains required for kinetochore localization and microtubule binding. The structure reveals a microtubule-binding interface containing a pair of tightly interacting calponin-homology (CH) domains with a previously unknown arrangement. The interaction with microtubules is cooperative and predominantly electrostatic. It involves positive charges in the CH domains and in the N-terminal tail of the Ndc80 subunit and negative charges in tubulin C-terminal tails and is regulated by the Aurora B kinase. We discuss our results with reference to current models of kinetochore-microtubule attachment and centromere organization.

The Saccharomyces cerevisiae DASH complex is an essential microtubule-binding component of the kinetochore. We coexpressed all ten subunits of this assembly in Escherichia coli and purified a single complex, a approximately 210-kDa heterodecamer with an apparent stoichiometry of one copy of each subunit. The hydrodynamic properties of the recombinant assembly are indistinguishable from those of the native complex in yeast extracts. The structure of DASH alone and bound to microtubules was visualized by EM. The free heterodecamer is relatively globular. In the presence of microtubules, DASH oligomerizes to form rings and paired helices that encircle the microtubules. We discuss potential roles for such collar-like structures in maintaining microtubule attachment and spindle integrity during chromosome segregation.

Kinetochores are multiprotein complexes that assemble on centromeric DNA and attach chromosomes to spindle microtubules. Over the past six years, the number of proteins known to localize to the Saccharomyces cerevisiae kinetochore has increased from around 10 to over 60. However, relatively little is known about the protein-protein interactions that mediate kinetochore assembly or about the overall structure of microtubule-attachment sites. Here we used biophysical techniques, affinity purification, mass spectrometry, and in vivo assays to examine the state of association of 31 centromere-binding proteins, including six proteins newly identified as kinetochore subunits. We found that yeast kinetochores resemble transcriptional enhancers in being composed of at least 17 discrete subcomplexes that assemble on DNA to form a very large structure with a mass in excess of 5 MD. Critical to kinetochore assembly are proteins that bridge subunits in direct contact with DNA and subunits bound to microtubules. We show that two newly identified kinetochore complexes, COMA (Ctf19p-Okp1p-Mcm21p-Ame1p) and MIND (Mtw1p including Nnf1p-Nsl1p-Dsn1p) function as bridges. COMA, MIND, and the previously described Ndc80 complex constitute three independent and essential platforms onto which outer kinetochore proteins assemble. In addition, we propose that the three complexes have different functions with respect to force generation and MT attachment.[Keywords: Kinetochore; chromosome segregation; mitosis; centromere; proteomics; hydrodynamics] Supplemental material is available at http://www.genesdev.org.

In Escherichia coli, the two-component Cpx system comprising the CpxA sensor kinase and the CpxR response regulator modulates gene expression in response to a variety of stresses including membrane-protein damage, starvation, and high osmolarity. To date, the few known CpxR-P target operons were mostly identified by genetic screens. To facilitate the discovery of all target operons, we derived a 15-bp weighted matrix for CpxR-P recognition that takes into account the relative base frequency at each nucleotide position. This matrix essentially consists of two tandem 5-GTAAA-3 motifs separated by a 5-bp linker. All of the 15-bp stretches on both strands of the E. coli MG1655 genome were then scored for their degree of matching with the matrix and classified in statistical deviation groups. The effectiveness of this screening is indicated by the identification of eight new target operons (ung, ompC, psd, mviA, aroK, rpoErseABC, secA, and aer) among eleven candidates tested. Moreover, the matrix score correlates with the likelihood that a site is a true target and with the relative site affinity for CpxR-P in vitro. Our data indicate that some 100 operons are under direct CpxR-P control and that the signal transduction pathway interacts with several other control circuits in manners hitherto unanticipated.

The ArcB/ArcA two-component signal transduction system of Escherichia coli regulates gene expression in response to the redox conditions of growth. Over the years, genetic screens have lead to the identification of about 30 ArcA-P-controlled operons that are involved in redox metabolism. However, the discovery of 3 targets that are not implicated in respiratory metabolism (the tra operon for plasmid conjugation, psi site for Xerbased recombination, and oriC site for chromosome replication) suggests that the Arc modulon may comprise additional operons that are involved in a myriad of functions. To identify these operons, we derived the ArcA-Pdependent transcription profile of E. coli using oligonucleotide-based microarray analysis. The findings indicated that 9% of all open reading frames in E. coli are affected either directly or indirectly by ArcA-P. To identify which operons are under the direct control of ArcA-P, we developed the ArcA-P recognition weight matrix from footprinting data and used it to scan the genome, yielding an ArcA-P sequence affinity map. By overlaying both methods, we identified 55 new Arc-regulated operons that are implicated in energy metabolism, transport, survival, catabolism, and transcriptional regulation. The data also suggest that the Arc response pathway, which translates into a net global downscaling of gene expression, overlaps partly with the FNR regulatory network. A conservative but reasonable assessment is that the Arc pathway recruits 100 -150 operons to mediate a role in cellular adaptation that is more extensive than hitherto anticipated.In Escherichia coli, gene expression in response to changing respiratory conditions of growth is partially mediated by the Arc two-component signal transduction system (1-6), which comprises the transmembrane ArcB sensor kinase and its cytosolic cognate response regulator ArcA (7,8). Under anaerobic or microaerobic conditions, ArcB undergoes autophosphorylation and then catalyzes the transphosphorylation of ArcA. Under aerobic conditions, oxidized forms of quinone electron carriers in the membrane inhibit the autophosphorylation of ArcB and therefore its mediation of the Arc metabolic response (9). Phospho-ArcA (ArcA-P) 1 represses certain target operons (e.g. glcDEFGB, Ref. . To date, some 30 operons are known to be controlled by ArcA-P, most of which are involved in respiratory metabolism (6). The 7-bp sequence 5Ј-TATTTaa-3Ј (the lowercase letters are less-conserved nucleotides) was proposed as a putative signature for promoter recognition by ArcA-P, based on DNaseI protection experiments at the pflA promoter (14). A subsequent homology search that included ArcA-P-protected promoter regions of cydAB, pflA, gltA, lldPRD, sdhCDAB, and sodA, plus the entire promoter regions of 16 additional operons whose expressions are ArcA-P-controlled, led to the suggestion of 5Ј-nGTTAATTAn-3Ј (n is A or T) as the ArcA-P binding consensus (15). This 10-bp consensus proved useful for locating ArcA-P binding sites at two novel targets that are not involve...