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We have cloned and sequenced complementary DNA encoding a Ca2+-ATPase of rabbit muscle sarcoplasmic reticulum. We propose a model of the protein which has 3 cytoplasmic domains joined to a set of 10 transmembrane helices by a narrow, penta-helical stalk. In this model, ATP bound to one cytoplasmic domain would phosphorylate an aspartate in an adjoining cytoplasmic domain, inducing translocation of Ca2+ from binding sites on the stalk.

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Post-translational acetylation of histone H4 N-terminal tail in chromatin has been associated with several nuclear processes including transcription. We report the purification and characterization of a native multisubunit complex (NuA4) from yeast that acetylates nucleosomal histone H4. NuA4 has an apparent molecular mass of 1.3 MDa. All four conserved lysines of histone H4 can be acetylated by NuA4. We have identified the catalytic subunit of the complex as the product of ESA1, an essential gene required for cell cycle progression in yeast. Antibodies against Esa1p specifically immunoprecipitate NuA4 activity whereas the complex purified from a temperature-sensitive esa1 mutant loses its acetyltransferase activity at the restrictive temperature. Additionally, we have identified another subunit of the complex as the product of TRA1, an ATM-related essential gene homologous to human TRRAP, an essential cofactor for c-Myc-and E2F-mediated oncogenic transformation. Finally, the ability of NuA4 to stimulate GAL4-VP16-driven transcription from chromatin templates in vitro is also lost in the temperature-sensitive esa1 mutant. The function of the essential Esa1 protein as the HAT subunit of NuA4 and the presence of Tra1p, a putative transcription activator-interacting subunit, supports an essential link between nuclear H4 acetylation, transcriptional regulation and cell cycle control.

We describe a new method for accurately defining the sequence recognition properties of DNA-binding proteins by selecting high-affinity binding sites from random-sequence DNA. The yeast transcriptional activator protein GCN4 was coupled to a Sepharose column, and binding sites were isolated by passing short, random-sequence oligonucleotides over the column and eluting them with increasing salt concentrations. Of 43 specifically bound oligonucleotides, 40 contained the symmetric sequence TGA(C/G)TCA, whereas the other 3 contained sequences matching six of these seven bases. The extreme preference for this 7-base-pair sequence suggests that each position directly contacts GCN4. The three nucleotide positions on each side of this core heptanucleotide also showed sequence preferences, indicating their effect on GCN4 binding. Interestingly, deviations in the core and a stronger sequence preference in the flanking region were found on one side of the central C G base pair. Although GCN4 binds as a dimer, this asymmetry supports a model in which interactions on each side of the binding site are not equivalent. The random selection method should prove generally useful for defining the specificities of other DNA-binding proteins and for identifying putative target sequences from genomic DNA.A frequent goal in molecular biology is to define the nucleotide sequences required for binding by a specific transcriptional regulatory protein. One approach is to collect wild-type binding sites which occur in biologically related contexts. Sequences responsible for a particular function may then be recognized by their occurrence in most of the identified sites. However, the collection and identification of these sequences can be difficult, and the results will be biased if some of the sequences encode functions other than the one of interest. Another approach is to analyze mutated versions of a given binding site to determine which positions are functionally important. However, this method requires a large number of mutant sites and is biased by the starting wild-type sequence; related sequences which may bind the protein will not be studied.Random-sequence oligonucleotides have proven to be a useful tool for identifying and defining the sequence requirements of other genetic elements (4,11,14,16,17). The isolation of functional elements from random-sequence DNA, termed random selection, can be done quickly and with many advantages not applicable to the study of wildtype elements. First, the number of elements generated is sufficiently large to define the element precisely. Second, each sequence is very likely to contain a highly functional example of the element of interest. Third, confounding (nonrelated) elements are unlikely to be present in the surrounding DNA. Fourth, elements are localized to short, easily sequenced segments of DNA and hence are more likely to be seen over random noise.In this paper, we describe a modification of the randomselection technique (16,17) GCN4 is a 281-amino-acid protein that binds to the promot...

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In a survey of the bilayer-spanning regions of integral membrane proteins, membrane-buried proline residues were found in nearly all transport proteins examined, whereas membrane-buried regions of nontransport proteins were largely devoid of intramembranous proline residues. When amino acids from the complete sequences of representative sets of transport and nontransport membrane proteins were analyzed for the distribution of proline residues between aqueous vs. membranous domains, proline was shown to be selectively excluded from membranous domains of the nontransport proteins, in accord with expectation from energetic and structural considerations. In contrast, proline residues in transport proteins were evenly distributed between aqueous and membranous domains, consistent with the notion that functional membrane-buried proline residues are selectively included in transport proteins. As cis peptide bonds involving proline arise in proteins and have been implicated in protein dynamic processes, the cis-trans isomerization of an Xaa-Pro peptide bond (Xaa = unspecified amino acid) buried within the membrane-and the resulting redirection of the protein chain-is proposed to provide the reversible conformational change requisite for the regulation (opening/closing) of a transport channel. Parallel to this function, the relatively negative character of the carbonyl groups of Xaa-Pro peptide bonds may promote their participation as intramembranous liganding sites for positive species in proton/cation transport processes.

The yeast Ada and TBP class of Spt proteins interact in multiple complexes that are required for transcriptional regulation. We have identified Tra1p as a component of these complexes through tandem mass spectrometry analysis of proteins that associate with Ngg1p/ Ada3p. TRA1 is an essential gene and encodes a 3744-amino acid protein that is a member of a group of proteins including the catalytic subunit of DNA-dependent protein kinase, ATM and TRRAP, with carboxylterminal regions related to phosphatidylinositol 3-kinases. The interaction between Tra1p and Ada/Spt components was verified by the reciprocal coimmunoprecipitation of Ada2p and Tra1p from whole cell extracts in one or more complexes containing Spt7p. Tra1p cofractionated with Ngg1p and Spt7p through consecutive chromatography on Mono Q, DNA-cellulose, and Superose 6 columns. Binding of Tra1p to DNA-cellulose required Ada components. The association of Tra1p with two Ada⅐Spt complexes was suggested by its cofractionation with Ngg1p and Spt7p in two peaks on the Mono Q column. In the absence of Ada2p, the elution profile of Tra1p shifted to a distinct peak. Despite the similarity of Tra1p to a group of putative protein kinases, we have not detected protein kinase activity within immunoprecipitates of Tra1p or the Ada⅐Spt complexes.The ADA genes were identified in Saccharomyces cerevisiae based on their requirement for the regulated activation and repression of transcription (1-3). In initial studies, Ada2p, Ngg1p/Ada3p, and Gcn5p/Ada4p were shown to function in a complex (4 -6). The identity of Gcn5p and its human homolog hGCN5 as histone acetyltransferases suggested that one role of the complex was to modulate nucleosome structure (reviewed in Ref. 7). Biochemical analyses of the Ada proteins demonstrated that they are found in at least four high molecular weight complexes, two with sizes of more than 2 MDa and others of 900 and 200 kDa (8 -10). Other proteins identified within these complexes include the product of ADA1 (1, 11) and the products of the TBP class of SPT genes, SPT3, SPT7, SPT8, and SPT20/ADA5 (9, 12, 13). The latter group of proteins are found within at least one of the ADA-containing complexes, the SAGA complex (9). The association between the Spt and Ada proteins agrees with the functional link between the Ada proteins and TBP which was suggested by immunoprecipitation and affinity chromatography (8, 14 -16).A detailed understanding of the mechanisms and regulation of the Ada and Spt protein-containing complexes (Ada⅐Spt complexes for simplicity) requires the identification of component proteins. We now identify Tra1p as a component of the Ada⅐Spt complexes. Tra1p is a member of a group of putative protein kinases, including the catalytic subunit of human DNA-dependent protein kinase (DNA-PK CS ) and ATM, that contain a carboxyl-terminal region related to phosphatidylinositol 3-kinases (PI3K 1 ; reviewed in . In addition, Tra1p shows extensive sequence similarity throughout its entire length to the human protein TRRAP that associates...