Solution structure of SWI1 AT-rich interaction domain from Saccharomyces cerevisiae and its nonspecific binding to DNA

SWI1 is a subunit of the SWI/SNF complex involved in chromatin remodeling. It contains an AT-rich interaction domain (ARID) which has the potential DNA binding activity. In this study, we determined the solution structure of the SWI1 ARID domain from Saccharomyces cerevisiae by nuclear magnetic resonance spectroscopy. Yeast SWI1 ARID domain is composed of seven alpha helices, six of which are conserved among the ARID family. In addition, the DNA-binding activity of the SWI1 ARID domain was confirmed by chemical shift perturbation assay. Similar to its human homolog, the yeast SWI1 ARID domain binds DNA nonspecifically.     

DNA binding properties of the Saccharomyces cerevisiae DAT1 gene product.

The DAT1 gene of Saccharomyces cerevisiae encodes a DNA binding protein (Dat1p) that specifically recognizes the minor groove of non-alternating oligo(A).oligo(T) tracts. Sequence-specific recognition requires arginine residues found within three perfectly repeated pentads (G-R-K-P-G) of the Dat1p DNA binding domain [Reardon, B. J., Winters, R. S., Gordon, D., and Winter, E. (1993) Proc. Natl. Acad. Sci. USA 90, 11327-1131]. This report describes a rapid and simple method for purifying the Dat1p DNA binding domain and the biochemical characterization of its interaction with oligo(A).oligo(T) tracts. Oligonucleotide binding experiments and the characterization of yeast genomic Dat1p binding sites show that Dat1p specifically binds to any 11 base sequence in which 10 bases conform to an oligo(A).oligo(T) tract. Binding studies of different sized Dat1p derivatives show that the Dat1p DNA binding domain can function as a monomer. Competition DNA binding assays using poly(I).poly(C), demonstrate that the minor groove oligo(A).oligo(T) constituents are not sufficient for high specificity DNA binding. These data constrain the possible models for Dat1p/oligo(A).oligo(T) complexes, suggest that the DNA binding domain is in an extended structure when complexed to its cognate DNA, and show that Dat1p binding sites are more prevalent than previously thought.     

Molecular cloning of the actin gene from yeast Saccharomyces cerevisiae.

Two overlapping DNA fragments from yeast Saccharomyces cerevisiae containing the actin gene have been inserted into pBR322 and cloned in E.coli. Clones were identified by hybridization to complementary RNA from a plasmid containing a copy of Dictyostelium actin mRNA. One recombinant plasmid obtained (pYA102) contains a 3.93-kb Hindlll fragment, the other (pYA208) a 5.1-kb Pstl fragment, both share a common 2.2-kb fragment harboring part of the actin gene. Cloned yeast actin DNA was identified by R-loop formation and translation of the hybridized actin mRNA and by DNA sequence analysis. Cytoplasmic actin mRNA has been estimated to be about 1250 nucleotides long. There is only one type of the actin gene in S.cerevisiae.     

Cloning and functional analysis of the ubiquitin-specific protease gene UBP1 of Saccharomyces cerevisiae

In eukaryotes, both natural and engineered fusions of ubiquitin to itself or other proteins are cleaved by processing proteases after the last (Gly76) residue of ubiquitin. Using the method of sib selection, and taking advantage of the fact that bacteria such as Escherichia coli lack ubiquitin-specific enzymes, we have cloned a gene, named UBP1, of the yeast Saccharomyces cerevisiae that encodes a ubiquitin-specific processing protease. With the exception of polyubiquitin, the UBP1 protease cleaves at the carboxyl terminus of the ubiquitin moiety in natural and engineered fusions irrespective of their size or the presence of an amino-terminal ubiquitin extension. These properties of UBP1 distinguish it from the previously cloned yeast protease YUH1, which deubiquitinates relatively short ubiquitin fusions but is virtually inactive with longer fusions such as ubiquitin-beta-galactosidase. The amino acid sequence of the 809-residue UBP1 lacks significant similarities to other known proteins, including the 236-residue YUH1 protease. Null ubp1 mutants are viable, and retain the ability to deubiquitinate ubiquitin-beta-galactosidase, indicating that the family of ubiquitin-specific proteases in yeast is not limited to UBP1 and YUH1.     

Cloning and functional analysis of the arginyl-tRNA-protein transferase gene ATE1 of Saccharomyces cerevisiae

Aminoacyl-tRNA-protein transferases (Arg-transferases) catalyze post-translational conjugation of specific amino acids to the amino termini of acceptor proteins. A function of these enzymes in eukaryotes has been shown to involve the conjugation of destabilizing amino acids to the amino termini of short-lived proteins, these reactions being a part of the N-end rule pathway of protein degradation (Gonda, D. K., Bachmair, A., Wünning, I., Tobias, J. W., Lane, W. S., and Varshavsky, A. (1989) J. Biol. Chem. 264, 16700-16712). We have cloned the ATE1 gene of the yeast Saccharomyces cerevisiae which encodes arginyl-tRNA-protein transferase. ATE1 gives rise to a approximately 1.6-kilobase mRNA and codes for a 503-residue protein. Expression of the yeast ATE1 gene in Escherichia coli, which lacks Arg-transferases, was used to show that the ATE1 protein possesses the Arg-transferase activity. Null ate1 mutants are viable but lack the Arg-transferase activity and are unable to degrade those substrates of the N-end rule pathway that start with residues recognized by the Arg-transferase.     

Nucleotide sequence and functional analysis of the RAD1 gene of Saccharomyces cerevisiae.

The RAD1 gene of Saccharomyces cerevisiae is involved in excision repair of damaged DNA. The nucleotide sequence of the RAD1 gene presented here shows an open reading frame of 3,300 nucleotides. Two ATG codons occur in the open reading frame at positions +1 and +334, respectively. Since a deletion of about 2.7 kilobases of DNA from the 5' region of the RAD1 gene, which also deletes the +1 ATG and 11 additional codons in the RAD1 open reading frame, partially complements UV sensitivity of a rad1 delta mutant, we examined the role of the +1 ATG and +334 ATG codons in translation initiation of RAD1 protein. Mutation of the +1 ATG codon to ATC affected the complementation ability of the RAD1 gene, whereas mutation of the +334 ATG codon to ATC showed no discernible effect on RAD1 function. These results indicate that translation of RAD1 protein is initiated from the +1 ATG codon. Productive in-frame RAD1-lacZ fusions showed that the RAD1 open reading frame is expressed in yeasts. The RAD1-encoded protein contains 1,100 amino acids with a molecular weight of 126,360.     

Identification and characterization of regulatory elements in the phosphoenolpyruvate carboxykinase gene PCK1 of Saccharomyces cerevisiae

Phosphoenolpyruvate carboxykinase is a key enzyme in gluconeogenesis. The expression of the PCK1 gene in Saccharomyces cerevisiae is strictly regulated and dependent on the carbon source provided. Two upstream activation sites (UAS1_PCK1 and UAS2_PCK1) and one upstream repression site (URS_PCK1) were localized by detailed deletion analysis. The efficacy of these three promoter elements when separated from each other was confirmed by investigations using heterologous promoter test plasmids. Activation mediated by UAS1_PCK1 or UAS2_PCK1 did not occur in the presence of glucose, indicating that these elements are essential for glucose derepression. The repressing effect caused by URS_PCK1 was much stronger in glucose-grown cells than in ethanol-grown cells.     

The population biology and evolutionary significance of Ty elements in Saccharomyces cerevisiae

The basic structure and properties of Ty elements are considered with special reference to their role as agents of evolutionary change. Ty elements may generate genetic variation for fitness by their action as mutagens, as well as by providing regions of portable homology for recombination. The mutational spectra generated by Ty 1 transposition events may, due to their target specificity and gene regulatory capabilities, possess a higher frequency of adaptively favorable mutations than spectra resulting from other types of mutational processes. Laboratory strains contain between 25–35 elements, and in both these and industrial strains the insertions appear quite stable. In contrast, a wide variation in Ty number is seen in wild isolates, with a lower average number/genome. Factors which may determine Ty copy number in populations include transposition rates (dependent on Ty copy number and mating type), and stabilization of Ty elements in the genome as well as selection for and against Ty insertions in the genome. Although the average effect of Ty transpositions are deleterious, populations initiated with a single clone containing a single Ty element steadily accumulated Ty elements over 1,000 generations. Direct evidence that Ty transposition events can be selectively favored is provided by experiments in which populations containing large amounts of variability for Ty1 copy number were maintained for 100 generations in a homogeneous environment. At their termination, the frequency of clones containing 0 Ty elements had decreased to 0.0, and the populations had became dominated by a small number of clones containing >0 Ty elements. No such reduction in variability was observed in populations maintained in a structured environment, though changes in Ty number were observed. The implications of genetic (mating type and ploidy) changes and environmental fluctuations for the long-term persistence of Ty elements within the S. cerevisiae species group are discussed.     

Organization of the ribosomal RNA gene cluster in the yeast Saccharomyces cerevisiae

The chromosomal organization of the ribosomal RNA gene cluster from Saccharomyces cerevisiae was investigated. 18 S rRNA R-loops were formed with unfractionated high molecular weight DNA crosslinked once per 2.7 × 10³ bases with trioxsalen and observed in the electron microscope. Almost all the R-loops were found in very long continuous 9.34 ± 0.18 × 10³ base repeating units. In addition, molecules were found at a frequency of one to two per genome equivalent of rDNA where several rRNA genes were linked to long stretches of non-rDNA. These results suggest that rDNA is arranged in a single tandem repetitive cluster of 100 to 140 genes flanked on one or both sides by non-rDNA.     

Condensin binding at distinct and specific chromosomal sites in the Saccharomyces cerevisiae genome

Mitotic chromosome condensation is chiefly driven by the condensin complex. The specific recognition (targeting) of chromosomal sites by condensin is an important component of its in vivo activity. We previously identified the rRNA gene cluster in Saccharomyces cerevisiae as an important condensin-binding site, but both genetic and cell biology data suggested that condensin also acts elsewhere. In order to characterize the genomic distribution of condensin-binding sites and to assess the specificity of condensin targeting, we analyzed condensin-bound sites using chromatin immunoprecipitation and hybridization to whole-genome microarrays. The genomic condensin-binding map shows preferential binding sites over the length of every chromosome. This analysis and quantitative PCR validation confirmed condensin-occupied sites across the genome and in the specialized chromatin regions: near centromeres and telomeres and in heterochromatic regions. Condensin sites were also enriched in the zones of converging DNA replication. Comparison of condensin binding in cells arrested in G(1) and mitosis revealed a cell cycle dependence of condensin binding at some sites. In mitotic cells, condensin was depleted at some sites while enriched at rRNA gene cluster, subtelomeric, and pericentromeric regions.