Reducing a DNA molecule's translocation speed in a solid-state nanopore is a key step toward rapid single molecule identification. Here we demonstrate that DNA translocation speeds can be reduced by an order of magnitude over previous results. By controlling the electrolyte temperature, salt concentration, viscosity, and the electrical bias voltage across the nanopore, we obtain a 3 base/micros translocation speed for 3 kbp double-stranded DNA in a 4-8 nm diameter silicon nitride pore. Our results also indicate that the ionic conductivity inside such a nanopore is smaller than it is in bulk.

Voltage biased solid-state nanopores are used to detect and characterize individual single stranded DNA molecules of fixed micrometer length by operating a nanopore detector at pH values greater than approximately 11.6. The distribution of observed molecular event durations and blockade currents shows that a significant fraction of the events obey a rule of constant event charge deficit (ecd) indicating that they correspond to molecules translocating through the nanopore in a distribution of folded and unfolded configurations. A surprisingly large component is unfolded. The result is an important milestone in developing solid-state nanopores for single molecule sequencing applications.

The authors measured ionic current blockages caused by protein translocation through voltage-biased silicon nitride nanopores in ionic solution. By calculating the mean amplitude, time duration, and the integral of current blockages, they estimated the relative charge and size of protein molecules at a single molecule level. The authors measured the change in protein charge of bovine serum albumin (BSA) protein induced by pH variation. They also confirmed that BSA molecules indeed traverse nanopores using an improved chemiluminescent analysis. They demonstrated that a larger protein fibrinogen could be distinguished from BSA by a solid-state nanopore measurement.

A prior genetic study indicated that activity of Sir silencing proteins at a hypothetical AGE locus is essential for long life span. In this model, the SIR4-42 mutation would direct the Sir protein complex to the AGE locus, giving rise to a long life span. We show by indirect immunofluorescence that Sir3p and Sir4p are redirected to the nucleolus in the SIR4-42 mutant. Furthermore, this relocalization is dependent on both UTH4 a novel yeast gene that extends life span, and its homologue YGL023. Strikingly, the Sir complex is relocalized from telomeres to the nucleolus in old wild-type cells. We propose that the rDNA is the AGE locus and that nucleolar function is compromised in old yeast cells in a way that may be mitigated by targeting of Sir proteins to the nucleolus.

The CCAAT-binding factor is a conserved heteromeric transcription factor that binds to CCAAT box-containing upstream activation sites (UASs) within the promoters of numerous eukaryotic genes. The The transcriptional activation of gene expression is mediated by the binding of distinct regulatory factors to specific cis-acting DNA sequence elements, referred to as enhancers or upstream activation sites (UASs), located within the promoters of eukaryotic genes. Many of these transcription factors are composed of multiple polypeptides that interact in a specific manner to form an oligomeric complex that is capable of recognizing specific cisacting DNA sequence elements. Several families of hetero-oligomeric transcription factors have been identified in eukaryotes (McKnight et al. 1987;Olesen et al. 1987;

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The CCAAT-binding factor is an evolutionarily conserved heteromeric transcription factor that binds to CCAAT box-containing upstream activation sites within the promoters of numerous eukaryotic genes. The CCAAT-binding factor from Saccharomyces cerevisiae is a heterotetramer that contains the subunits Hap2p, Hap3p, Hap4p, and Hap5p and that functions in the activation of genes involved in respiratory metabolism. Here we describe the isolation of the cDNA encoding the Schizosaccharomyces pombe homolog of Hap5p, designated php5؉ . We have shown that Php5p is a subunit of the CCAAT-binding factor in fission yeast and is required for transcription of the S. pombe cyc1 ؉ gene. Analysis of the evolutionarily conserved regions of Hap5p, Php5p, and the mammalian homolog CBF-C revealed two essential domains within Hap5p that are required for DNA binding and transcriptional activation. One is an 87-amino-acid core domain that is conserved among Hap5p, Php5p, and CBF-C and that is required for the assembly of the Hap2p-Hap3p-Hap5p heterotrimer both in vitro and in vivo. A second domain that is essential for the recruitment of Hap4p into the CCAAT-binding complex was identified in Hap5p and Php5p.The transcriptional activation of gene expression is mediated by the binding of distinct regulatory factors to specific cis-acting DNA sequence elements, referred to as enhancers or upstream activation sites, located within the promoters of eukaryotic genes. Many of the DNA-binding transcriptional activators interact with specific DNA sequence elements as either homodimers or heterodimers and can be classified into several groups according to the structural motifs that are necessary for dimerization (41). Other transcriptional activators are heteromeric complexes consisting of multiple polypeptides that interact in a specific manner to recognize cis-acting promoter sequences. Examples of such protein complexes include the herpes simplex virus activator VP16 that forms a complex with cellular DNA-binding proteins, including Oct-1, to activate the transcriptional machinery (13,34,44); the interferonstimulated complex that binds specifically to the interferonstimulated response element (9,24,28,29); the GA-binding protein complex that is required for VP16-mediated activation of herpes simplex virus immediate-early genes (27, 55); and the core binding protein complex that promotes T-cell-specific expression of several genes (57). In all of these cases, one polypeptide from the complex can bind to DNA in a sitespecific manner in the absence of the other proteins from the complex.The CCAAT-binding factor is a heteromeric DNA-binding complex that binds to promoter elements containing the pentanucleotide CCAAT sequence. In the yeast Saccharomyces cerevisiae, the CCAAT-binding factor contains four subunits, designated Hap2p, Hap3p, Hap4p, and Hap5p (11,18,35,38), that are required for the transcriptional activation of numerous nuclear genes whose products are involved in mitochondrial functions (12, 61). The HAP2, HAP3, and HAP4 genes were ...

The CCAAT-binding factor (CBF) is an evolutionarily conserved multimeric transcriptional activator in eukaryotes. In Saccharomyces cerevisiae, the CCAAT-binding factor is composed of four subunits, termed Hap2p, Hap3p, Hap4p, and Hap5p. The Hap2p/Hap3p/Hap5p heterotrimer is the DNA-binding component of the complex that binds to the consensus 5-CCAAT-3 sequence in the promoter of target genes. The Hap4p subunit contains the transcriptional activation domain necessary for stimulating transcription after interacting with Hap2p/Hap3p/Hap5p. In this report, we demonstrate that Hap2p, Hap3p, and Hap5p assemble via a one-step pathway requiring all three subunits simultaneously, as opposed to the mammalian CCAAT-binding factor which has been shown to assemble via a two-step pathway with CBF-A (Hap3p homolog) and CBF-C (Hap5p homolog) forming a stable dimer before CBF-B (Hap2p homolog) can interact. We have also found that the interaction of Hap4p with Hap2p/Hap3p/Hap5p requires DNA binding as a prerequisite. To further understand the protein-protein and protein-DNA interactions of this transcription factor, we identified the minimal domain of Hap4p necessary for interaction with the Hap2p/Hap3p/Hap5p-DNA complex, and we demonstrate that this domain is sufficient to complement the respiratory deficiency of a hap4⌬ mutant and activate transcription when fused with the VP16 activation domain. These studies provide a further understanding of the assembly of the yeast CCAAT-binding factor at target promoters and raise a number of questions concerning the protein-protein and protein-DNA interactions of this multisubunit transcription factor.Saccharomyces cerevisiae is a respirofermentative yeast that represses respiratory metabolism when growing in medium containing glucose as the sole carbon source, even in an oxygenated environment (14, 53). Following glucose depletion, cells undergo a major reprogramming of gene expression, known as the diauxic shift, to activate the genes that encode proteins needed for respiration and gluconeogenesis (12,16,30,45). Thus, the organism can utilize the ethanol that was generated during the fermentative metabolism. The CCAAT-binding factor (CBF; the Hap2p/ Hap3p/Hap4p/Hap5p complex) is one of the transcriptional activators responsible for the activation of many of the genes involved in respiratory metabolism (12,16,45,57), as well as other genes needed for other metabolic functions, such as ammonia assimilation (10, 11, 41).The CCAAT-binding factor is a multisubunit transcriptional activator that binds to the 5Ј-CCAAT-3Ј consensus elements within promoters (6, 36). This activator is unique among DNAbinding proteins in that it requires three heterologous subunits, termed Hap2p, Hap3p, and Hap5p, for DNA-binding activity (35,38). The Hap2p/Hap3p/Hap5p trimer has been shown to be sufficient for CCAAT-specific binding at target promoters (38); however, this complex lacks the ability to activate transcription. A fourth subunit of the complex, termed Hap4p, is necessary for transcriptional activat...