Molecular mechanism by which the nucleoid occlusion factor, SlmA, keeps cytokinesis in checkNucleoid occlusion (NO) restricts bacterial cell division to prevent chromosome guillotining in the cell midzone when replication or segregation is delayed. Structural work suggests that the NO factor SlmA (synthetic lethal with a defective Min system) interferes with formation of the cytokinetic Z-ring by altering associations between FtsZ protofilaments.

We report a direct technique for determining the binding sites of small molecules on naturally occurring heterogeneous DNA. Methidiumpropyl-EDTAFe(II) [MPE-Fe(II)] cleaves double helical DNA with low sequence specificity. Using a combination of MPE-Fe(H) cleavage of drug-protected DNA fragments and Maxam-Gilbert gel methods of sequence analysis, we have determined the preferred binding sites on a Rsa I-EcoRI restriction fragment from pBR322 for the intercalator actinomycin D and the minor groove binders netropsin and distamycin A. Netropsin and distamycin A gave identical DNA cleavage-inhibition patterns and bound preferentially to A+T-rich regions with a minimal protected site offour base pairs. We were able to observe the effect ofincreasing concentration on site selection by netropsin and distamycin A. Actinomycin D afforded a completely different cleavage-inhibition pattern, with 4-to 16-base-pair-long protected regions centered around one or more G-C base pairs.

AP-2 is a retinoic acid-inducible and developmentally regulated activator of transcription. We have cloned an alternative AP-2 transcript (AP-2B) from the human teratocarcinoma cell line PA-1, which encodes a protein differing in the C terminus from the previously isolated AP-2 protein (AP-2A). This protein contains the activation domain of AP-2 and part of the DNA binding domain but lacks the dimerization domain which is necessary for DNA binding. Analysis of overlapping genomic clones spanning the entire AP-2 gene proves that AP-2A and AP-2B transcripts are alternatively spliced from the same gene. Both transient and stable transfection experiments show that AP-2B inhibits AP-2 transactivator function, as measured by an AP-2-responsive chloramphenicol acetyltransferase reporter plasmid. Furthermore, constitutive AP-2B expression in PA-1 cells causes a retinoic acid-resistant phenotype, anchorage-independent growth in soft agar, and tumorigenicity in nude mice, in a fashion similar to transformation of these cells by oncogenes. To determine the mechanism by which AP-2B exerts its inhibitory function, we purified bacterially expressed AP-2A and AP-2B proteins. While bacterial AP-2B does not bind an AP-2 consensus site, it strongly inhibits binding of the endogenous AP-2 present in PA-1 cell nuclear extracts. However, DNA sequence-specific binding of bacterially expressed AP-2A cannot be inhibited by bacterially expressed AP-2B. Therefore, inhibition of AP-2 activity by the protein AP-2B may require an additional factor or modification supplied by nuclear extracts.

The binding of a 19-mer guanosine-rich oligodeoxyribonucleotide, TG3TG4TG4TG3T (ODN 1), to a complementary polypurine DNA target was investigated by DNase I footprinting and restriction endonuclease protection assays. Monovalent cations inhibited intermolecular purine-purine-pyrimidine triple-helical DNA formation, with K+ and Rb+ being most effective, followed by NH4+ and Na+. Li+ and Cs+ had little to no effect. Similar results were observed with the G/A-rich oligonucleotide AG3AG4AG4AG3AGCT. Kinetic studies indicated that monovalent cations interfered with oligonucleotide-duplex DNA association but did not significantly promote triplex dissociation. The observed order of monovalent cation inhibition of triplex formation is reminiscent of their effect on tetraplex formation with G/T-rich oligonucleotides. However, using electrophoretic mobility shift assays we found that the oligonucleotide ODN 1 did not appear to form a four-stranded species under conditions promoting tetraplex formation. Taken together, our data suggest that processes other than the self-association of oligonucleotides into tetraplexes might be involved in the inhibitory effect of monovalent cations on purine-pyrimidine-purine triplex formation.

Histone deacetylases (HDACs), enzymes involved in chromatin remodeling, are promising targets for anticancer drug development. Several HDAC inhibitors (HDACi) are in clinical trials. One limitation of present HDACi is their nonspecificity, affecting many HDACs with similar effectiveness. We have identified a small molecule, the sesquiterpene lactone parthenolide (PN), which specifically depletes HDAC1 protein without affecting other class I/II HDACs. HDAC1 depletion occurred through proteasomal degradation and resulted in transcriptional consequences comparable to those observed with pan-HDACi. Surprisingly, HDAC1 depletion did not occur through the inflammation mediator IKK2, a known PN target and regulator of HDAC1. Rather, PN promoted HDAC1 depletion and cell death through the DNA-damage-transducer ataxia telangiectasia mutated. Our study suggests that modulating cellular HDAC protein levels with small molecules provides an alternative approach to specific HDAC inhibition and effective cancer treatment.

Background: MicroRNA processing is a tightly controlled multistep process involving accessory proteins. Results: Expression of microRNAs 15a and 16 is controlled by nucleolin expression and localization. Conclusion: Nucleolin facilitates the biogenesis of microRNAs 15a and 16 through direct interaction with the microprocessor complex. Significance: Nucleolin represents a novel component of the microRNA processing pathway.

No abstract

Commitment of a TATA box-driven class II gene to transcription requires binding of only one transcription factor, TFIID. Additional factors (TFIIB, TFIIE, and RNA polymerase U) do not remain associated with the TFIID-promoter complex during the course of transcription. This indicates that there are two intermediates along the transcription reaction pathway which may be potential targets for the regulation of gene expression.A basal level of specific transcription from many class II genes requires the coordinated interaction of several cellular proteins and the TATA box region within the minimal promoter (for reviews, see references 1 and 10). Biochemical dissection of HeLa cell nuclear extracts has defined three chromatographic fractions, termed transcription factors TFIIB, TFIID, and TFIIE, which in addition to RNA polymerase II are required for reconstituting transcription in vitro (9,12). TFIID has been shown by DNase I footprinting to bind specifically to the TATA box (10, 13), this step being a prerequisite for functional preinitiation complex assembly (14). The exact roles of the other transcription factors remain unclear.Significant headway towards understanding the mechanism of class III gene transcription was achieved after an analysis of transcription factor-promoter interactions by a template challenge assay (8). This assay monitors the ability of one gene, upon preincubation with a subset of the transcription factors, to stably sequester one or more factors and thus exclude transcription of a second gene added subsequently (along with the remaining transcription factors). Through these experiments, it was possible to determine both the minimal number and identities of the transcription factors required to form both stable and metastable preinitiation complexes.We have applied a template challenge analysis to investigate the assembly of preinitiation complexes on class II genes. Our model system consisted of the core promoter from the major late promoter of adenovirus (-53 to +10, relative to the start of initiation), which drives transcription of a synthetic DNA template (C2AT, or G-less cassette) for which a rapid in vitro transcription assay has been described (12). Differentiation between the products of the two templates was achieved by employing cassettes of different length. This has allowed us to determine the minimum factor requirement necessary for commitment of a class II gene to transcription (template commitment) and the stability of subsequent transcription complexes.Factor Fig. 1A. Preincubation of a single template with a complete set of required transcription factors completely prevented transcription from a second template during the course of a 60-min reaction. Such an effect reflects sequestration of a limiting component(s) within a complex which is resistant to challenge by an initially naked DNA molecule. In order to determine the minimal requirement for template commitment, subsets of transcription factors were preincubated with the major late promoter. TFIID alone was sufficie...