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Claudio Rivetti

Publications and source records attributed to Claudio Rivetti.

11 recordsLinked to original sources
PubMed2006

DNA condensation and cell transfection properties of guanidinium calixarenes: dependence on macrocycle lipophilicity, size, and conformation.

Calix[n]arenes functionalized with guanidinium groups at the upper rim and alkyl chains at the lower rim bind to DNA, condense it, and in some cases, promote cell transfection depending on their structure and lipophilicity. Atomic force microscopy (AFM) studies indicate that upon DNA binding the hydrophobic association of the lipophilic chains of cone guanidinium calix[4]arenes drives the formation of intramolecular DNA condensates, characterized by DNA loops emerging from a dense core. Furthermore, hexyl and octyl chains confer to these calixarenes cell transfection capabilities. Conversely, larger and conformationally mobile calix[6]- and calix[8]arene methoxy derivatives form intermolecular aggregates characterized by "gorgonlike" structures composed of multiple plectomenes. These adducts, in which interstrand connections are dominated by electrostatic interactions, fail to promote cell transfection. Finally, calix[4]arenes in a 1,3-alternate conformation show an intermediate behavior because they condense DNA, but the process is driven by charge-charge interactions.

Calixarenes↗
PubMed2006

Collision events between RNA polymerases in convergent transcription studied by atomic force microscopy.

Atomic force microscopy (AFM) has been used to image, at single molecule resolution, transcription events by Escherichia coli RNA polymerase (RNAP) on a linear DNA template with two convergently aligned lambda(pr) promoters. For the first time experimentally, the outcome of collision events during convergent transcription by two identical RNAP has been studied. Measurement of the positions of the RNAP on the DNA, allows distinction of open promoter complexes (OPCs) and elongating complexes (EC) and collided complexes (CC). This discontinuous time-course enables subsequent analysis of collision events where both RNAP remain bound on the DNA. After collision, the elongating RNAP has caused the other (usually stalled) RNAP to back-track along the template. The final positions of the two RNAP indicate that these are collisions between an EC and a stalled EC (SEC) or OPC (previously referred to as sitting-ducks). Interestingly, the distances between the two RNAP show that they are not always at closest approach after 'collision' has caused their arrest.

DNA↗
PubMed2006

Structural and functional properties of lengsin, a pseudo-glutamine synthetase in the transparent human lens.

Lengsin (LGS) is an abundant transcript in the human lens, encoding a predicted polypeptide similar to glutamine synthetase (GS). We show that a major alternatively spliced product of LGS codes for a 57kDa polypeptide that assembles into a catalytically inactive dodecamer, cross-reacts with anti-GS antibodies, and is expressed at high levels in transparent, but not cataractous, human lenses. Based on this characteristic oligomeric organization, preferential expression in the transparent lens, and amyloid-beta association previously reported for GS, a potential chaperone-like role of LGS has been investigated. We find that LGS has six binding sites for the hydrophobic surface probe bis-ANS and relieves cellular toxicity caused by amyloid-beta expression in a folding-impaired yeast mutant. While documenting the structural similarity between LGS and prokaryotic GS-I, the data rule out any involvement of lengsin in glutamine biosynthesis and suggest an unrelated role that may be important for lens homeostasis and transparency.

Alternative Splicing↗
PubMed2004

DNA condensation and self-aggregation of Escherichia coli Dps are coupled phenomena related to the properties of the N-terminus.

Escherichia coli Dps (DNA-binding proteins from starved cells) is the prototype of a DNA-protecting protein family expressed by bacteria under nutritional and oxidative stress. The role of the lysine-rich and highly mobile Dps N-terminus in DNA protection has been investigated by comparing the self-aggregation and DNA-condensation capacity of wild-type Dps and two N-terminal deletion mutants, DpsDelta8 and DpsDelta18, lacking two or all three lysine residues, respectively. Gel mobility and atomic force microscopy imaging showed that at pH 6.3, both wild type and DpsDelta8 self-aggregate, leading to formation of oligomers of variable size, and condense DNA with formation of large Dps-DNA complexes. Conversely, DpsDelta18 does not self-aggregate and binds DNA without causing condensation. At pH 8.2, DpsDelta8 and DpsDelta18 neither self-aggregate nor cause DNA condensation, a behavior also displayed by wild-type Dps at pH 8.7. Thus, Dps self-aggregation and Dps-driven DNA condensation are parallel phenomena that reflect the properties of the N-terminus. DNA protection against the toxic action of Fe(II) and H2O2 is not affected by the N-terminal deletions either in vitro or in vivo, in accordance with the different structural basis of this property.

Bacterial Proteins↗
PubMed2004

Distinct roles of transcription factors TFIIIB and TFIIIC in RNA polymerase III transcription reinitiation.

Eukaryotic RNA polymerase (Pol) III is recruited to target promoters by a stable preinitiation complex containing transcription factors TFIIIC and TFIIIB. After the first transcription cycle, reinitiation proceeds through facilitated recycling, a process by which the terminating Pol III rapidly reloads onto the same transcription unit. Here, we show that Pol III is repeatedly recaptured in vitro by the first transcribed gene, even in the presence of a juxtaposed competitor promoter complex, thus suggesting that facilitated recycling is not merely due to a stochastic reassociation process favored by the small size of class III genes. The transcription factor requirements for facilitated reinitiation were investigated by taking advantage of Pol III templates that support both TFIIIC-dependent and TFIIIC-independent transcription. A TFIIIC-less transcription system, in which TFIIIB was reconstituted from recombinant TATA box-binding protein and Brf1 proteins and a crude fraction containing the Bdp1 component, was sufficient to direct efficient Pol III recycling on short ( approximately 100 bp) class III genes. Unexpectedly, however, on longer (>300 bp) transcription units, reinitiation in the presence of TFIIIB alone was compromised, and TFIIIC was further required to reestablish a high reinitiation rate. Transcription reinitiation was also severely impaired when recombinant Bdp1 protein replaced the corresponding crude fraction in reconstituted TFIIIB. The data reveal an unexpected complexity in the Pol III reinitiation mechanism and suggest the existence of a handing-back network between Pol III, TFIIIC, and TFIIIB on actively transcribed class III genes.

DNA-(Apurinic or Apyrimidinic Site) Lyase↗
PubMed2004

Transcription reinitiation properties of bacteriophage T7 RNA polymerase.

We have analyzed the kinetics of transcription initiation and reinitiation in vitro by one of the simplest and best characterized transcription machineries, bacteriophage T7 RNA polymerase (T7 RNAP). We used a short transcription unit with T7-specific promoter and terminator elements as a template, and a heparin challenge assay to distinguish the first transcription cycle from the subsequent ones. When present at sub-saturating concentrations with respect to template DNA, T7 RNAP could find its promoter and initiate the first transcription cycle in less than 1min. Reinitiation under the same conditions proceeded more slowly, with only three new transcription cycles being completed in 10min; after that time, reinitiation practically ceased. When the polymerase was in large excess over template DNA, however, reinitiation proceeded linearly for longer times, at a rate of 1cycle/min. Our data suggest that polymerase recycling represents a critical step in T7 RNAP transcription, and that such a step may become rate-limiting for transcription at sub-saturating polymerase concentrations.

DNA↗
PubMed2003

Gene expression profiling in human age-related nuclear cataract.

PURPOSE: To identify genes that are differentially expressed in age-related nuclear cataracts compared to transparent human lenses. METHODS: Total RNA was extracted from pools of central 5 mm capsulorrhexis epithelial samples microdissected at surgery from eyes with nuclear cataract or from age-matched transparent lenses (post-mortem). mRNA levels in the two samples were compared by hybridization to DNA microarrays (GeneFilter GF211) containing 4,132 known human genes. Only mRNAs consistently modulated over four comparisons were retained for analysis. A subset of the mRNA expression differences thus identified were verified and confirmed by Real-Time RT-PCR. Expressed and modulated genes were categorized according to the Gene Ontology classification. RESULTS: The data revealed 262 genes that are downregulated and 7 that are upregulated by a factor of 2.5 or more in epithelial samples from cataractous lenses compared with transparent lenses. The highest content of downregulated genes was found in the functional classes "Signal transduction", "Regulation of cell proliferation", and "Protein modification". The "Response to oxidative stress" class was one of the least modulated. Among downregulated genes, we found several mRNAs coding for transcription/translation-related proteins, heat shock proteins 70 and 27, two ubiquitin-conjugating enzymes, two subunits of the cytoskeletal/chaperone protein, tubulin, betaA4-crystallin, and a group of Alzheimer-related proteins, including presenilin 1 and presenilin 2. CONCLUSIONS: An extensive mRNA downregulation accompanies the development of the nuclear type of human age-related cataract. A few genes and classes of genes more prominently displaying this type of response have been identified. Altogether, the data indicate the tendency, in age-related nuclear cataract, towards a shutdown of de novo RNA and protein biosynthesis rather than an upregulation of cell defense components such as chaperones and various kinds of antioxidative or detoxifying proteins.

Aged↗
PubMed2003

Visualizing RNA extrusion and DNA wrapping in transcription elongation complexes of bacterial and eukaryotic RNA polymerases.

Transcription ternary complexes of Escherichia coli RNA polymerase and yeast RNA polymerase III have been analyzed by atomic force microscopy. Using the method of nucleotide omission and different DNA templates, E.coli RNAP has been stalled at position +24, +70 and +379 and RNAP III at position +377 from the starting site. Conformational analysis of E.coli RNAP elongation complexes reveals an average DNA compaction of 22nm and a DNA deformation compatible with approximately 180 degrees DNA wrapping against the enzyme. The extent of protein-DNA interaction attributed to wrapping, however, is less than that of corresponding open promoter complexes. DNA wrapping was also observed for RNAP III elongation complexes, which showed a DNA compaction of 30nm. When the RNA polymerases were stalled far from the promoter (+379 and +377), the growing RNA transcript was often visible and it was prevalently seen exiting from the enzyme on the opposite side relative to the smallest angle subtended by the upstream and downstream DNA arms. Surprisingly, we found that many complexes had a second RNAP, not involved in transcription, bound to the growing RNA of a ternary complex. DNA wrapping in the elongation complex suggests a possible mechanism by which the polymerase may overcome the physical barrier to transcription imposed by the nucleosomes.

Bacterial Proteins↗
PubMed2003

Imaging transcription complexes with the Atomic Force Microscope.

Recent developments in sample deposition and image analysis have shown that the Atomic Force Microscope is a valuable tool for the structural investigation of transcription complexes. When deposited under conditions that allow molecular equilibration onto the substrate, transcription complexes behave as worm-like chains and the mean square end-to-end distance can readily be used to determine the protein induced DNA bend angle. Measurements of the DNA contour length by means of accurate image processing procedures have revealed a DNA compaction in transcription complexes which is compatible with wrapping of the DNA against the surface of the RNA Polymerase. The methods presented have to be considered of general practical use for imaging protein-DNA complexes.

DNA↗
PubMed2002

A nick-sensing DNA 3'-repair enzyme from Arabidopsis.

DNA single-strand breaks, a major cause of genome instability, often produce unconventional end groups that must be processed to restore terminal moieties suitable for reparative DNA gap filling or ligation. Here, we describe a bifunctional repair enzyme from Arabidopsis (named AtZDP) that recognizes DNA strand breaks and catalyzes the removal of 3'-end-blocking lesions. The isolated C-terminal domain of AtZDP is by itself competent for 3'-end processing, but not for strand break recognition. The N-terminal domain instead contains three Cys(3)-His zinc fingers and recognizes various kinds of damaged double-stranded DNA. Gapped DNA molecules are preferential targets of AtZDP, which bends them by approximately 73 degrees upon binding, as measured by atomic force microscopy. Potential partners of AtZDP were identified in the Arabidopsis genome using the human single-strand break repairosome as a reference. These data identify a novel pathway for single-strand break repair in which a DNA-binding 3'-phosphoesterase acts as a "nick sensor" for damage recognition, as the catalyst of one repair step, and possibly as a nucleation center for the assembly of a fully competent repair complex.

Amino Acid Sequence↗