Claudio Rivetti

Direct Observation of One-Dimensional Diffusion and Transcription by Escherichia coli RNA Polymerase

The dynamics of nonspecific and specific Escherichia coli RNA polymerase (RNAP)-DNA complexes have been directly observed using scanning force microscopy operating in buffer. To this end, imaging conditions had to be found in which DNA molecules were adsorbed onto mica strongly enough to be imaged, but loosely enough to be able to diffuse on the surface. In sequential images of nonspecific complexes, RNAP was seen to slide along DNA, performing a one-dimensional random walk. Heparin, a substance known to disrupt nonspecific RNAP-DNA interactions, prevented sliding. These observations suggest that diffusion of RNAP along DNA constitutes a mechanism for accelerated promoter location. Sequential images of single, transcribing RNAP molecules were also investigated. Upon addition of 5 microM nucleoside triphosphates to stalled elongation complexes in the liquid chamber, RNAP molecules were seen to processively thread their template at rates of 1.5 nucleotide/s in a direction consistent with the promoter orientation. Transcription assays, performed with radiolabeled, mica-bound transcription complexes, confirmed this rate, which was about three times smaller than the rate of complexes in solution. This assay also showed that the pattern of pause sites and the termination site were affected by the surface. By using the Einstein-Sutherland friction-diffusion relation the loading force experienced by RNAP due to DNA-surface friction is estimated and discussed.

Wrapping of DNA around the E.coli RNA polymerase open promoter complex

High‐resolution atomic force microscopy (AFM) and biochemical methods were used to analyze the structure of Escherichia coli RNA polymerase·σ70 (RNAP) open promoter complex (RPo). A detailed analysis of a large number of molecules shows that the DNA contour length of RPo is reduced by ∼30 nm (∼90 bp) relative to the free DNA. The DNA bend angle measured with different methods varied from 55 to 88°. The contour length reduction and the DNA bend angle were much less in inactive RNAP–DNA complexes. These results, together with previously published observations, strongly support the notion that during transcription initiation, the promoter DNA wraps nearly 300° around the polymerase. This amount of DNA bending requires an energy of 60 kJ/mol. The structural analysis of the open promoter complexes revealed that two‐thirds of the DNA wrapped around the RNAP is part of a region upstream of the transcription start site, whereas the remaining one‐third is part of the downstream region. Based on these data, a model of the σ70·RPo conformation is proposed.

Scanning force microscopy under aqueous solutions

Merely ten years after its invention, the scanning force microscope is becoming a powerful method to investigate the structure and dynamics of biological molecules under aqueous environments. From the visualization of transcription in real time to the mechanical manipulation of individual proteins, the advances made during the past year open up a vast number of exciting applications of this technique in biology.

Conformation-sensitive Antibodies against Alzheimer Amyloid-β by Immunization with a Thioredoxin-constrained B-cell Epitope Peptide

Immunotherapy against the amyloid-β (Aβ) peptide is a valuable potential treatment for Alzheimer disease (AD). An ideal antigen should be soluble and nontoxic, avoid the C-terminally located T-cell epitope of Aβ, and yet be capable of eliciting antibodies that recognize Aβ fibrils and neurotoxic Aβ oligomers but not the physiological monomeric species of Aβ. We have described here the construction and immunological characterization of a recombinant antigen with these features obtained by tandem multimerization of the immunodominant B-cell epitope peptide Aβ1-15 (Aβ15) within the active site loop of bacterial thioredoxin (Trx). Chimeric Trx(Aβ15)n polypeptides bearing one, four, or eight copies of Aβ15 were constructed and injected into mice in combination with alum, an adjuvant approved for human use. All three polypeptides were found to be immunogenic, yet eliciting antibodies with distinct recognition specificities. The anti-Trx(Aβ15)4 antibody, in particular, recognized Aβ42 fibrils and oligomers but not monomers and exhibited the same kind of conformational selectivity against transthyretin, an amyloidogenic protein unrelated in sequence to Aβ. We have also demonstrated that anti-Trx(Aβ15)4, which binds to human AD plaques, markedly reduces Aβ pathology in transgenic AD mice. The data indicate that a conformational epitope shared by oligomers and fibrils can be mimicked by a thioredoxin-constrained Aβ fragment repeat and identify Trx(Aβ15)4 as a promising new tool for AD immunotherapy.

Accurate length determination of DNA molecules visualized by atomic force microscopy : evidence for a partial B- to A-form transition on mica

Achieving the most correct estimate of the contour length of digitized DNA molecules is a key aspect of the microscopic analysis of nucleic acids by either electron microscopy (EM) or atomic force microscopy (AFM). Six different methods, that are mathematically not too complex and suited for common, practical use, have been tested here using simulated polymers in two dimensions and real DNA molecules (564, 1054, 2049 and 4297 bp long) imaged in air by AFM. The main result is that the frequently used Freeman estimator (L(F) = n(e) + square root 2n(o)) overestimates the real contour length of the polymers by about 4%. More accurate estimates are obtained with the Kulpa estimator (L(K) = 0.948n(e) + 1.343n(o)) or with the corner count estimator (L(C) = 0.980)n(e) + 1.406n(o) - 0.091n(c)). In the range of the DNA sizes and magnifications we have considered, however, the best results are obtained with an ad hoc developed routine that smoothes the DNA trace by a polynomial fitting of degree 3 over a moving window of 5 points. Under these conditions, the difference between the measured and the real contour length of the molecules is less than 0.4%. The accuracy of this procedure allowed us to reveal a discrete, size-dependent, shortening of DNA molecules deposited onto mica under low salt conditions and imaged in air by AFM. Awareness of this structural alteration, that can be attributed to a partial transition from B- to A-form DNA, may lead to a more correct interpretation of DNA molecules or protein-DNA complexes imaged by AFM.

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 λpr 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.

Genetic analysis and morphological identification of pilus-like structures in members of the genus Bifidobacterium

BackgroundCell surface pili in Gram positive bacteria have been reported to orchestrate the colonization of host tissues, evasion of immunity and the development of biofilms. So far, little if any information is available on the presence of pilus-like structures in human gut commensals like bifidobacteria.Results and discussionIn this report, Atomic Force Microscopy (AFM) of various bifidobacterial strains belonging to Bifidobacterium bifidum, Bifidobacterium longum subsp. longum, Bifidobacterium dentium, Bifidobacterium adolescentis and Bifidobacterium animalis subsp. lactis revealed the existence of appendages resembling pilus-like structures. Interestingly, these microorganisms harbour two to six predicted pilus gene clusters in their genome, with each organized in an operon encompassing the major pilin subunit-encoding gene (designated fim A or fim P) together with one or two minor pilin subunit-encoding genes (designated as fim B and/or fim Q), and a gene encoding a sortase enzyme (str A). Quantitative Real Time (qRT)-PCR analysis and RT-PCR experiments revealed a polycistronic mRNA, encompassing the fim A/P and fim B/Q genes, which are differentially expressed upon cultivation of bifidobacteria on various glycans.

High and low oxygen affinity conformations of T state hemoglobin

To understand the interplay between tertiary and quaternary transitions associated with hemoglobin function and regulation, oxygen binding curves were obtained for hemoglobin A fixed in the T quaternary state by encapsulation in wet porous silica gels. At pH 7.0 and 15°C, the oxygen pressure at half saturation (p50) was measured to be 12.4 ± 0.2 and 139 ± 4 torr for hemoglobin gels prepared in the absence and presence of the strong allosteric effectors inositol hexaphosphate and bezafibrate, respectively. Both values are in excellent agreement with those found for the binding of the first oxygen to hemoglobin in solution under similar experimental conditions. The corresponding Hill coefficients of hemoglobin gels were 0.94 ± 0.02 and 0.93 ± 0.03, indicating, in the frame of the Monod, Wyman, and Changeux model, that high and low oxygen‐affinity tertiary T‐state conformations have been isolated in a pure form. The values, slightly lower than unity, reflect the different oxygen affinity of α‐ and β‐hemes. Significantly, hemoglobin encapsulated in the presence of the weak effector phosphate led to gels that show intermediate oxygen affinity and Hill coefficients of 0.7 to 0.8. The heterogeneous oxygen binding results from the presence of a mixture of the high and low oxygen‐affinity T states. The Bohr effect was measured for hemoglobin gels containing the pure conformations and found to be more pronounced for the high‐affinity T state and almost absent for the low‐affinity T state. These findings indicate that the functional properties of the T quaternary state result from the contribution of two distinct, interconverting conformations, characterized by a 10‐fold difference in oxygen affinity and a different extent of tertiary Bohr effect. The very small degree of T‐state cooperativity observed in solution and in the crystalline state might arise from a ligand‐induced perturbation of the distribution between the high‐ and low‐affinity T‐state conformations.

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.

Time course of chemical and structural events in protein crystals measured by microspectrophotometry

The functional properties of proteins in the crystalline state have been investigated over the past 30 years by a variety of methods, including single crystal polarized absorption spectroscopy. This technique has provided information on the accumulation and equilibrium distribution of protein-ligand complexes in the crystal and, in a few cases, on the rates of interconversion of catalytic intermediates. It has been possible to detect synergistic effects in the binding of different ligands, cooperativity and half-site reactivity and even formation of active multiprotein complexes, obtained by diffusion of one small protein in the pre-formed crystals of the other. Lattice interactions restrain the conformational transitions of some proteins existing in multiple states in solution. The crystal offers the unique opportunity to analyse not only the structure but also the function of a single form of the protein. The relevance of these data to the planning and interpretation of structural studies, especially in the perspectives of time-resolved crystallography, will be discussed with reference to well-characterized systems.