Measuring the kinetics of amyloid fibril elongation using quartz crystal microbalances.

Kinetic measurements of amyloid growth provide insight into the free energy landscape of this supramolecular process and are crucial in the search for potent inhibitors of the main disorders with which it is associated, including Alzheimer's and Parkinson's diseases and Type II diabetes. In recent years, a new class of surface-bound biosensor assays, e.g., those based on surface plasmon resonance (SPR) and the quartz crystal microbalance (QCM) have been established as extremely valuable tools for kinetic measurements of amyloid formation. Here we describe detailed protocols of how QCM techniques can be used to monitor the elongation of amyloid fibrils in real time and to study the influence of external factors on the kinetics of amyloid growth with unprecedented accuracy.

Elongation Factor ELL (Eleven-Nineteen Lysine-rich Leukemia) Acts as a Transcription Factor for Direct Thrombospondin-1 Regulation

The eleven-nineteen lysine-rich leukemia (ELL) gene undergoes translocation and fuses in-frame to the multiple lineage leukemia gene in a substantial proportion of patients suffering from acute forms of leukemia. Studies show that ELL indirectly modulates transcription by serving as a regulator for transcriptional elongation as well as for p53, U19/Eaf2, and steroid receptor activities. Our in vitro and in vivo data demonstrate that ELL could also serve as a transcriptional factor to directly induce transcription of the thrombospondin-1 (TSP-1) gene. Experiments using ELL deletion mutants established that full-length ELL is required for the TSP-1 up-regulation and that the trans-activation domain likely resides in the carboxyl terminus. Moreover, the DNA binding domain may localize to the first 45 amino acids of ELL. Not surprisingly, multiple lineage leukemia-ELL, which lacks these amino acids, did not induce expression from the TSP-1 promoter. In addition, the ELL core-response element appears to localize in the -1426 to -1418 region of the TSP-1 promoter. Finally, studies using zebrafish confirmed that ELL regulates TSP-1 mRNA expression in vivo, and ELL could inhibit zebrafish vasculogenesis, at least in part, through up-regulating TSP-1. Given the importance of TSP-1 as an anti-angiogenic protein, our findings may have important ramifications for better understanding cancer.

Xenopus cytoplasmic linker-associated protein 1 (XCLASP1) promotes axon elongation and advance of pioneer microtubules

Dynamic microtubules (MTs) are required for neuronal guidance, in which axons extend directionally toward their target tissues. We found that depletion of the MT-binding protein Xenopus cytoplasmic linker-associated protein 1 (XCLASP1) or treatment with the MT drug Taxol reduced axon outgrowth in spinal cord neurons. To quantify the dynamic distribution of MTs in axons, we developed an automated algorithm to detect and track MT plus ends that have been fluorescently labeled by end-binding protein 3 (EB3). XCLASP1 depletion reduced MT advance rates in neuronal growth cones, very much like treatment with Taxol, demonstrating a potential link between MT dynamics in the growth cone and axon extension. Automatic tracking of EB3 comets in different compartments revealed that MTs increasingly slowed as they passed from the axon shaft into the growth cone and filopodia. We used speckle microscopy to demonstrate that MTs experience retrograde flow at the leading edge. Microtubule advance in growth cone and filopodia was strongly reduced in XCLASP1-depleted axons as compared with control axons, but actin retrograde flow remained unchanged. Instead, we found that XCLASP1-depleted growth cones lacked lamellipodial actin organization characteristic of protrusion. Lamellipodial architecture depended on XCLASP1 and its capacity to associate with MTs, highlighting the importance of XCLASP1 in actin-microtubule interactions.

Quantitative regulation of FLC via coordinated transcriptional initiation and elongation

The basis of quantitative regulation of gene expression is still poorly understood. In Arabidopsis thaliana, quantitative variation in expression of FLOWERING LOCUS C (FLC) influences the timing of flowering. In ambient temperatures, FLC expression is quantitatively modulated by a chromatin silencing mechanism involving alternative polyadenylation of antisense transcripts. Investigation of this mechanism unexpectedly showed that RNA polymerase II (Pol II) occupancy changes at FLC did not reflect RNA fold changes. Mathematical modeling of these transcriptional dynamics predicted a tight coordination of transcriptional initiation and elongation. This prediction was validated by detailed measurements of total and chromatin-bound FLC intronic RNA, a methodology appropriate for analyzing elongation rate changes in a range of organisms. Transcription initiation was found to vary ∼ 25-fold with elongation rate varying ∼ 8- to 12-fold. Premature sense transcript termination contributed very little to expression differences. This quantitative variation in transcription was coincident with variation in H3K36me3 and H3K4me2 over the FLC gene body. We propose different chromatin states coordinately influence transcriptional initiation and elongation rates and that this coordination is likely to be a general feature of quantitative gene regulation in a chromatin context.

Root elongation rate is correlated with the length of the bare root apex of maize and lupin roots despite contrasting responses of root growth to compact and dry soils

Background We investigated interacting effects of matric potential and soil strength on root elongation of maize and lupin, and relations between root elongation rates and the length of bare (hairless) root apex. Methods Root elongation rates and the length of bare root apexwere determined formaize and lupin seedlings in sandy loam soil of various matric potentials (−0.01 to −1.6 MPa) and bulk densities (0.9 to 1.5 Mg m−3). Results Root elongation rates slowed with both decreasing matric potential and increasing penetrometer resistance. Root elongation of maize slowed to 10 % of the unimpeded rate when penetrometer resistance increased to 2 MPa, whereas lupin elongated at about 40 % of the unimpeded rate. Maize root elongation rate was more sensitive to changes in matric potential in loosely packed soil (penetrometer resistances <1 MPa) than lupin. Despite these differing responses, root elongation rate of both species was linearly correlated with length of the bare root apex (r2 0.69 to 0.97). Conclusion Maize root elongation was more sensitive to changes in matric potential and mechanical impedance than lupin. Robust linear relationships between elongation rate and length of bare apex suggest good potential for estimating root elongation rates for excavated roots.

eIF5A has a function in the elongation step of translation in yeast

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

NATURAL ELONGATION OF SPHERICAL TOKAMAKS

The variation of the elongation of axisymmetric plasma columns in vertical equilibrium magnetic fields is investigated as a function of the aspect ratio using the Solov'ev equilibrium model and the principle of virtual casing.

Cooperative RNA Polymerase Molecules Behavior on a Stochastic Sequence-Dependent Model for Transcription Elongation

The transcription process is crucial to life and the enzyme RNA polymerase (RNAP) is the major component of the transcription machinery. The development of single-molecule techniques, such as magnetic and optical tweezers, atomic-force microscopy and single-molecule fluorescence, increased our understanding of the transcription process and complements traditional biochemical studies. Based on these studies, theoretical models have been proposed to explain and predict the kinetics of the RNAP during the polymerization, highlighting the results achieved by models based on the thermodynamic stability of the transcription elongation complex. However, experiments showed that if more than one RNAP initiates from the same promoter, the transcription behavior slightly changes and new phenomenona are observed. We proposed and implemented a theoretical model that considers collisions between RNAPs and predicts their cooperative behavior during multi-round transcription generalizing the Bai et al. stochastic sequence-dependent model. In our approach, collisions between elongating enzymes modify their transcription rate values. We performed the simulations in Mathematica® and compared the results of the single and the multiple-molecule transcription with experimental results and other theoretical models. Our multi-round approach can recover several expected behaviors, showing that the transcription process for the studied sequences can be accelerated up to 48% when collisions are allowed: the dwell times on pause sites are reduced as well as the distance that the RNAPs backtracked from backtracking sites. © 2013 Costa et al.

A macroscopic kinetic model for DNA polymerase elongation and high-fidelity nucleotide election

The enzymatically catalyzed template-directed extension of ssDNA/primer complex is an impor-tant reaction of extraordinary complexity. The DNA polymerase does not merely facilitate the insertion of dNMP, but it also performs rapid screening of substrates to ensure a high degree of fidelity. Several kinetic studies have determined rate constants and equilibrium constants for the elementary steps that make up the overall pathway. The information is used to develop a macro-scopic kinetic model, using an approach described by Ninio [Ninio J., 1987. Alternative to the steady-state method: derivation of reaction rates from first-passage times and pathway probabili-ties. Proc. Natl. Acad. Sci. U.S.A. 84, 663–667]. The principle idea of the Ninio approach is to track a single template/primer complex over time and to identify the expected behavior. The average time to insert a single nucleotide is a weighted sum of several terms, in-cluding the actual time to insert a nucleotide plus delays due to polymerase detachment from ei-ther the ternary (template-primer-polymerase) or quaternary (+nucleotide) complexes and time delays associated with the identification and ultimate rejection of an incorrect nucleotide from the binding site. The passage times of all events and their probability of occurrence are ex-pressed in terms of the rate constants of the elementary steps of the reaction pathway. The model accounts for variations in the average insertion time with different nucleotides as well as the in-fluence of G+C content of the sequence in the vicinity of the insertion site. Furthermore the model provides estimates of error frequencies. If nucleotide extension is recognized as a compe-tition between successful insertions and time delaying events, it can be described as a binomial process with a probability distribution. The distribution gives the probability to extend a primer/template complex with a certain number of base pairs and in general it maps annealed complexes into extension products.