Changes in DNA bending and flexing due to tethered cations detected by fluorescence resonance energy transfer

Local DNA deformation arises from an interplay among sequence-related base stacking, intrastrand phosphate repulsion, and counterion and water distribution, which is further complicated by the approach and binding of a protein. The role of electrostatics in this complex chemistry was investigated using tethered cationic groups that mimic proximate side chains. A DNA duplex was modified with one or two centrally located deoxyuracils substituted at the 5-position with either a flexible 3-aminopropyl group or a rigid 3-aminopropyn-1-yl group. End-to-end helical distances and duplex flexibility were obtained from measurements of the time-resolved Förster resonance energy transfer between 5′- and 3′-linked dye pairs. A novel analysis utilized the first and second moments of the G(t) function, which encompasses only the energy transfer process. Duplex flexibility is altered by the presence of even a single positive charge. In contrast, the mean 5′–3′ distance is significantly altered by the introduction of two adjacently tethered cations into the double helix but not by a single cation: two adjacent aminopropyl groups decrease the 5′–3′ distance while neighboring aminopropynyl groups lengthen the helix

TATA-Binding Protein Recognition and Bending of a Consensus Promoter Are Protein Species Dependent

The structure and behavior of full-length human TBP binding the adenovirus major late promoter (AdMLP) have been characterized using biophysical methods. The human protein induces a 97° bend in DNAAdMLP. The high-resolution functional data provide a quantitative energetic and kinetic description of the partial reaction sequence as native human TBP binds rapidly to a consensus promoter with high affinity. The reaction proceeds with successive formation of three bound species, all having strongly bent DNA, with the concurrence of binding and bending demonstrated by both fluorescence and anisotropy stopped flow. These results establish the protein species dependence of the TBP−DNAAdMLP structure and recognition mechanism. Additionally, the strong correlation between the DNA bend angle and transcription efficiency demonstrated previously for yeast TBP is shown to extend to human TBP. The heterologous NH2-terminal domains are the apparent source of the species-specific differences. Together with previous studies the present work establishes that TBPwt−DNATATA function and structure depend both on the TATA box sequence and on the TBP species

TATA-Binding Protein Recognition and Bending of a Consensus Promoter Are Protein Species Dependent

The structure and behavior of full-length human TBP binding the adenovirus major late promoter (AdMLP) have been characterized using biophysical methods. The human protein induces a 97° bend in DNAAdMLP. The high-resolution functional data provide a quantitative energetic and kinetic description of the partial reaction sequence as native human TBP binds rapidly to a consensus promoter with high affinity. The reaction proceeds with successive formation of three bound species, all having strongly bent DNA, with the concurrence of binding and bending demonstrated by both fluorescence and anisotropy stopped flow. These results establish the protein species dependence of the TBP−DNAAdMLP structure and recognition mechanism. Additionally, the strong correlation between the DNA bend angle and transcription efficiency demonstrated previously for yeast TBP is shown to extend to human TBP. The heterologous NH2-terminal domains are the apparent source of the species-specific differences. Together with previous studies the present work establishes that TBPwt−DNATATA function and structure depend both on the TATA box sequence and on the TBP species

The association of NADPH with the guanine nucleotide exchange factor from rabbit reticulocytes: A role of pyridine dinucleotides in eukaryotic polypeptide chain initiation

The guanine nucleotide exchange factor (GEF) was purified to apparent homogeneity from postribosomal supernatants of rabbit reticulocytes by chromatography on DEAE-celiulose and phosphocellulose, fractionation by glycerol gradients, and chromatography on Mono S and Mono Q (Pharmacia). At the Mono S step GEF is isolated as a complex with the eukaryotic polypeptide chain initiation factor 2 (eIF-2) and is separated from this factor by column chromatography on Mono Q. An emission spectrum characteristic of a reduced pyridine dinucleotide was observed when GEF was subjected to fluorescence analysis. By both coupled enzymatic analysis and chromatography on reverse-phase or Mono Q columns, the bound dinucleotide associated with GEF was determined to be NADPH. The GEF-catalyzed exchange of eIF-2-bound GDP for GTP was markedly inhibited by NAD+ and NADP+. This inhibition was not observed in the presence of equimolar concentrations of NADPH. Similarly, the stimulation of ternary complex (eIF-2•GTP•Met-tRNAf) formation by GEF in the presence of 1 mM Mg2+ was abolished in the presence of oxidized pyridine dinucleotide. These results demonstrate that pyridine dinucleotides may be directly involved in the regulation of polypeptide chain initiation by acting as allosteric regulators of GEF activity

Detailed characterization of the solution kinetics and thermodynamics of biotin, biocytin and HABA binding to avidin and streptavidin

The high affinity (KD ~ 10−15 M) of biotin for avidin and streptavidin is the essential component in a multitude of bioassays with many experiments using biotin modifications to invoke coupling. Equilibration times suggested for these assays assume that the association rate constant (kon) is approximately diffusion limited (109 M-1s-1) but recent single molecule and surface binding studies indicate that they are slower than expected (105 to 107 M-1s-1). In this study, we asked whether these reactions in solution are diffusion controlled, which reaction model and thermodynamic cycle describes the complex formation, and if there are any functional differences between avidin and streptavidin. We have studied the biotin association by two stopped-flow methodologies using labeled and unlabeled probes: I) fluorescent probes attached to biotin and biocytin; and II) unlabeled biotin and HABA, 2-(4’-hydroxyazobenzene)- benzoic acid. Both native avidin and streptavidin are homo-tetrameric and the association data show no cooperativity between the binding sites. The kon values of streptavidin are faster than avidin but slower than expected for a diffusion limited reaction in both complexes. Moreover, the Arrhenius plots of the kon values revealed strong temperature dependence with large activation energies (6–15 kcal/mol) that do not correspond to a diffusion limited process (3–4 kcal/mol). Accordingly, we propose a simple reaction model with a single transition state for non-immobilized reactants whose forward thermodynamic parameters complete the thermodynamic cycle, in agreement with previously reported studies. Our new understanding and description of the kinetics, thermodynamics, and spectroscopic parameters for these complexes will help to improve purification efficiencies, molecule detection, and drug screening assays or find new applications

March 1955

Presidents Report - Frank P. Dunlap (page 1) The Student Purdue Serves - Dr. N. M. Parkhurst (2) A Student Reports - Bill Roach (4) Corrective Management of Shrubs - H. W. Gilbert (5) Nutrient Absorption by Plants - J. R. Watson Jr. (7) Disease Development is Slow - Dr. Wm. Klomparens (10) Poa Annua Control with Arsenic Materials - W. H. Daniel (11) Labor Relations - Cincinnati Country Club - John McCoy (14) Labor Policies at my Course - Ernest Schneider (15) Labor Policies at my Course - Don Strand (16) Nitrogen Use and Why - Robert M. Williams (17) Fertilizing Greens and Why - Don Likes(18) Nitrogen Use and Why - Lawrence Huber (20) Report on Experimental Green - Taylor Boyd (21) Experiences with Fairway Improvements - Ray Davis (23) Fairway Improvement Program - Bert Rost (24) Experiences on Merion Bluegrass - Carl Habenicht (24) Merion Bluegrass Experiences - P. E. Drachman (25) Zoysia for Lawns and Nurseries - P. E. Drachman (27) Preparation for Mortorized Carts - James W. Brandt (28) Preparing for Motorized Carts - Carl Bretzlaff (29) Merion Bluegrass Experiences - Ben O. Warren (31) Pennlu Distribution - W. H. Daniel(32) Zoysia for Midwest Lawns - W. H. Daniel (34) Crabgrass Prevention and Control - W. H. Daniel (36) Plant Carbohydrates Must Balance Nitrogen - M. R. Teel (39) Put Yourself in His Place - Fred Grau (40) Nitrogen Use and Why Wm. E. Lyons (45) The Management of Bentgrass Fairways - O. J. Noer (49) Fairway Improvement Program O. W. Young (52) Experiences with Zoysia - Ferank Dinelli (53

Determinants of infant growth in Eastern Uganda: a community-based cross-sectional study

<p>Abstract</p> <p>Background</p> <p>Child under-nutrition is a leading factor underlying child mortality and morbidity in Sub-Saharan Africa. Several studies from Uganda have reported impaired growth, but there have been few if any community-based infant anthropometric studies from Eastern Uganda. The aim of this study was to describe current infant growth patterns using WHO Child Growth Standards and to determine the extent to which these patterns are associated with infant feeding practices, equity dimensions, morbidity and use of primary health care for the infants.</p> <p>Methods</p> <p>A cross-sectional survey of infant feeding practices, socio-economic characteristics and anthropometric measurements was conducted in Mbale District, Eastern Uganda in 2003; 723 mother-infant (0–11 months) pairs were analysed. Infant anthropometric status was assessed using z-scores for weight-for-length (WLZ), length-for-age (LAZ) and weight-for-age (WAZ). Dependent dichotomous variables were constructed using WLZ < -2 (wasting) and LAZ < -2 (stunting) as cut-off values. A conceptual hierarchical framework was used as the basis for controlling for the explanatory factors in multivariate analysis. Household wealth was assessed using principal components analysis.</p> <p>Results</p> <p>The prevalences of wasting and stunting were 4.2% and 16.7%, respectively. Diarrhoea during the previous 14 days was associated with wasting in the crude analysis, but no factors were significantly associated with wasting in the adjusted analysis. The adjusted analysis for stunting showed associations with age and gender. Stunting was more prevalent among boys than girls, 58.7% versus 41.3%. Having brothers and/or sisters was a protective factor against stunting (OR 0.4, 95% CI 0.2–0.8), but replacement or mixed feeding was not (OR 2.7, 95% CI 1.0–7.1). Lowest household wealth was the most prominent factor associated with stunting with a more than three-fold increase in odds ratio (OR 3.5, 95% CI 1.6–7.8). This pattern was also seen when the mean LAZ was investigated across household wealth categories: the adjusted mean difference between the top and the bottom wealth categories was 0.58 z-scores, p < 0.001. Those who had received pre-lacteal feeds had lower adjusted mean WLZ than those who had not: difference 0.20 z-scores, p = 0.023.</p> <p>Conclusion</p> <p>Sub-optimal infant feeding practices after birth, poor household wealth, age, gender and family size were associated with growth among Ugandan infants.</p

Phylogeny of Echinoderm Hemoglobins

Recent genomic information has revealed that neuroglobin and cytoglobin are the two principal lineages of vertebrate hemoglobins, with the latter encompassing the familiar myoglobin and α-globin/β-globin tetramer hemoglobin, and several minor groups. In contrast, very little is known about hemoglobins in echinoderms, a phylum of exclusively marine organisms closely related to vertebrates, beyond the presence of coelomic hemoglobins in sea cucumbers and brittle stars. We identified about 50 hemoglobins in sea urchin, starfish and sea cucumber genomes and transcriptomes, and used Bayesian inference to carry out a molecular phylogenetic analysis of their relationship to vertebrate sequences, specifically, to assess the hypothesis that the neuroglobin and cytoglobin lineages are also present in echinoderms.The genome of the sea urchin Strongylocentrotus purpuratus encodes several hemoglobins, including a unique chimeric 14-domain globin, 2 androglobin isoforms and a unique single androglobin domain protein. Other strongylocentrotid genomes appear to have similar repertoires of globin genes. We carried out molecular phylogenetic analyses of 52 hemoglobins identified in sea urchin, brittle star and sea cucumber genomes and transcriptomes, using different multiple sequence alignment methods coupled with Bayesian and maximum likelihood approaches. The results demonstrate that there are two major globin lineages in echinoderms, which are related to the vertebrate neuroglobin and cytoglobin lineages. Furthermore, the brittle star and sea cucumber coelomic hemoglobins appear to have evolved independently from the cytoglobin lineage, similar to the evolution of erythroid oxygen binding globins in cyclostomes and vertebrates.The presence of echinoderm globins related to the vertebrate neuroglobin and cytoglobin lineages suggests that the split between neuroglobins and cytoglobins occurred in the deuterostome ancestor shared by echinoderms and vertebrates

Intermediate species possessing bent DNA are present along the pathway to formation of a final TBP-TATA complex

Binding of the TATA-binding protein (TBP) to the “TATA” sequences present in the promoters of eukaryotic class II genes is the first step in the sequential assembly of transcription pre-initiation complexes. Myriad structural changes, including severe bending of the DNA, accompany TBP-TATA complex formation. A detailed kinetic study has been conducted to elucidate the mechanistic details of TBP binding and DNA bending. The binding of Saccharomyces cerevisiae TBP to the adenovirus major late promoter (AdMLP) was followed in real-time through a range of temperatures and TBP concentrations using fluorescence resonance energy transfer (FRET) and stopped-flow mixing. The results of association and relaxation kinetics and equilibrium binding experiments were analyzed globally to obtain the complete kinetic and energetic profile of the reaction. This analysis reveals a complex mechanism with two intermediate species, with the DNA in the intermediates apparently bent similarly to the DNA in the final complex. TBP binding and DNA bending occur simultaneously through the multiple steps of the reaction. The first and third steps in this sequential process show nearly identical large increases in both enthalpy and entropy, whereas the middle step is highly exothermic and proceeds with a large decrease in entropy. The first intermediate is significantly populated at equilibrium and resembles the final complex both structurally and energetically. It is postulated that both this intermediate and the final complex bind transcription factor IIB in the second step of pol II pre-initiation complex assembly. A consequence of such a reactive intermediate is that the rate of assembly of transcriptionally competent pre-initiation complexes from bi-directionally bound TBP is greatly increased