Oligonucleotides containing 2'-O-[2-(2,3-dihydroxypropyl)amino-2-oxoethyl]uridine as suitable precursors of 2'-aldehyde oligonucleotides for chemoselective ligation

2'-O-[2-(2,3-Diacetoxypropyl)amino-2-oxoethyl]uridine 3'-phosphoramidite was prepared and used in solid-phase synthesis to obtain oligonucleotides containing a 1,2-diol group, which may then be converted into a 2'-aldehyde group. The oligonucleotides were conjugated efficiently to various molecules by chemoselective ligation that involves an addition-elimination reaction between the 2'-aldehyde group and a suitable nucleophile, such as a hydrazine, a O-alkylhydroxylamine or an 1,2-aminothiol. The method was applied successfully to the conjugation of peptides to oligonucleotides at the 2'-position.     

An analysis of core deformations in protein superfamilies

An analysis is presented on how structural cores modify their shape across homologous proteins, and whether or not a relationship exists between these structural changes and the vibrational normal modes that proteins experience as a result of the topological constraints imposed by the fold. A set of 35 representative, well-populated protein families is studied. The evolutionary directions of deformation are obtained by using multiple structural alignments to superimpose the structures and extract a conserved core, together with principal components analysis to extract the main deformation modes from the three-dimensional superimposition. In parallel, a low-resolution normal mode analysis technique is employed to study the properties of the mechanical core plasticity of these same families. We show that the evolutionary deformations span a low dimensional space of 4-5 dimensions on average. A statistically significant correspondence exists between these principal deformations and the approximately 20 slowest vibrational modes accessible to a particular topology. We conclude that, to a significant extent, the structural response of a protein topology to sequence changes takes place by means of collective deformations along combinations of a small number of low-frequency modes. The findings have implications in structure prediction by homology modeling.     

Binding of EMSY to HP1beta: implications for recruitment of HP1beta and BS69

EMSY is a large nuclear protein that binds to the transactivation domain of BRCA2. EMSY contains an approximately 100-residue segment at the amino terminus called the ENT (EMSY N-terminal) domain. Plant proteins containing ENT domains also contain members of the royal family of chromatin-remodelling domains. It has been proposed that EMSY may have a role in chromatin-related processes. This is supported by the observation that a number of chromatin-regulator proteins, including HP1beta and BS69, bind directly to EMSY by means of a conserved motif adjacent to the ENT domain. Here, we report the crystal structure of residues 1-108 of EMSY at 2.0 A resolution. The structure contains both the ENT domain and the HP1beta/BS69-binding motif. This binding motif forms an extended peptide-like conformation that adopts distinct orientations in each subunit of the dimer. Biophysical and nuclear magnetic resonance analyses show that the main complex formed by EMSY and the chromoshadow domain of HP1 (HP1-CSD) consists of one EMSY dimer sandwiched between two HP1-CSD dimers. The HP1beta-binding motif is necessary and sufficient for EMSY to bind to the chromoshadow domain of HP1beta.     

Comparative kinetic analysis reveals that inducer-specific ion release precedes the mitochondrial permeability transition

Relationships among the multiple events that precede the mitochondrial membrane permeability transition (MPT) are not yet clearly understood. A combination of newly developed instrumental and computational approaches to this problem is described. The instrumental innovation is a high-resolution digital apparatus for the simultaneous, real-time measurement of four mitochondrial parameters as indicators of the respiration rate, membrane potential, calcium ion transport, and mitochondrial swelling. A computational approach is introduced that tracks the fraction of mitochondria that has undergone pore opening. This approach allows multiple comparisons on a single time scale. The validity of the computational approach for studying complex mitochondrial phenomena was evaluated with mitochondria undergoing an MPT induced by Ca(2+), phenylarsine oxide or alamethicin. Selective ion leaks were observed that precede the permeability transition and that are inducer specific. These results illustrate the occurrence of inducer-specific sequential changes associated with the induction of the permeability transition. Analysis of the temporal relationship among the multiple mitochondrial parameters of isolated mitochondria should provide insights into the mechanisms underlying these responses.     

Efficient suppression of the amber codon in E. coli in vitro translation system

An mRNA encoding the esterase from Alicyclobacillus acidocaldarius with catalytically essential serine codon (ACG) replaced by an amber (UAG) codon was used to study the suppression in in vitro translation system. Suppression of UAG by tRNA(Ser(CUA)) was monitored by determination of the full-length and active esterase. It was shown that commonly used increase of suppressor tRNA concentration inhibits protein production and therefore limits suppression. In situ deactivation of release factor by specific antibodies leads to efficient suppression already at low suppressor tRNA concentration and allows an in vitro synthesis of fully active enzyme in high yield undistinguishable from wild-type protein.     

SREBP-1 dimerization specificity maps to both the helix-loop-helix and leucine zipper domains: use of a dominant negative

The mammalian SREBP family contains two genes that code for B-HLH-ZIP proteins that bind sequence-specific DNA to regulate the expression of genes involved in lipid metabolism. We have designed a dominant negative (DN), termed A-SREBP-1, that inhibits the DNA binding of either SREBP protein. A-SREBP-1 consists of the dimerization domain of B-SREBP-1 and a polyglutamic acid sequence that replaces the basic region. A-SREBP-1 heterodimerizes with either B-SREBP-1 or B-SREBP-2, and both heterodimers are more stable than B-SREBP-1 bound to DNA. Circular dichroism thermal denaturation studies show that the B-SREBP-1.A-SREBP-1 heterodimer is -9.8 kcal mol(-1) dimer(-1) more stable than the B-SREBP-1 homodimer. EMSA assays demonstrate that A-SREBP-1 can inhibit the DNA binding of either B-SREBP-1 or B-SREBP-2 in an equimolar competition but does not inhibit the DNA binding of the three B-HLH-ZIP proteins MAX, USF, or MITF, even at 100 molar eq. Chimeric proteins containing the HLH domain of SREBP-1 and the leucine zipper from either MAX, USF, or MITF indicate that both the HLH and leucine zipper regions of SREBP-1 contribute to its dimerization specificity. Transient co-transfection studies demonstrate that A-SREBP-1 can inhibit the transactivation of SREBP-1 and SREBP-2 but not USF. A-SREBP-1 may be useful in metabolic diseases where SREBP family members are overexpressed.     

Membrane fluidity and its roles in the perception of environmental signals

Poikilothermic organisms are exposed to frequent changes in environmental conditions and their survival depends on their ability to acclimate to such changes. Changes in ambient temperature and osmolarity cause fluctuations in the fluidity of cell membranes. Such fluctuations are considered to be critical to the initiation of the regulatory reactions that ultimately lead to acclimation. The mechanisms responsible for the perception of changes in membrane fluidity have not been fully characterized. However, the analysis of genome-wide gene expression using DNA microarrays has provided a powerful new approach to studies of the contribution of membrane fluidity to gene expression and to the identification of environmental sensors. In this review, we focus on the mechanisms that regulate membrane fluidity, on putative sensors that perceive changes in membrane fluidity, and on the subsequent expression of genes that ensures acclimation to a new set of environmental conditions.     

Discrimination against deoxyribonucleotide substrates by bacterial RNA polymerase

Nucleic acid polymerases have evolved elaborate mechanisms that prevent incorporation of the non-cognate substrates, which are distinguished by both the base and the sugar moieties. While the mechanisms of substrate selection have been studied in single-subunit DNA and RNA polymerases (DNAPs and RNAPs, respectively), the determinants of substrate binding in the multisubunit RNAPs are not yet known. Molecular modeling of Thermus thermophilus RNAP-substrate NTP complex identified a conserved beta' subunit Asn(737) residue in the active site that could play an essential role in selection of the substrate ribose. We utilized the Escherichia coli RNAP model system to assess this prediction. Functional in vitro analysis demonstrates that the substitutions of the corresponding beta' Asn(458) residue lead to the loss of discrimination between ribo- and deoxyribonucleotide substrates as well as to defects in RNA chain extension. Thus, in contrast to the mechanism utilized by the single-subunit T7 RNAP where substrate selection commences in the inactive pre-insertion site prior to its delivery to the catalytic center, the bacterial RNAPs likely recognize the sugar moiety in the active (insertion) site.