mRNA turnover meets RNA interference

By using two very different approaches, recent work by Gazzani et al. (2004) and Souret et al. (2004) reveal a fundamental link between mRNA degradation and RNA silencing pathways in Arabidopsis.     

Global position and recruitment of HATs and HDACs in the yeast genome

Chromatin regulators play fundamental roles in the regulation of gene expression and chromosome maintenance, but the regions of the genome where most of these regulators function has not been established. We explored the genome-wide occupancy of four different chromatin regulators encoded in Saccharomyces cerevisiae. The results reveal that the histone acetyltransferases Gcn5 and Esa1 are both generally recruited to the promoters of active protein-coding genes. In contrast, the histone deacetylases Hst1 and Rpd3 are recruited to specific sets of genes associated with distinct cellular functions. Our results provide new insights into the association of histone acetyltransferases and histone deacetylases with the yeast genome, and together with previous studies, suggest how these chromatin regulators are recruited to specific regions of the genome.     

Ribonucleic acids of human milk

The milk feeding is the most essential process laying the foundation of human health at the postnatal development. However little is known about nucleic acids secreted into mother's milk during lactation. In order to investigate the composition and abundance of human milk NA we adapted the conventional isolation method to achieve high yield of total nucleic acids from milk samples. Concentration of total NA in milk samples of different donors varies from 20 to 68 mkg/ml at early stages of lactation. The average concentration tends to fall down to the end of lactation. The chain length of the major forms of NA varies from mononucleotides up to approximately 100 bases. Compositions of milk oligonucleotides are similar in samples of different donors. Major milk oligonucleotides are formed of RNA. Human milk contains the set of long-chain oligonucleotides with a developed secondary structure. Sequences of some oligo-RNAs correspond to the 3'-part of 5.8 S human ribosomal RNA and to the 3'-parts of tRNAVal and tRNATyr Primary structures of some others oligo-RNAs were related to fragments of human 18S and 28S rRNAs.     

Cell cycle regulation of the murine 8-oxoguanine DNA glycosylase (mOGG1): mOGG1 associates with microtubules during interphase and mitosis

8-Oxoguanine DNA glycosylase (OGG1) is a major DNA repair enzyme in mammalian cells. OGG1 participates in the repair of 8-oxoG, the most abundant known DNA lesion induced by endogenous reactive oxygen species in aerobic organisms. In this study, antibodies directed against purified recombinant human OGG1 (hOGG1) or murine (mOGG1) protein were chemically conjugated to either the photosensitizer Rose Bengal or the fluorescent dye Texas red. These dye-protein conjugates, in combination with binding assays, were used to identify associations between mOGG1 and the cytoskeleton of NIH3T3 fibroblasts. Results from these binding studies showed that mOGG1 associates with the cytoskeleton by specifically binding to the centriole and microtubules radiating from the centrosome at interphase and the spindle assembly at mitosis. Similar results were obtained with hOGG1. Together results reported in this study suggest that OGG1 is a microtubule-associated protein itself or that OGG1 utilizes yet to be identified motor proteins to ride on microtubules as tracks facilitating the movement and redistribution of cytoplasmic OGG1 pools during interphase and mitosis and in response to oxidative DNA damage.     

Recognition of damaged DNA by Escherichia coli Fpg protein: insights from structural and kinetic data

Formamidopyrimidine-DNA glycosylase (Fpg) excises oxidized purines from damaged DNA. The recent determination of the three-dimensional structure of the covalent complex of DNA with Escherichia coli Fpg, obtained by reducing the Schiff base intermediate formed during the reaction [Gilboa et al., J. Biol. Chem. 277 (2002) 19811] has revealed a number of potential specific and non-specific interactions between Fpg and DNA. We analyze the structural data for Fpg in the light of the kinetic and thermodynamic data obtained by the method of stepwise increase in ligand complexity to estimate relative contributions of individual nucleotide units of lesion-containing DNA to its total affinity for this enzyme [Ishchenko et al., Biochemistry 41 (2002) 7540]. Stopped-flow kinetic analysis that has allowed the dissection of Fpg catalysis in time [Fedorova et al., Biochemistry 41 (2002) 1520] is also correlated with the structural data.     

Computer simulation of water in cytochrome c oxidase

Statistical mechanics and molecular dynamics simulations have been carried out to study the distribution and dynamics of internal water molecules in bovine heart cytochrome c oxidase (CcO). CcO is found to be capable of holding plenty of water, which in subunit I alone amounts to about 165 molecules. The dynamic characterization of these water molecules is carried out. The nascent water molecules produced in the redox reaction at the heme a(3)-CuB binuclear site form an intriguing chain structure. The chain begins at the position of Glu242 at the end of the D channel, and has a fork structure, one branch of which leads to the binuclear center, and the other to the propionate d of heme a(3). The branch that leads to the binuclear center has dynamic access both to the site where the formation of water occurs, and to delta-nitrogen of His291. From the binuclear center, the chain continues to run into the K channel. The stability of this hydrogen bond network is examined dynamically. The catalytic site is located at the hydrophobic region, and the nascent water molecules are produced at the top of the energy hill. The energy gradient is utilized as the mechanism of water removal from the protein. The water exit channels are explored using high-temperature dynamics simulations. Two putative channels for water exit from the catalytic site have been identified. One is leading directly toward Mg(2+) site. However, this channel is only open when His291 is dissociated from CuB. If His291 is bound to CuB, the only channel for water exit is the one that originates at E242 and leads toward the middle of the membrane. This is the same channel that is presumably used for oxygen supply.     

Production and purification of recombinant human oxytocin overexpressed as a hybrid protein in Escherichia coli

The plasmid DNA pERilox4 containing the gene of the recombinant protein, which included the leader sequence and the oxytocinoyl lysine tetramer, was constructed. The high level of gene expression in E. coli was achieved. The method for purification of the recombinant protein and its isolation in the soluble form was developed. The conditions for digestion of the hybrid protein by trypsin and carboxypeptidase B were matched. The effective method for transformation of oxytocinic acid to oxytocin was worked out. The scheme suggested allowed obtaining oxytocin in high yield.     

The regulation of subtilisin-cleaved actin by tropomyosin/troponin

Vertebrate striated muscle contraction is regulated in a Ca(2+)-dependent fashion by tropomyosin (Tm) and troponin (Tn). This regulation involves shifts in the position of Tm and Tn on actin filaments and may include conformational changes in actin that are then communicated to myosin subfragment 1 (S1). To determine whether subdomain 2 of actin plays a role in this regulation, the DNase-I loop 38-52 of this subdomain was cleaved by subtilisin between residues Met(47) and Gly(48). Despite impaired unregulated function, the potentiation and regulation of cleaved actin movement in the in vitro motility assay was not significantly different from that of uncleaved actin. Stopped-flow measurements of ADP release from regulated and unregulated cleaved acto-S1 showed a marked increase in ADP release from acto-S1 in the presence of the regulatory complex. The enhancement of the actin affinity for S1 in the presence of regulatory proteins was greater for uncleaved than for cleaved F-actin. Finally, both cleaved and uncleaved actins protect myosin loop 1 from papain cleavage equally well. Our results suggest that the potentiation of actin function in the in vitro motility assay by regulatory proteins stems from changes in cross-bridge cycle kinetics. In addition, the unimpaired calcium-sensitive regulation of cleaved actin indicates that subdomain 2 conformation does not play an essential role in the regulation process.     

ATP binding by glutamyl-tRNA synthetase is switched to the productive mode by tRNA binding

Aminoacyl-tRNA synthetases catalyze the formation of an aminoacyl-AMP from an amino acid and ATP, prior to the aminoacyl transfer to tRNA. A subset of aminoacyl-tRNA synthetases, including glutamyl-tRNA synthetase (GluRS), have a regulation mechanism to avoid aminoacyl-AMP formation in the absence of tRNA. In this study, we determined the crystal structure of the 'non-productive' complex of Thermus thermophilus GluRS, ATP and L-glutamate, together with those of the GluRS.ATP, GluRS.tRNA.ATP and GluRS.tRNA.GoA (a glutamyl-AMP analog) complexes. In the absence of tRNA(Glu), ATP is accommodated in a 'non-productive' subsite within the ATP-binding site, so that the ATP alpha-phosphate and the glutamate alpha-carboxyl groups in GluRS. ATP.Glu are too far from each other (6.2 A) to react. In contrast, the ATP-binding mode in GluRS.tRNA. ATP is dramatically different from those in GluRS.ATP.Glu and GluRS.ATP, but corresponds to the AMP moiety binding mode in GluRS.tRNA.GoA (the 'productive' subsite). Therefore, tRNA binding to GluRS switches the ATP-binding mode. The interactions of the three tRNA(Glu) regions with GluRS cause conformational changes around the ATP-binding site, and allow ATP to bind to the 'productive' subsite.     

Protein kinase network in the regulation of phosphorylation and dephosphorylation of smooth muscle myosin light chain

The contraction of smooth muscle is regulated primarily by intracellular Ca²⁺ signal. It is well established that the elevation of the cytosolic Ca²⁺ level activates myosin light chain kinase, which phosphorylates 20 kDa regulatory myosin light chain and activates myosin ATPase. The simultaneous measurement of cytosolic Ca²⁺ concentration and force development revealed that the alteration of the Ca²⁺-sensitivity of the contractile apparatus as well as the Ca²⁺ signal plays a critical role in the regulation of smooth muscle contraction. The fluctuation of an extent of myosin phosphorylation for a given change in Ca²⁺ concentration is considered to contribute to the major mechanisms regulating the Ca²⁺-sensitivity. The level of myosin phosphorylation is determined by the balance between phosphorylation and dephosphorylation. The phosphorylation level for a given Ca²⁺ elevation is increased either by Ca²⁺-independent activation of phosphorylation process or inhibition of dephosphorylation. In the last decade, the isolation and cloning of myosin phosphatase facilitated the understanding of regulatory mechanism of dephosphorylation process at the molecular level. The inhibition of myosin phosphatase can be achieved by (1) alteration of hetrotrimeric structure, (2) phosphorylation of 110 kDa regulatory subunit MYPT1 at the specific site and (3) inhibitory protein CPI-17 upon its phosphorylation. Rho-kinase was first identified to phosphorylate MYPT1, and later many kinases were found to phosphorylate MYPT1 and inhibit dephosphorylation of myosin. Similarly, the phosphorylation of CPI-17 can be catalysed by multiple kinases. Moreover, the myosin light chain can be phosphorylated by not only authentic myosin light chain kinase in a Ca²⁺-dependent manner but also by multiple kinases in a Ca²⁺-independent manner, thus adding a novel mechanism to the regulation of the Ca²⁺-sensitivity by regulating the phosphorylation process. It is now clarified that the protein kinase network is involved in the regulation of myosin phosphorylation and dephosphorylation. However, the physiological role of each component remains to be determined. One approach to accomplish this purpose is to investigate the effects of the dominant negative mutants of the signalling molecule on the smooth muscle contraction. In this regards, a protein transduction technique utilizing the cell-penetrating peptides would provide a useful tool. In the preliminary study, we succeeded in introducing a fragment of MYPT1 into the arterial strips, and found enhancement of contraction.