High-resolution structures of bacterial 70S ribosomes have provided atomic details about mRNA and tRNA binding to the decoding center during elongation, but such information is lacking for preinitiation complexes (PICs). We identified residues in yeast 18S rRNA critical in vivo for recruiting methionyl tRNA i Met to 40S subunits during initiation by isolating mutations that derepress GCN4 mRNA translation. Several such Gcd − mutations alter the A928:U1389 base pair in helix 28 (h28) and allow PICs to scan through the start codons of upstream ORFs that normally repress GCN4 translation. The A928U substitution also impairs TC binding to PICs in a reconstituted system in vitro. Mutation of the bulge G926 in h28 and certain other residues corresponding to direct contacts with the P-site codon or tRNA in bacterial 70S complexes confer Gcd In protein synthesis, each nucleotide triplet in mRNA is decoded by the cognate aminoacyl-tRNA in the aminoacyl (A) site of the ribosome, while peptidyl-tRNA containing the growing polypeptide chain base-pairs with the preceding triplet in the peptidyl (P) site. During initiation, in contrast, the AUG start codon is decoded in the P-site by Met-tRNA i Met. In bacteria, the Shine-Dalgarno sequence in mRNA base-pairs with 16S rRNA to help position the AUG start codon in the P-site. Accurate start codon selection also depends on translation initiation factors IF1, IF2, and IF3, of which IF1 and IF3 stimulate binding of Met-tRNA i Met to the preinitiation complex (PIC) in preference to elongator tRNA species (Antoun et al. 2006). The ribosomal determinants of tRNA binding to the P-site in bacteria are being illuminated by high-resolution crystal structures of 70S ribosomes bound to mRNA with cognate tRNA in the P-site. On the small (30S) subunit, 11 residues of 16S rRNA directly contact the P-site codon, the P-tRNA anticodon, or the P-tRNA anticodon stem-loop (ASL) (Berk et al. 2006;Korostelev et al. 2006;Selmer et al. 2006). In particular, G1338 and A1339 interact with G-C base pairs in the ASL of tRNA i Met , and functional studies have implicated these 16S residues in promoting recruitment of initiator versus elongator tRNA and accurate selection of AUG as start codon (Lancaster and Noller 2005;Qin et al. 2007).Initiation in eukaryotes is more complex, with 13 different initiation factors (eIFs) participating in a multistep pathway. The eIF2, in its GTP-bound state, forms a ternary complex (TC) with Met-tRNA i Met and binds to the 40S subunit to form the 43S PIC in a manner stimulated by eIF1, eIF1A, eIF5, and eIF3. The 43S PIC then binds to the 5Ј end of mRNA, preactivated by eIF4F bound to the cap. The AUG codon is selected as the 43S PIC scans the leader, with the anticodon of Met-tRNA i Met inspecting successive triplets, presumably, as they enter the P-site. eIF1 and eIF1A promote the scanning process at least partly by stabilizing an open conformation of the 40S subunit when non-AUGs occupy the P-site. The GTP

In this study, we investigated drug profile of 24 anticancer drugs tested against a large number of cell lines in order to understand the relation between drug resistance and altered genomic features of a cancer cell line. We detected frequent mutations, high expression and high copy number variations of certain genes in both drug resistant cell lines and sensitive cell lines. It was observed that a few drugs, like Panobinostat, are effective against almost all types of cell lines, whereas certain drugs are effective against only a limited type of cell lines. Tissue-specific preference of drugs was also seen where a drug is more effective against cell lines belonging to a specific tissue. Genomic features based models have been developed for each anticancer drug and achieved average correlation between predicted and actual growth inhibition of cell lines in the range of 0.43 to 0.78. We hope, our study will throw light in the field of personalized medicine, particularly in designing patient-specific anticancer drugs. In order to serve the scientific community, a webserver, CancerDP, has been developed for predicting priority/potency of an anticancer drug against a cancer cell line using its genomic features (http://crdd.osdd.net/raghava/cancerdp/).

Swi6/HP1, an evolutionarily conserved protein, is critical for heterochromatin assembly in fission yeast and higher eukaryotes. In fission yeast, histone deacetylation by histone deacetylases is thought to be followed by H3-Lys-9 methylation by the histone methyltransferase Clr4/Suv39H1. H3-Lys-9-Me2 interacts with the chromodomain of Swi6/HP1. Swi6/HP1 is thought to act downstream of Clr4/Suv39, and further self-association of Swi6/HP1 is assumed to stabilize the heterochromatin structure. Here, we show that the self-association-defective mutant of Swi6 does not interact with Clr4. It not only fails to localize to heterochromatin loci but also interferes with heterochromatic localization of H3-Lys-9-Me2 (and thereby Clr4) and the endogenous Swi6 in a dominant negative manner. Thus, self-association of Swi6/HP1 helps in binding to and recruitment of Clr4 and thereby in establishment and maintenance of heterochromatin by a concerted rather than a sequential mechanism.Distinct sets of histone modifications organize chromatin into transcriptionally expressed or repressed structures. As in higher eukaryotes, fission yeast euchromatic regions are associated with hyperacetylated histones, particularly Lys-9 and Lys-14 acetylated and Lys-4 methylated histone H3. Conversely, heterochromatin assembly (silent mating type, centromere, rDNA, and telomere loci) involves deacetylation of histone H3 at Lys-9 and Lys-14 by Clr3, Clr6, and Sir2, followed by H3-Lys-9 methylation by the histone methyltransferase Clr4/ Suv39H1. H3-Lys-9-Me2 is recognized by the chromodomain in Swi6/HP1 (1, 2). The property of Swi6 to form multimers is thought to cause folding of chromatin into a transcriptionally inactive structure (3). The RNAi pathway is also involved in heterochromatin assembly. The dcr1⌬, rdp1⌬, and ago1⌬ mutants are defective in H3-Lys-9 methylation and Swi6 recruitment at heterochromatin loci (4).Structural studies show that the chromoshadow domain (CSD) 3 in Swi6/HP1 contains a dimerization motif that creates a cleft for binding a pentapeptide (5, 6). Mutations in the conserved residues in the cleft inhibit interaction with the pentapeptide motif in proteins (6, 7). We found that mutation of one such residue (L315E) severely compromises the self-association property of Swi6. is not only defective in complementing the silencing defect in the swi6⌬ mutant but also exerts a dominant negative effect on silencing, accompanied by loss of heterochromatic localization not only of itself but also that of native Swi6 and H3-Lys-9-Me2 and, by implication, of Clr4. These results support a concerted rather than a sequential action of Clr4 and Swi6 in establishment and maintenance of heterochromatin. EXPERIMENTAL PROCEDURESStrains and Plasmids Used-All strains and plasmids used are listed in supplemental Tables 1 and 2, respectively.Media Compositions-All fission yeast media were prepared according to Moreno et al. (8). The serial dilution plate assay has been described earlier (9). Iodine staining assays for switching and silencing have ...

Findings indicate that this CAI game was not effective in improving asthma symptoms in this group of children.

We have developed a database called dbEM (database of Epigenetic Modifiers) to maintain the genomic information of about 167 epigenetic modifiers/proteins, which are considered as potential cancer targets. In dbEM, modifiers are classified on functional basis and comprise of 48 histone methyl transferases, 33 chromatin remodelers and 31 histone demethylases. dbEM maintains the genomic information like mutations, copy number variation and gene expression in thousands of tumor samples, cancer cell lines and healthy samples. This information is obtained from public resources viz. COSMIC, CCLE and 1000-genome project. Gene essentiality data retrieved from COLT database further highlights the importance of various epigenetic proteins for cancer survival. We have also reported the sequence profiles, tertiary structures and post-translational modifications of these epigenetic proteins in cancer. It also contains information of 54 drug molecules against different epigenetic proteins. A wide range of tools have been integrated in dbEM e.g. Search, BLAST, Alignment and Profile based prediction. In our analysis, we found that epigenetic proteins DNMT3A, HDAC2, KDM6A, and TET2 are highly mutated in variety of cancers. We are confident that dbEM will be very useful in cancer research particularly in the field of epigenetic proteins based cancer therapeutics. This database is available for public at URL: http://crdd.osdd.net/raghava/dbem.