The naturally trans-acting ribozyme RNase P RNA has leadzyme properties

Divalent metal ions promote hydrolysis of RNA backbones generating 5′OH and 2′;3′P as cleavage products. In these reactions, the neighboring 2′OH act as the nucleophile. RNA catalyzed reactions also require divalent metal ions and a number of different metal ions function in RNA mediated cleavage of RNA. In one case, the LZV leadzyme, it was shown that this catalytic RNA requires lead for catalysis. So far, none of the naturally isolated ribozymes have been demonstrated to use lead to activate the nucleophile. Here we provide evidence that RNase P RNA, a naturally trans-acting ribozyme, has leadzyme properties. But, in contrast to LZV RNA, RNase P RNA mediated cleavage promoted by Pb(2+) results in 5′ phosphate and 3′OH as cleavage products. Based on our findings, we infer that Pb(2+) activates H(2)O to act as the nucleophile and we identified residues both in the substrate and RNase P RNA that most likely influenced the positioning of Pb(2+) at the cleavage site. Our data suggest that Pb(2+) can promote cleavage of RNA by activating either an inner sphere H(2)O or a neighboring 2′OH to act as nucleophile

The Genomic Pattern of tDNA Operon Expression in E. coli

In fast-growing microorganisms, a tRNA concentration profile enriched in major isoacceptors selects for the biased usage of cognate codons. This optimizes translational rate for the least mass invested in the translational apparatus. Such translational streamlining is thought to be growth-regulated, but its genetic basis is poorly understood. First, we found in reanalysis of the E. coli tRNA profile that the degree to which it is translationally streamlined is nearly invariant with growth rate. Then, using least squares multiple regression, we partitioned tRNA isoacceptor pools to predicted tDNA operons from the E. coli K12 genome. Co-expression of tDNAs in operons explains the tRNA profile significantly better than tDNA gene dosage alone. Also, operon expression increases significantly with proximity to the origin of replication, oriC, at all growth rates. Genome location explains about 15% of expression variation in a form, at a given growth rate, that is consistent with replication-dependent gene concentration effects. Yet the change in the tRNA profile with growth rate is less than would be expected from such effects. We estimated per-copy expression rates for all tDNA operons that were consistent with independent estimates for rDNA operons. We also found that tDNA operon location, and the location dependence of expression, were significantly different in the leading and lagging strands. The operonic organization and genomic location of tDNA operons are significant factors influencing their expression. Nonrandom patterns of location and strandedness shown by tDNA operons in E. coli suggest that their genomic architecture may be under selection to satisfy physiological demand for tRNA expression at high growth rates

Substrate discrimination in RNase P RNA-mediated cleavage: importance of the structural environment of the RNase P cleavage site

Like the translational elongation factor EF-Tu, RNase P interacts with a large number of substrates where RNase P with its RNA subunit generates tRNAs with matured 5′ termini by cleaving tRNA precursors immediately 5′ of the residue at +1, i.e. at the position that corresponds to the first residue in tRNA. Most tRNAs carry a G(+1)C(+72) base pair at the end of the aminoacyl acceptor-stem whereas in tRNA(Gln) G(+1)C(+72) is replaced with U(+1)A(+72). Here, we investigated RNase P RNA-mediated cleavage as a function of having G(+1)C(+72) versus U(+1)A(+72) in various substrate backgrounds, two full-size tRNA precursors (pre-tRNA(Gln) and pre-tRNA(Tyr)Su3) and a model RNA hairpin substrate (pATSer). Our data showed that replacement of G(+1)C(+72) with U(+1)A(+72) influenced ground state binding, cleavage efficiency under multiple and single turnover conditions in a substrate-dependent manner. Interestingly, we observed differences both in ground state binding and rate of cleavage comparing two full-size tRNA precursors, pre-tRNA(Gln) and pre-tRNA(Tyr)Su3. These findings provide evidence for substrate discrimination in RNase P RNA-mediated cleavage both at the level of binding, as previously observed for EF-Tu, as well as at the catalytic step. In our experiments where we used model substrate derivatives further indicated the importance of the +1/+72 base pair in substrate discrimination by RNase P RNA. Finally, we provide evidence that the structural architecture influences Mg(2+) binding, most likely in its vicinity

Unexpected diversity of RNase P, an ancient tRNA processing enzyme: challenges and prospects

For an enzyme functioning predominantly in a seemingly housekeeping role of 5′ tRNA maturation, RNase P displays a remarkable diversity in subunit make-up across the three domains of life. Despite the protein complexity of this ribonucleoprotein enzyme increasing dramatically from bacteria to eukarya, the catalytic function rests with the RNA subunit during evolution. However, the recent demonstration of a protein-only human mitochondrial RNase P has added further intrigue to the compositional variability of this enzyme. In this review, we discuss some possible reasons underlying the structural diversity of the active sites, and use them as thematic bases for elaborating new directions to understand how functional variations might have contributed to the complex evolution of RNase P

Draft genome sequence of Saccharopolyspora rectivirgula

We have sequenced the genome of Saccharopolyspora rectivirgula, the causative agent of farmer’s lung disease. The draft genome consists of 182 contigs totaling 3,977,051 bp, with a GC content of 68.9%

PENGEMBANGAN MODEL MANAJEMEN BERBASIS SEKOLAH YANG LEBIH MENGEDEPANKAN PELIBATAN PARTISIPASI MASYARAKAT UNTUK IMPLEMENTASI KURIKULUM 2013 DI BENGKULU

Penelitian ini bertujuan untuk mengembangkan model manajemen berbasis sekolah yang lebih mengedepankan pelibatan partisipasi masyarakat dalam rangka implementtasi kurikulum 2013 di Bengkulu. Penelitian tahun-1 ditujukan untuk mendeskripsikan faktor ekonomi, sosial, dan budaya masyarakat yang potensial berkontribusi terhadap pelaksanaan program sekolah. Berdasarkan data ekonomi, sosial, dan budaya tersebut maka pada tahun ke-2 peneliti akan memberikan penguatan pelibatan partisipasi masyarakat guna memberikan dukungan terhadap implementasi program sekolah, mengajak masyarakat untuk mengidentifikasi apa yang dapat mereka sumbangkan untuk kepentingan pendidikan di sekolah, dan sekolah menemukan cara yang tepat untuk meningkatkan partisipasi masyarakat. Pendekatan yang digunakan dalam mencapai tujuan tersebut antara lain dengan jalan: (1) menetapkan sekolah yang relevan dengan masalah dan bersedia menjadi subjek penelitian; (2) mengidentifikasi faktor ekonomi, sosial dan budaya masyarakat yang berpeluang memberikan kontribusi dalam pelaksanaan program sekolah; dan (3) memberikan penguatan terhadap komite sekolah agar dapat meningkatkan partisipasi masyarakat guna mendukung implementasi kurikulum 2013. Luaran penelitian tahun-1 antara lain berupa: (1) tersusun instrumen identifikasi potensi sekolah dan faktor ekonomi, sosial, dan budaya masyarakat yang potensial memberikan kontribusi pada pelaksanaan program sekolah; (2) deskripsi potensi sekolah yang dapat digunakan sebagai media pelibatan partisipasi masyarakat; (3) deskripsi faktor ekonomi, sosial, dan budaya masyarakat yang potensial memberikan kontribusi pada pelaksanaan program sekolah; dan (4) tersusun standar prosedur pelibatan partisipasi masyarakat dalam implementasi program sekolah. Semua hasil tesebut disajikan dalam (A) Laporan Penelitian; (B) Poster; (C) Makalah Seminar Internasional; (D) Proposal Penelitian Tahun-2

Cleavage mediated by the P15 domain of bacterial RNase P RNA

Independently folded domains in RNAs frequently adopt identical tertiary structures regardless of whether they are in isolation or are part of larger RNA molecules. This is exemplified by the P15 domain in the RNA subunit (RPR) of the universally conserved endoribonuclease P, which is involved in the processing of tRNA precursors. One of its domains, encompassing the P15 loop, binds to the 3′-end of tRNA precursors resulting in the formation of the RCCA–RNase P RNA interaction (interacting residues underlined) in the bacterial RPR–substrate complex. The function of this interaction was hypothesized to anchor the substrate, expose the cleavage site and result in re-coordination of Mg2+ at the cleavage site. Here we show that small model-RNA molecules (~30 nt) carrying the P15-loop mediated cleavage at the canonical RNase P cleavage site with significantly reduced rates compared to cleavage with full-size RPR. These data provide further experimental evidence for our model that the P15 domain contributes to both substrate binding and catalysis. Our data raises intriguing evolutionary possibilities for ‘RNA-mediated’ cleavage of RNA

Growth, cell division and sporulation in mycobacteria

Bacteria have the ability to adapt to different growth conditions and to survive in various environments. They have also the capacity to enter into dormant states and some bacteria form spores when exposed to stresses such as starvation and oxygen deprivation. Sporulation has been demonstrated in a number of different bacteria but Mycobacterium spp. have been considered to be non-sporulating bacteria. We recently provided evidence that Mycobacterium marinum and likely also Mycobacterium bovis bacillus Calmette–Guérin can form spores. Mycobacterial spores were detected in old cultures and our findings suggest that sporulation might be an adaptation of lifestyle for mycobacteria under stress. Here we will discuss our current understanding of growth, cell division, and sporulation in mycobacteria