Proton-guided movements of tRNA within the Leishmania mitochondrial RNA import complex

The RNA import complex (RIC) from the mitochondrion of the kinetoplastid protozoan Leishmania tropica contains two subunits that directly bind to import signals on two distinct subsets of tRNA and interact with each other allosterically. What happens to the tRNA subsequent to its loading on the complex is unknown. A third subunit—RIC9—has intrinsic affinity for both types of tRNA and is essential for import in vivo. Here we show that antibody against RIC9 inhibited the import of both types of tRNA into mitoplasts in vitro, but failed to inhibit the binding of these tRNAs to their respective receptors, indicating that RIC9 acts in a subsequent step. Using photoaffinity crosslinking-immunoprecipitation to detect translocation intermediates, it was observed that tRNA was transferred from its cognate receptor to RIC9, followed by translocation across the membrane and release as free tRNA in the inner compartment. Transfer required elevated temperatures and ATP, but ATP was substituted by acid pH. These tRNA movements were sensitive to uncouplers and inhibitors, suggesting distinct roles of the electrical and chemical components of the proton motive force generated by vectorial proton translocation accompanying ATP hydrolysis. By analysis of partially assembled complexes in L. tropica depleted of various subunits, and in vitro assembly assays, RIC9 was shown to make stable contacts with RIC8A, a tRNA receptor and RIC6, a membrane-embedded component. The results have implications for the mechanism of tRNA import

Mini-exon derived RNA gene of Leishmania donovani: structure, organization and expression

Mini-exon derived RNA is a small nuclear RNA of trypanosomatid protozoa such as Leishmania which donates its 5'-terminal 39 nucleotides to the 5'-ends of cellular messenger RNAs by trans-splicing. We have cloned a mini-exon derived RNA gene from Leishmania donovani and studied its organization and expression. About 200 copies of the gene per haploid genome are organized as a tandem repeat on a single chromosome. The gene is transcribed as a 95-nucleotide RNA. The first 39 nucleotides of mini-exon derived RNA is also found at the 5'-terminus of a cellular mRNA (β-tubulin), thus confirming its identity. Sequence analysis of the gene and its flanking regions showed that while classical RNA polymerase II promoter elements such as TATA and CAAT are absent from the 5'-upstream region, intragenic sequence motifs resembling RNA polymerase III promoter elements are present. The implications of this finding for mini-exon derived RNA expression are discussed

The effect of β-tubulin-specific antisense oligonucleotide encapsulated in different cationic liposomes on the supression of intracellular L. Donovani parasites in vitro

An antisense oligonucleotide (20mer) targeted to the parasite β-tubulin gene and encapsulated in cationic liposomes, was used to test its antileishmanial activity in vitro. Cationic liposomes containing dioleyl trimethyl ammonium propane (DOTAP) were found to have higher antileishmanial activity (88% at 4µM oligonucleotide) compared to two other liposomes with stearyl amine (SA) and cetyl trimethyl ammonium bromide (CTAB) as cations. Dot-blot experiments were performed to analyse the expression of β-tubulin mRNA using β-tubulin-specific radiolabelled DNA as a probe. When compared with their respective controls, β-tubulin-specific gene expression was found to be diminished by treatment with a specific antisense oligonuclotide encapsulated in cationic liposomes (CTAB: DOPE) in a concentration-dependent manner. These experiments show that antisense oligonucleotides targeted to the β-tubulin gene of Leishmania donovani inhibit β-tubulin synthesis leading to the arrest of multiplication of intracellular parasites

Targeted mRNA degradation by complex-mediated delivery of antisense RNAs to intracellular human mitochondria

Mitochondrial dysfunction underlies a large number of acute or progressive diseases, as well as aging. However, proposed therapies for mitochondrial mutations suffer from poor transformation of mitochondria with exogenous DNA, or lack of functionality of the transferred nucleic acid within the organelle. We show that a transfer RNA import complex (RIC) from the parasitic protozoon Leishmania tropica rapidly and efficiently delivered signal-tagged antisense (STAS) RNA or DNA to mitochondria of cultured human cells. STAS-induced specific degradation of the targeted mitochondrial mRNA, with downstream effects on respiration. These results reveal the existence of a novel small RNA-mediated mRNA degradation pathway in mammalian mitochondria, and suggest that RIC-mediated delivery could be used to target therapeutic RNAs to the organelle within intact cells

Identification of new genes in human chromosome 3 contig 7 by graphical representation technique

The rapidly growing library of genomic length sequences and the working draft of the human genome sequence imply a concomitant need to determine new methods to analyse the sequences for rapid identification of new genes and their functions. We have developed a graphical technique for quick determination of probable coding regions in DNA sequences. In this article we apply this technique to the new sequence data from human chromosome 3 contig 7 to test the efficacy of the proposed system in a live case, and also compare with results from other genomic sequences. We report here a sampling of sequence segments that pass theoretical tests of likelihood of being genes and list several that have close homology with sequences from the expressed sequence tag (EST) databases. We also comment on the possible use of the graphical representation technique in the shotgun method of gene sequencing

Allosteric regulation of tRNA import: interactions between tRNA domains at the inner membrane of Leishmania mitochondria

Import of nucleus‐encoded tRNAs into the mitochondria of the kinetoplastid protozoon Leishmania involves recognition of specific import signals by the membrane‐bound import machinery. Multiple signals on different tRNA domains may be present, and further, importable RNAs interact positively (Type I) or negatively (Type II) with one another at the inner membrane in vitro. By co‐transfection assays, it is shown here that tRNATyr (Type I) transiently stimulates the rate of entry of tRNAIle (Type II) into Leishmania mitochondria in transfected cells, and conversely, is inhibited by tRNAIle. Truncation and mutagenesis experiments led to the co‐localization of the effector and import activities of tRNATyr to the D domain and those of tRNAIle to the variable region–T domain (V‐T region), indicating that both activities originate from a single RNA–receptor interaction. A third tRNA, human tRNALys, is imported into Leishmania mitochondria in vitro as well as in vivo. This tRNA has Type I and Type II motifs in the D domain and the V‐T region, respectively, and shows both Type I and Type II effector activities. Such dual‐type tRNAs may interact simultaneously with the Type I and Type II binding sites of the inner membrane import machinery

A mosaic of RNA binding and protein interaction motifs in a bifunctional mitochondrial tRNA import factor from Leishmania tropica

Proteins that participate in the import of cytosolic tRNAs into mitochondria have been identified in several eukaryotic species, but the details of their interactions with tRNA and other proteins are unknown. In the kinetoplastid protozoon Leishmania tropica, multiple proteins are organized into a functional import complex. RIC8A, a tRNA-binding subunit of this complex, has a C-terminal domain that functions as subunit 6b of ubiquinol cytochrome c reductase (complex III). We show that the N-terminal domain, unique to kinetoplastid protozoa, is structurally similar to the appended S15/NS1 RNA-binding domain of aminoacyl tRNA synthetases, with a helix–turn–helix motif. Structure-guided mutagenesis coupled with in vitro assays showed that helix α1 contacts tRNA whereas helix α2 targets the protein for assembly into the import complex. Inducible expression of a helix 1-deleted variant in L. tropica resulted in formation of an inactive import complex, while the helix 2-deleted variant was unable to assemble in vivo. Moreover, a protein-interaction assay showed that the C-terminal domain makes allosteric contacts with import receptor RIC1 complexed with tRNA. These results help explain the origin of the bifunctionality of RIC8A, and the allosteric changes accompanying docking and release of tRNA during import

Role of terminal dipole charges in aggregation of α-helix pair in the voltage gated K+ channel

AbstractThe voltage sensor domain (VSD) of the potassium ion channel KvAP is comprised of four (S1–S4) α-helix proteins, which are encompassed by several charged residues. Apart from these charges, each peptide α-helix having two inherent equal and opposite terminal dipolar charges behave like a macrodipole. The activity of voltage gated ion channel is electrostatic, where all the charges (charged residues and dipolar terminal charges) interact with each other and with the transmembrane potential. There are evidences that the role of the charged residues dominate the stabilization of the conformation and the gating process of the ion channel, but the role of the terminal dipolar charges are never considered in such analysis. Here, using electrostatic theory, we have studied the role of the dipolar terminal charges in aggregation of the S3b–S4 helix pair of KvAP in the absence of any external field (V=0). A system attains stability, when its potential energy reaches minimum values. We have shown that the presence of terminal dipole charges (1) change the total potential energy of the charges on S3b–S4, affecting the stabilization of the α-helix pair within the bilayer lipid membrane and (2) the C- and the N-termini of the α-helices favor a different dielectric medium for enhanced stability. Thus, the dipolar terminal charges play a significant role in the aggregation of the two neighboring α-helices