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Characterization of a trna interacting component of the Leishmania mitochondrial tRNA import complex

IR@IICB: CSIR-Indian Institute of Chemical Biology, Kolkata

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Title Characterization of a trna interacting component of the Leishmania mitochondrial tRNA import complex
Creator Dhar, Gunjan
Subject Molecular & Human Genetics
Description There is a remarkable diversity in the scope and mechanism of mitochondrial tRNA import (reviewed in reference 268).Human mitochondria do not import tRNA, but a number of neuromuscular degenerative and metabolic diseases are caused by mutations in mitochondrial tRNA genes (321).In yeast a single tRNA is imported,apparently through protein import channels and requiring at least two soluble factors, including the mitochondrial form of the cognate aminoacyl-tRNA synthetase(274).By contrast , in kinetoplastid protozoa (Leishmania and trypanosomes),import of a whole spectrum of tRNAs is necessitated by the complete lack of mitochondrial tRNA genes(277,322). In this system,membrane-bound tRNAbinding proteins recognize specific structural motifs(import signals) on tRNA, soluble factors are not required , and the translocation pathway appears to be distinct from that of protein import(293,302,323).Moreover, the sequence and bioenergetic requirements for outer and inner membrane transfer are nonidentical(304),indicating the presence of a distinct transport machinery(theRNA import complex [RIC]) at the inner membrane, a situation similar to the TOM and TIM complexes for protein import (324).A 15-kDa polypeptide has been shown to be required for import into Leishmania mitochondria(295); otherwise,the import machinery remains undefined. Using an in vitro evolution protocol, it was recently shown that Leishmania mitochondria recognize a number of short sequence motifs homologous to multiple domains in tRNAs, suggesting the presence of several import signals (305).Moreover, novel positive and negative allosteric interactions between these aptamers , as well as between intact tRNAs, at the inner membrane were described (305).The RNAs could be classified into two types: type I RNAs are efficiently transferred through the inner membrane but are inhibited by type II . In contrast, type II RNAs have poor inner membrane transfer efficiencies and are stimulated by type I . For example, tRNATyr (GUA) is a type I RNA containing the conserved motif UAGAGC in the D domain , while tRNAIle (UAU) is type II with the sequence UCGCGGGUU in the variable loop-T domain (V-T) region(305).The mechanism of these allosteric interactions is unknown, but there are several possibilities . A single conformationally flexible dimeric or multimeric receptor could bind to either a type I or a type II motif. Alternatively, distinct type I and type II receptors may interact directly or indirectly through a third subunit. A related issue is whether the effector and substrate binding subunits for either RNA are identical or different (306). 2 To begin to define the molecular components of the import machinery , the group has recently reported the isolation of a multi-protein complex (the RNA Import Complex, or RIC) that is sufficient to induce import of tRNAs into artificial phospholipid vesicles (268). This reconstituted system retains all the properties of import in intact mitoplasts, including ATP dependence, and sensitivity to respiratory uncouplers and inhibitors (306). Two tRNA-binding proteins were identified within this complex by photo-crosslinking and immunochemistry: a 45-kDa protein that binds tRNATyr directly , and another 21-kDa protein that binds tRNAIle only in the presence of tRNATyr, suggesting allosteric changes within RIC leading to modulation of tRNA affinities (305-307). The identities of these tRNA-binding proteins are presently unknown. Ongoing work in the laboratory suggests that the 45-kDa band of RIC resolved by SDS-PAGE contains more than one protein species .E xpression of these open reading frames (ORFs) in bacteria , and the generation of ORF –specific antibodies , is in progress. These reagents will be useful in the identification of the tRNA- binding component. In the light of the above, the objectives of the proposed research are: (i) To obtain and analyze the genes corresponding to the protein species present in the 45 kDa band of the Leishmania RNA Import Complex; (ii) To study the organization & expression of these genes in Leishmania; and (iii) To study the effect of conditional knockout of these genes on mitochondrial function in vivo.
Date 2009
Type Thesis
Format application/pdf
Identifier http://www.eprints.iicb.res.in/522/1/merged_document.pdf
Dhar, Gunjan (2009) Characterization of a trna interacting component of the Leishmania mitochondrial tRNA import complex. PhD thesis, Jadavpur University.
Relation http://www.eprints.iicb.res.in/522/