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

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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
NonPeerReviewed
 
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/