Nuclear gene for mitochondrial leucyl-tRNA synthetase of Neurospora crassa: isolation, sequence, chromosomal mapping, and evidence that the leu-5 locus specifies structural information.

Abstract

We have isolated and characterized the nuclear gene for the mitochondrial leucyl-tRNA synthetase (LeuRS) of Neurospora crassa and have established that a defect in this structural gene is responsible for the keu-5 phenotype. We have purified mitochondrial LeuRS protein, determined its N-terminal sequence, and used this sequence information to identify and isolate a full-length genomic DNA clone. The 3.7-kilobase-pair region representing the structural gene and flanking regions has been sequenced. The 5' ends of the mRNA were mapped by Si nuclease protection, and the 3' ends were determined from the sequence of cDNA clones. The gene contains a single short intron, 60 base pairs long. The methionine-initiated open reading frame specifies a 52-amino-acid mitochondrial targeting sequence followed by a 942-amino-acid protein. Restriction fragment length polymorphism analyses mapped the mitochondrial LeuRS structural gene to linkage group V, exactly where the ku-S mutation had been mapped before. We show that the ku-S strain has a defect in the structural gene for mitochondrial LeuRS by restoring growth under restrictive conditions for this strain after transformation with a wild-type copy of the mitochondrial LeuRS gene. We have cloned the mutant allele present in the ku-S strain and identified the defect as being due to a Thr-to-Pro change in mitochondrial LeuRS. Finally, we have used immunoblotting to show that despite the apparent lack of mitochondrial LeuRS activity in ku-S extracts, the ku-S strain contains levels of mitochondrial LeuRS protein to similar to those of the wild-type strain.The mitochondria of eucaryotic cells possess a DNA genome which is distinct from that in the nucleus. The mitochondrial DNA is replicated, transcribed, and used to synthesize proteins within the mitochondrial matrix. However, the coding capacity of most mitochondrial genomes is limited to the tRNAs and rRNAs required for protein synthesis and the mRNAs for a few proteins, most of which are involved in electron transport (8). The mitochondria are almost completely dependent upon the nuclear genome for the proteins that function within them. These nucleusencoded proteins are synthesized in the cytoplasm and imported into the mitochondrion. Thus, the nuclear genome encodes proteins which are required for analogous functions in the mitochondria and the cytoplasm (protein synthesis and energy metabolism) and in the mitochondria and the nucleus (DNA replication, RNA synthesis, most tRNA modifications, etc.).Work with the yeast Saccharomyces cerevisiae has suggested two strategies for specifying differential localization of proteins performing the same basic functions. For histidyl-tRNA synthetase (43), valyl-tRNA synthetase (10), and several of the tRNA-modifying enzymes (18,20,29), differential transcription of single genes with alternate in-frame protein synthesis initiation sites allows for two forms to be made and localized to the appropriate compartment. The 5' end of the mRNA for these enzymes determines which form of the enzyme...

A mutant cysteinyl-tRNA synthetase affecting timing of chromosomal replication initiation in B. subtilis and conferring resistance to a protein kinase C inhibitor.

“…Conserved amino acids are shaded and HXGH and KMSKS are boxed. Sequences used in this alignment were: LeuRS Neurospora crassa (Chow et al, 1989), Leu RS S.cerevisiae (Hohmann and Thevelin, 1992), CysRS E.coli (Eriani et al, 1991), MetRS B.stearothermophilus (Mechulam et al, 1991), MetRS E.coli (Dardel et al, 1990) and LeuRS E.coli (Hartlein and Madern, 1987 (Leach et al, 1988 (Donachie, 1968), there are few reports in the literature where the initiation mass has been directly measured. Rather, the related parameter DNA/mass has been taken as an indicator of initiation mass.…”

Intron locations and functional deletions in relation to the design and evolution of a subgroup of class I tRNA synthetases

“…Both the LeuRS sequences from Neurospora crassa cytoplasm (Nc-L) and mitochondria (Ncm-L) contain a single intron (Benarous et al, 1988;Chow et al, 1989). The locations of the eight introns in the structure of T therrnophila isoleucine tRNA synthetase (which is modeled after the E. coli methionine tRNA synthetase) are shown in the upper portion of Figure 1.…”

“…thermophila) isoleucyltRNA synthetase, and this is the first to contain multiple introns. The possibility of introns separating structural or functional domains was mentioned by Csank and Martindale (1992).…”

“…The ' I : thermophila intron positions (Csank & Martindale, 1992) were based on an alignment between the E. coli and ' I : thermophila IleRS sequences. The positions of the single introns in the N. crassa mitochondria (Ncm) and cytoplasmic (Nc) LeuRS genes are also indicated (Benarous et al, 1988;Chow et al, 1989). Lower panel: The alignment for the 21 sequences of the class I subgroup are shown for the CPI region.…”

“…Ten aminoacyl tRNA synthetases belong to a distinct class, based on two characteristic sequence motifs that are part of an N-terminal nucleotide-binding fold held in common (Webster et al, 1984;Hountondji et al, 1986;Cusack et al, 1990;Eriani et al, 1990;Burbaum & Schimmel, 1991;Nagel & Doolittle, 1991;Moras, 1992). These 10 enzymes are further subdivided into a subclass of 5 , which consists of Cys-, Ile-, Leu-, Met-, and ValtRNA synthetases (Hou et al, 1991;Shiba & Schimmel, 1992).…”