Molecular and functional dissection of a putative RNA-binding region in yeast mitochondrial leucyl-tRNA synthetase.
Aminoacylation and editing by leucyl-tRNA synthetases (LeuRS) require migration of the tRNA acceptor stem end between the canonical aminoacylation core and a separate domain called CP1 that is responsible for amino acid editing. The LeuRS CP1 domain can also support group I intron RNA splicing in the yeast mitochondria, although splicing-sensitive sites have been identified on the main body. The RDW peptide, a highly conserved peptide within an RDW-containing motif, resides near one of the beta-strand linkers that connects the main body to the CP1 domain. We hypothesized that the RDW peptide was important for interactions of one or more of the LeuRS-RNA complexes. An assortment of X-ray crystallography structures suggests that the RDW peptide is dynamic and forms unique sets of interactions with the aminoacylation and editing complexes. Mutational analysis identified specific sites within the RDW peptide that failed to support protein synthesis activity in complementation experiments. In vitro enzymatic investigations of mutations at Trp445, Arg449, and Arg451 in yeast mitochondrial LeuRS suggested that these sites within the RDW peptide are critical to the aminoacylation complex, but impacted amino acid editing activity to a much less extent. We propose that these highly conserved sites primarily influence productive tRNA interactions in the aminoacylation complex.
Nawaz
Mir Hussain
MH
Department of Biochemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801-3732, USA.
Pang
Yan Ling Joy
YL
Martinis
Susan A
SA
D017398:Alternative Splicing; D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D005816:Genetic Complementation Test; D007935:Leucine-tRNA Ligase / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D024101:Mitochondrial Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D010455:Peptides / Q000737:chemistry; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D046249:Transfer RNA Aminoacylation
D024101:Mitochondrial Proteins; D010455:Peptides; D029701:Saccharomyces cerevisiae Proteins; D007935:Leucine-tRNA Ligase