ABSTRACT
Glutamyl-tRNA synthetase and prolyl-tRNA synthetase belong to different classes of aminoacyl-tRNA synthetases that are thought to have evolved along independent evolutionary pathways. However, both enzymes are on one polypeptide chain encoded by a single human gene, the EPRS locus, which is transcribed as one long mRNA. We report the structure of the human EPRS gene, which consists of 29 exons spread over at least 90 kb of genomic DNA. The exons, encoding the glutamyl-specific and the prolyl-specific parts of the enzyme, are each clustered in 10-kb sections located at opposite ends of the gene. These two exon clusters are separated by a long intervening DNA section with a number of exons, encoding functions that may be involved in the organization of the mammalian multienzyme synthetase complex. The upstream gene region shows structural features of a regulated gene, and preliminary experiments suggest that the gene is expressed at specific times in growth-stimulated cultured cells. We have localized the gene to the distal long arm of human chromosome 1 and to a corresponding site in mouse chromosome 1.
Subject(s)
Amino Acyl-tRNA Synthetases/genetics , Chromosomes, Human, Pair 1 , Genes , Glutamate-tRNA Ligase/genetics , Amino Acid Sequence , Animals , Base Sequence , Biological Evolution , Chromosome Mapping , Exons , Gene Expression Regulation , Genetic Linkage , Humans , Mice/genetics , Molecular Sequence Data , Regulatory Sequences, Nucleic Acid , Repetitive Sequences, Nucleic Acid , Species SpecificityABSTRACT
Polyclonal antibodies were raised against a multiprotein 'holoenzyme' form of calf thymus DNA polymerase alpha-primase and used to probe a human cDNA-protein expression library constructed in the lambda gt11 vector. The probe identified a series of cDNA clones derived from a 3.2 kb mRNA which encodes a novel 105 kDa polypeptide, the P1 protein. In intact cells, the P1 protein was specifically associated with the nucleus, and in cell extracts, it was associated with complex forms of DNA polymerase alpha-primase. The synthesis of human P1-specific mRNA was stimulated upon addition of fresh serum to growth-arrested cells, and RNA blot analyses with the human P1-cDNA probe indicated that P1 is encoded by a strictly conserved mammalian gene. The amino acid sequence deduced from a 240-codon open reading frame resident in the largest human P1-cDNA (0.84 kb) displayed greater than 96% identity with that deduced from the equivalent segment of a 795-codon open reading frame of a larger mouse P1-cDNA (2.8 kb). Throughout its length, the primary structure of mammalian P1 displayed strong homology with that of Mcm3, a 125 kDa yeast protein thought to be involved in the initiation of DNA replication (Gibson et al. 1990. Mol. Cell. Biol. 10: 5707-5720). The P1-Mcm3 homology, the strong conservation of P1 among mammals, its nuclear localization, and its association with the replication-specific DNA polymerase alpha strongly suggest an important role of the P1 protein in the replication of mammalian DNA.
Subject(s)
Cell Cycle Proteins , DNA-Binding Proteins , Fungal Proteins/genetics , Nuclear Proteins/genetics , Transcription Factors , Amino Acid Sequence , Animals , Blotting, Northern , Blotting, Western , Cell Line , Cloning, Molecular , DNA Primase , Fungal Proteins/chemistry , Humans , Mice , Minichromosome Maintenance Complex Component 3 , Molecular Sequence Data , Nuclear Proteins/chemistry , Nuclear Proteins/metabolism , RNA Nucleotidyltransferases/metabolism , Sequence Homology, Nucleic AcidABSTRACT
The human glutaminyl-tRNA synthetase is able to bind to its own mRNA. The enzyme contains two binding regions. One is located in the central section of the enzyme which includes its most hydrophilic portion with ten lysine residues in a block of 20 amino acids. This part of the enzyme binds unspecifically to all RNA sequences tested. A second binding region is located in that part of the enzyme which shows high degrees of sequence similarities with the bacterial and yeast glutaminyl-tRNA synthetases, and which is most likely responsible for the charging of tRNA with glutamine. This second RNA binding region specifically interacts with a site in the 3' noncoding region of the synthetase's mRNA. The binding site in the mRNA is characterized by an extended secondary structure that includes elements of the 'identity set' of nucleotides recognized by the enzyme when interacting with tRNA. We discuss possible physiological implications of the interaction between glutaminyl-tRNA synthetase and its mRNA.
Subject(s)
Glutamate-tRNA Ligase/metabolism , Nucleic Acid Conformation , RNA, Messenger/metabolism , Base Sequence , Binding Sites/physiology , Glutamate-tRNA Ligase/chemistry , Glutamine/metabolism , Humans , Lysine/metabolism , Molecular Sequence Data , Peptide Fragments/metabolism , RNA, Transfer, Glu/metabolism , Recombinant Fusion Proteins/metabolism , Ribonuclease T1/metabolismABSTRACT
We have used a cDNA encoding the core region of the human glutaminyl-tRNA synthetase to determine the chromosomal localization of the corresponding gene. Southern blots of restricted DNA from a panel of rodent-human cell lines and in situ chromosome hybridization gave identical results showing that the human gene locus for glutaminyl-tRNA synthetase resides on the distal long arm of chromosome 1. There are now nine mapped aminoacyl-tRNA synthetase genes in the human genome.
Subject(s)
Chromosomes, Human, Pair 1 , Glutamate-tRNA Ligase/genetics , Animals , Blotting, Southern , Chromosome Mapping , DNA Probes/genetics , Electrophoresis, Polyacrylamide Gel , Genetic Linkage , Humans , Hybrid Cells , Nucleic Acid Hybridization/geneticsABSTRACT
The human glutaminyl-tRNA synthetase is three times larger than the corresponding bacterial and twice as large as the yeast enzyme. It is possible that the additional sequences of the human glutaminyl-tRNA synthetase are required for the formation of the multienzyme complex which is known to include several of aminoacyl-tRNA synthetases in mammalian cells. To address this point we prepared antibodies against three regions of the human glutaminyl-tRNA synthetase, namely against its enzymatically important core region, and against two sections in its large C-terminal extension. In intact multienzyme complexes the core region was accessible to specific antibody binding. However, the C-terminal sections became available to specific antibody binding only when certain components of the multienzyme complex were either absent or degraded. These findings allow first conclusions as to the relative position of some components in the mammalian aminoacyl-tRNA synthetase complex.
Subject(s)
Amino Acyl-tRNA Synthetases/metabolism , Multienzyme Complexes , Amino Acyl-tRNA Synthetases/immunology , Antibody Specificity , Cells, Cultured , Centrifugation , Fluorescent Antibody Technique , HeLa Cells , Humans , Molecular Weight , Precipitin TestsABSTRACT
We have isolated from a Lambda-gt 11 library a human cDNA clone with one open reading frame of about 2400 bases. A stretch of about 350 amino acids in the deduced amino acid sequence is up to 40 percent identical with parts of the known amino acid sequences of E. coli and yeast glutaminyl (Gln)-tRNA synthetase. The isolated cDNA sequence corresponds to an internal section of a 5500 bases long mRNA that codes for a 170 kDa polypeptide associated with Gln-tRNA synthetase. Thus, the human enzyme is about three times larger than the E. coli and two times larger than the yeast Gln-tRNA synthetase. The three enzymes share an evolutionarily conserved core but differ in amino acid sequences linked to the N-terminal and C-terminal side of the core.
