Although previous studies have already shown that both cytoplasmic and mitochondrial activities of glycyltRNA synthetase are provided by a single gene, GRS1, in the yeast Saccharomyces cerevisiae, the mechanism by which this occurs remains unclear. Evidence presented here indicates that this bifunctional property is actually a result of two distinct translational products alternatively generated from a single transcript of this gene. Except for an amino-terminal 23-amino acid extension, these two isoforms have the same polypeptide sequence and function exclusively in their respective compartments under normal conditions. Reporter gene assays further suggest that this leader peptide can function independently as a mitochondrial targeting signal and plays the major role in the subcellular localization of the isoforms. Additionally, whereas the short protein is translationally initiated from a traditional AUG triplet, the longer isoform is generated from an upstream inframe UUG codon. To our knowledge, GRS1 appears to be the first example in the yeast wherein a functional protein isoform is initiated from a naturally occurring non-AUG codon. The results suggest that non-AUG initiation might be a mechanism existing throughout all kingdoms.
We describe the use of a phylogenetic approach to analyze the modular organization of the single-chained (898 amino acids) and multifunctional DNA polymerase of phage T4. We have identified, cloned in expression vectors, and sequenced the DNA polymerase gene (gene 43) of phage RB69, a distant relative of T4. The deduced primary structure of the RB69 protein (RB69 gp43) differs from that of T4 gp43 in discrete clusters of short sequence that are interspersed with clusters of high similarity between the two proteins. Despite these differences, the two enzymes can substitute for each other in phage DNA replication, although T4 gp43 does exhibit preference to its own genome. A 55-amino acid internal gp43 segment of high sequence divergence between T4 and RB69 could be replaced in RB69 gp43 with the corresponding segment from T4 without loss of replication function. The reciprocal chimera and a deletion mutant of the T4 gp43 segment were both inactive for replication and specifically inhibitory ("dominant lethal") to the T4 wild-type allele. The results show that phylogenetic markers can be used to construct chimeric and truncated forms of gp43 that, although inactive for replication, can still exhibit biological specificity.In DNA replication, DNA polymerases bear the major responsibility for copying genomes with high accuracy. As a group, these enzymes display a variety of molecular types, but most are unified by exhibiting two catalytic functions that control fidelity: primer/template-dependent nucleotidyl transferase (polymerase) and DNA 3Ј exonuclease (proofreading function) (Kornberg and Baker, 1992). In bacteriophage T4, the two functions are part of the same polypeptide chain, product of phage gene 43 (gp43), whereas in some biological systems the polymerase and DNA 3Ј exonuclease activities are specified by separate protein subunits, e.g. Escherichia coli DNA polymerase III holoenzyme (Kelman and O'Donnell, 1995). Another E. coli enzyme, DNA polymerase I, resembles T4 gp43 in size and in possessing polymerase and DNA 3Ј exonuclease functions in the same polypeptide chain; however, unlike T4 gp43, polymerase I also has an N-terminal 5Ј to 3Ј exonuclease function. A third E. coli DNA polymerase, polymerase II, resembles T4 gp43 in biochemical properties and amino acid sequence motifs but is a little smaller in size than the phage enzyme (Cai et al., 1995). One group of DNA polymerases, the reverse transcriptases, lack editing function altogether (Skalka and Geoff, 1993). T4 gp43 also bears a sequence-specific RNA-binding autogenous translational repressor function (Andrake et al., 1988) that only partially overlaps the DNA binding function of the enzyme (Pavlov and Karam, 1994).Typically, replication DNA polymerases work in complex with other proteins, which provide accessory functions that help meet a number of requirements and overcome a variety of constraints inherent to the semiconservative duplication of long supercoiled and condensed double-helical DNA genomes. In the case of T4, the interfacing of repli...
We show here that nonspecific RNA-protein interactions can significantly enhance the biological activity of an essential RNA⅐protein complex. Bacterial glutaminyltRNA synthetase poorly aminoacylates yeast tRNA and, as a consequence, cannot rescue a knockout allele of the gene for the yeast homologue. In contrast to the bacterial protein, the yeast enzyme has an extra appended domain at the N terminus. Previously, we showed that fusion of this yeast-specific domain to the bacterial protein enabled it to function as a yeast enzyme in vivo and in vitro. We suggested that the novel yeast-specific domain contributed to RNA interactions in a way that compensated for the poor fit between the yeast tRNA and bacterial enzyme. Here we establish that the novel appended domain by itself binds nonspecifically to different RNA structures. In addition, we show that fusion of an unrelated yeast protein, Arc1p, to the bacterial enzyme also converts it into a functional yeast enzyme in vivo and in vitro. A small C-terminal segment of Arc1p is necessary and sufficient for this conversion. This segment was shown by others to have nonspecific tRNA binding properties. Thus, nonspecific RNA binding interactions in general can compensate for barriers to formation of a specific and essential RNA⅐protein complex.Interactions between proteins and nucleic acids contain both specific and nonspecific components. The former include interactions between amino acid side chains and functional groups on bases, whereas nonspecific contacts with nucleic acids are generally directed toward the sugar and phosphate backbone (1-3). From the standpoint of functional relevance, the relative importance of nonspecific versus specific interactions has been difficult to evaluate. Most work has demonstrated the importance of particular specific interactions, generally by experiments with mutants where well defined contacts are replaced or manipulated (4, 5). However, less is known about the role of nonspecific contacts and their contribution toward the formation and function of specific complexes. With this in mind, we asked whether, in general, nonspecific protein-RNA interactions could restore function in vivo and in vitro to a weak complex that required high specificity to be functional.We show here that nonspecific RNA binding domains can significantly influence the functional activity of a highly specific enzyme-RNA interaction. This influence is simply achieved by fusing a nonspecific RNA binding domain to a heterologous target protein whose function requires a specific RNA interaction. The heterologous target protein is itself inactive in the organism chosen. The idea was to rescue its activity by fusion of a new RNA binding element.For this work, we used an aminoacyl-tRNA synthetase system that could be investigated both in vivo and in vitro (6). Because the synthetases are essential proteins, the investigation of specific interactions in vivo provided us with a stringent test for functional enzyme⅐RNA complexes. For example, complexes that were eithe...
Trbp111 is a 111 amino acid Aquifex aeolicus structure‐specific tRNA‐binding protein that has homologous counterparts distributed throughout evolution. A dimer is the functional unit for binding a single tRNA. Here we report the 3D structures of the A.aeolicus protein and its Escherichia coli homolog at resolutions of 2.50 and 1.87 Å, respectively. The structure shows a symmetrical dimer of two core domains and a central dimerization domain where the N‐ and C‐terminal regions of Trbp111 form an extensive dimer interface. The core of the monomer is a classical oligonucleotide/oligosaccharide‐binding (OB) fold with a five‐stranded β‐barrel and a small capping helix. This structure is similar to that seen in the anticodon‐binding domain of three class II tRNA synthetases and several other proteins. Mutational analysis identified sites important for interactions with tRNA. These residues line the inner surfaces of two clefts formed between the β‐barrel of each monomer and the dimer interface. The results are consistent with a proposed model for asymmetrical docking of the convex side of tRNA to the dimer.
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