Chien-Chia Wang

Modular Organization of T4 DNA Polymerase EVIDENCE FROM PHYLOGENETICS

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.

Translation of a Yeast Mitochondrial tRNA Synthetase Initiated at Redundant non-AUG Codons

Although initiation of translation at non-AUG codons occurs occasionally in prokaryotes and higher eukaryotes, it has not been reported in yeast until very recently. Evidence presented here shows that redundant ACG codons are recognized as alternative translation start sites for ALA1, the only gene in Saccharomyces cerevisiae coding for alanyl-tRNA synthetase. ALA1 is shown to be a bifunctional gene that provides both cytoplasmic and mitochondrial activities. Unlike most bifunctional genes that contain alternative in-frame AUG initiators, there is only one AUG codon, designated AUG1, close to the 5′-end of the ALA1 open reading frame. Transcriptional mapping identified three overlapping transcripts, with 5′-ends at positions 54, 105, and 117 nucleotides upstream of AUG1, respectively. Site-specific mutagenesis demonstrated that the cytoplasmic and mitochondrial functions of ALA1 are provided by two protein isoforms with distinct amino termini; that is, a short cytoplasmic form initiated at AUG1 and a longer mitochondrial isoform initiated at two upstream in-frame ACG codons, i.e. ACG–25 and ACG–24. These two ACG codons function redundantly in initiation of translation. Either codon can function in the absence of the other. The short transcript appears to serve as the template for the cytoplasmic form, whereas the longer transcripts are likely to code for both isoforms via alternative initiation. Because yeast ribosomes in general cannot efficiently recognize a non-AUG initiator, this unique feature of redundancy of non-AUG initiators in a single mRNA may in itself represent a novel paradigm for translation initiation from poor initiators.

Species barrier to RNA recognition overcome with nonspecific RNA binding domains.

We show here that nonspecific RNA-protein interactions can significantly enhance the biological activity of an essential RNA·protein complex. Bacterial glutaminyl-tRNA 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 enzymein 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 RNA·protein complex.

Translational Efficiency of a Non-AUG Initiation Codon Is Significantly Affected by Its Sequence Context in Yeast

Previous studies have shown that translation of mrna for yeast glycyl-tRNA synthetase is alternatively initiated from UUG and a downstream AUG initiation codon. Evidence presented here shows that unlike an AUG initiation codon, efficiency of this non-AUG initiation codon is significantly affected by its sequence context, in particular the nucleotides at positions –3 to –1 relative to the initiation codon. A/A/R (R represents A Or G) and C/G/C appear to be the most and least favorable sequences at these positions, respectively. Mutation of the native context sequence –3 to –1 from AAA to CGC reduced translation initiation from the UUG codon up to 32-fold and resulted in loss of mitochondrial respiration. although an AUG initiation codon is, in general, unresponsive to context changes in yeast, an AAA (–3 to –1) to CGC mutation still reduced its initiating activity up to 8-fold under similar conditions. these results suggest that sequence context is more important for translation initiation in yeast than previously appreciated.

Crystal structure of Trbp111: a structure-specific tRNA-binding protein

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.

Construction and uncertainty evaluation of a calibration system for GPS receivers

GPS is already a main method of positioning measurement in geodesy and is applied widely in many fields. In order to maintain and ensure the accuracy of positioning, an accurate and efficient system for calibrating the GPS receivers must be established. A highly accurate GPS calibration network, tied to the ITRF coordinates of IGS stations, can be effectively used to evaluate the performance of GPS receivers. This study addresses the feasibility of establishing a system for calibrating GPS receivers and the systems traceability in metrology. Uncertainties of the GPS calibration networks were established and maintained by NML (National Measurement Laboratory, Taiwan). Furthermore, the networks are evaluated based on the method suggested by the ISO (International Organization for Standardization). The uncertainties of NML network coordinates are obtained and used as a basis for calibration. The results of the slope distances between pillars measured by the GPS processing units and the precise EDM units are discussed. Analytical results indicate that the 3D expanded uncertainty of the main station TNML of the network in the ITRF system is around 33.2 mm at the 95% confidence level. The 3D expanded uncertainties of the calibration points of the ultra-short distance network and the short distance network are evaluated to be about 2.2 mm and 3.4 mm, respectively in relation to the main station TNML at the 95% confidence level. The precision of the NML network coordinates suffices to calibrate the geodetic and navigational GPS receivers of regional users and is available through the Internet.

Redundancy of Non-AUG Initiators A CLEVER MECHANISM TO ENHANCE THE EFFICIENCY OF TRANSLATION IN YEAST

It was recently shown that ALA1, the only alanyl-tRNA synthetase gene in Saccharomyces cerevisiae, uses two successive ACG triplets as the translation initiators for its mitochondrial form. Evidence presented here argues that the second ACG triplet not only acts as a remedial initiation site for scanning ribosomes that skip the first ACG, but also enhances the activity of the preceding initiator by providing a preferable “A” at its relative position +4. Therefore, ALA1 constructs with redundant ACG initiators exhibit stronger complementing activity and express a higher level of protein than do those with a single ACG initiator. A similar scenario is seen when a single or redundant ACG triplets are placed in the positions of the first and second AUG initiators of VAS1, which serve as the start sites of the mitochondrial and cytoplasmic forms of valyl-tRNA synthetase, respectively. Cumulatively, the results suggest that this feature of redundancy of non-AUG initiators in a single mRNA per se may represent a novel paradigm for improving the efficiency of a poor or otherwise nonproductive initiation event.

Promoting the formation of an active synthetase/tRNA complex by a nonspecific tRNA-binding domain.

Previous studies showed that valyl-tRNA synthetase of Saccharomyces cerevisiae contains an N-terminal polypeptide extension of 97 residues, which is absent from its bacterial relatives, but is conserved in its mammalian homologues. We showed herein that this appended domain and its human counterpart are both nonspecific tRNA-binding domains (Kd ∼ 0.5 μm). Deletion of the appended domain from the yeast enzyme severely impaired its tRNA binding, aminoacylation, and complementation activities. This N-domain-deleted yeast valyl-tRNA synthetase mutant could be rescued by fusion of the equivalent domain from its human homologue. Moreover, fusion of the N-domain of the yeast enzyme or its human counterpart to Escherichia coli glutaminyl-tRNA synthetase enabled the otherwise “inactive” prokaryotic enzyme to function as a yeast enzyme in vivo. Different from the native yeast enzyme, which showed different affinities toward mixed tRNA populations, the fusion enzyme exhibited similar binding affinities for all yeast tRNAs. These results not only underscore the significance of nonspecific tRNA binding in aminoacylation, but also provide insights into the mechanism of the formation of aminoacyl-tRNAs.

EVOLUTION OF RNA-BINDING SPECIFICITY IN T4 DNA POLYMERASE

DNA polymerase of phage T4 (T4 gp43), an essential component of the T4 DNA replicase, is a multifunctional single-chained (898-amino acid) protein that catalyzes the highly accurate synthesis of DNA in phage replication. The enzyme functions both as a DNA-binding replication protein and as a sequence-specific RNA-binding autogenous translational repressor. We have utilized a phylogenetic approach to study the relationships between the two nucleic acid-binding functions of the protein. We found that autogenous translational control of gp43 biosynthesis has been conserved in phage RB69, a distant relative of T4, although we also found that the RB69 system differs from its T4 counterpart in two regards: (a) nucleotide sequence and predicted secondary structure of the RNA target (translational operator), and (b) RNA specificity of the protein. T4 gp43 is specific to the RNA operator sequence of the T4 genome whereas RB69 gp43 can bind and repress operator RNA from both phages equally well. In studies with T4-RB69 gp43 chimeras, we mapped T4 gp43 RNA-binding specificity to a protein segment that also harbors important determinants for DNA binding and the polymerase catalytic function. Our results suggest that RNA functions as a regulator of both the dosage and activity of this DNA replication enzyme.

A single sequence context cannot satisfy all non-AUG initiator codons in yeast†

BackgroundPrevious studies in Saccharomyces cerevisiae showed that ALA1 (encoding alanyl-tRNA synthetase) and GRS1 (encoding glycyl-tRNA synthetase) respectively use ACG and TTG as their alternative translation initiator codons. To explore if any other non-ATG triplets can act as initiator codons in yeast, ALA1 was used as a reporter for screening.ResultsWe show herein that except for AAG and AGG, all triplets that differ from ATG by a single nucleotide were able to serve as initiator codons in ALA1. Among these initiator codons, TTG, CTG, ACG, and ATT had ~50% initiating activities relative to that of ATG, while GTG, ATA, and ATC had ~20% initiating activities relative to that of ATG. Unexpectedly, these non-AUG initiator codons exhibited different preferences toward various sequence contexts. In particular, GTG was one of the most efficient non-ATG initiator codons, while ATA was essentially inactive in the context of GRS1.ConclusionThis finding indicates that a sequence context that is favorable for a given non-ATG initiator codon might not be as favorable for another.