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.     

Mitogenomes Reveal Alternative Initiation Codons and Lineage-Specific Gene Order Conservation in Echinoderms

The mitochondrial genetic code is much more varied than the standard genetic code. The invertebrate mitochondrial code, for instance, comprises six initiation codons, including five alternative start codons. However, only two initiation codons are known in the echinoderm and flatworm mitochondrial code, the canonical ATG and alternative GTG. Here, we analyzed 23 Asteroidea mitogenomes, including ten newly sequenced species and unambiguously identified at least two other start codons, ATT and ATC, both of which also initiate translation of mitochondrial genes in other invertebrates. These findings underscore the diversity of the genetic code and expand upon the suite of initiation codons among echinoderms to avoid erroneous annotations. Our analyses have also uncovered the remarkable conservation of gene order among asteroids, echinoids, and holothuroids, with only an interchange between two gene positions in asteroids over ∼500 Ma of echinoderm evolution.     

Cross-species and cross-compartmental aminoacylation of isoaccepting tRNAs by a class II tRNA synthetase

It was previously shown that ALA1, the only alanyl-tRNA synthetase gene in Saccharomyces cerevisiae, codes for two functionally exclusive protein isoforms through alternative initiation at two consecutive ACG codons and an in-frame downstream AUG. We reported here the cloning and characterization of a homologous gene from Candida albicans. Functional assays show that this gene can substitute for both the cytoplasmic and mitochondrial functions of ALA1 in S. cerevisiae and codes for two distinct protein isoforms through alternative initiation from two in-frame AUG triplets 8-codons apart. Unexpectedly, although the short form acts exclusively in cytoplasm, the longer form provides function in both compartments. Similar observations are made in fractionation assays. Thus, the alanyl-tRNA synthetase gene of C. albicans has evolved an unusual pattern of translation initiation and protein partitioning and codes for protein isoforms that can aminoacylate isoaccepting tRNAs from a different species and from across cellular compartments.     

Identification of the initiation codon for the atpB gene in Chlamydomonas chloroplasts excludes translation of a precursor form of the beta subunit of the ATP synthase

The chloroplast atpB gene of Chlamydomonas reinhardtii, which encodes the beta subunit of the ATP synthase, contains three in-frame ATGs that are candidate translation initiation codons. An earlier study revealed that the N terminus of the assembled beta subunit maps at the +2 position with respect to the second in-frame methionine codon (Fiedler et al. 1995). Using chloroplast transformation, we have examined the possibility that either of the two additional in-frame ATG codons is competent for translation initiation. We provide evidence that translation of atpB is initiated exclusively at the second ATG codon. We conclude that the beta subunit is not synthesized with an N-terminal leader before its assembly into a functional ATP synthase complex.     

Initiation of herpes simplex virus thymidine kinase polypeptides.

When employed as a transgene reporter, the herpes simplex type 1 virus (HSV1) thymidine kinase gene (tk) is ectopically expressed in mouse testis. The principal testicular mRNA lacks the 5'-end of the tk reading frame. As a result the principal translation products, P2 and P3, are N-terminally truncated. These co-migrate in SDS-PAGE with polypeptides synthesised during HSV1 infection that were previously thought to be initiated at methionine codons ATG46 and ATG60. Prompted by these observations we generated modified tk genes each carrying only one of the first three ATG codons. Transfected cells expressed both full-length enzyme (P1) and P2 when only ATG1 was unmodified, P2 and P3 when only ATG46 was unmodified or P2 and a fourth polypeptide (P4) when only ATG60 was unmodified. Our observations indicate that P3 is initiated at ATG46 rather than ATG60, while P2 is initiated at a non-ATG codon rather than ATG46 and P4 is initiated at ATG60. When either of two putative non-ATG initiation codons was modified P2 was no longer produced. Cells mainly expressing either P1 or P3 exhibited the same sensitivity to Ganciclovir as cells transfected with the unaltered tk gene. P1 and P3 both have TK activity while P4 probably has none.     

The human DNA ligase III gene encodes nuclear and mitochondrial proteins

We provide evidence that the human DNA ligase III gene encodes a mitochondrial form of this enzyme. First, the DNA ligase III cDNA contains an in-frame ATG located upstream from the putative translation initiation start site. The DNA sequence between these two ATG sites encodes an amphipathic helix similar to previously identified mitochondrial targeting peptides. Second, recombinant green fluorescent protein harboring this sequence at its amino terminus was efficiently targeted to the mitochondria of Cos-1 monkey kidney cells. In contrast, native green fluorescent protein distributed to the cytosol. Third, a series of hemagglutinin-DNA ligase III minigene constructs were introduced into Cos-1 cells, and immunocytochemistry was used to determine subcellular localization of the epitope-tagged DNA ligase III protein. These experiments revealed that inactivation of the upstream ATG resulted in nuclear accumulation of the DNA ligase III protein, whereas inactivation of the downstream ATG abolished nuclear localization and led to accumulation within the mitochondrial compartment. Fourth, mitochondrial protein extracts prepared from human cells overexpressing antisense DNA ligase III mRNA possessed substantially less DNA ligase activity than did mitochondrial extracts prepared from control cells. DNA end-joining activity was also substantially reduced in extracts prepared from antisense mRNA-expressing cells. From these results, we conclude that the human DNA ligase III gene encodes both nuclear and mitochondrial enzymes. DNA ligase plays a central role in DNA replication, recombination, and DNA repair. Thus, identification of a mitochondrial form of this enzyme provides a tool with which to dissect mammalian mitochondrial genome dynamics.     

Nucleotide sequence and functional analysis of the RAD1 gene of Saccharomyces cerevisiae.

The RAD1 gene of Saccharomyces cerevisiae is involved in excision repair of damaged DNA. The nucleotide sequence of the RAD1 gene presented here shows an open reading frame of 3,300 nucleotides. Two ATG codons occur in the open reading frame at positions +1 and +334, respectively. Since a deletion of about 2.7 kilobases of DNA from the 5' region of the RAD1 gene, which also deletes the +1 ATG and 11 additional codons in the RAD1 open reading frame, partially complements UV sensitivity of a rad1 delta mutant, we examined the role of the +1 ATG and +334 ATG codons in translation initiation of RAD1 protein. Mutation of the +1 ATG codon to ATC affected the complementation ability of the RAD1 gene, whereas mutation of the +334 ATG codon to ATC showed no discernible effect on RAD1 function. These results indicate that translation of RAD1 protein is initiated from the +1 ATG codon. Productive in-frame RAD1-lacZ fusions showed that the RAD1 open reading frame is expressed in yeasts. The RAD1-encoded protein contains 1,100 amino acids with a molecular weight of 126,360.