Non-standard translational events in Candida albicans mediated by an unusual seryl-tRNA with a 5'-CAG-3' (leucine) anticodon.

From in vitro translation studies we have previously demonstrated the existence of an apparent efficient UAG (amber) suppressor tRNA in the dimorphic fungus Candida albicans (Santos et al., 1990). Using an in vitro assay for termination codon readthrough the tRNA responsible was purified to homogeneity from C.albicans cells. The determined sequence of the purified tRNA predicts a 5'-CAG-3' anticodon that should decode the leucine codon CUG and not the UAG termination codon as originally hypothesized. However, the tRNA(CAG) sequence shows greater nucleotide homology with seryl-tRNAs from the closely related yeast Saccharomyces cerevisiae than with leucyl-tRNAs from the same species. In vitro tRNA-charging studies demonstrated that the purified tRNA(CAG) is charged with Ser. The gene encoding the tRNA was cloned from C.albicans by a PCR-based strategy and DNA sequence analysis confirmed both the structure of the tRNA(CAG) and the absence of any introns in the tRNA gene. The copy number of the tRNA(CAG) gene (1-2 genes per haploid genome) is in agreement with the relatively low abundance (< 0.5% total tRNA) of this tRNA. In vitro translation studies revealed that the purified tRNA(CAG) could induce apparent translational bypass of all three termination codons. However, peptide mapping of in vitro translation products demonstrated that the tRNA(CAG) induces translational misreading in the amino-terminal region of two RNA templates employed, namely the rabbit alpha- and beta-globin mRNAs. These results suggest that the C.albicans tRNA(CAG) is not an 'omnipotent' suppressor tRNA but rather may mediate a novel non-standard translational event in vitro during the translation of the CUG codon. The possible nature of this non-standard translation event is discussed in the context of both the unusual structural features of the tRNA(CAG) and its in vitro behaviour.     

Rhizobium meliloti nifN (fixF) gene is part of an operon regulated by a nifA-dependent promoter and codes for a polypeptide homologous to the nifK gene product.

An essential gene for symbiotic nitrogen fixation (fixF) is located near the common nodulation region of Rhizobium meliloti. A DNA fragment carrying fixF was characterized by hybridization with Klebsiella pneumoniae nif DNA and by nucleotide sequence analysis. The fixF gene was found to be related to K. pneumoniae nifN and was therefore renamed as the R. meliloti nifN gene. Upstream of the nifN coding region a second open reading frame was identified coding for a putative polypeptide of 110 amino acids (ORF110). By fragment-specific Tn5 mutagenesis it was shown that the nifN gene and ORF110 form an operon. The control region of this operon contains a nif promoter and also the putative nifA-binding sequence. For the deduced amino acid sequence of the nifN gene product a striking homology to the R. meliloti nifK protein was found. One cysteine residue and its adjacent amino acid sequence, which are highly conserved in the R. meliloti nifK, R. meliloti nifN, and K. pneumoniae nifN proteins, may play a role in binding the FeMo cofactor.     

The nucleotide sequence of the tobacco chloroplast gene for the large subunit of ribulose-l,5-bisphosphate carboxylase/oxygenase

The gene for the large subunit (LS) of ribulose-l,5-bisphosphate carboxylase/oxygenase (RuBPCase/ Oase) from tobacco has been cloned in pBR322 and sequenced. The coding region contains 1431 bp (477 codons). The deduced arnino acid sequence of tobacco LS protein shows 90% homology with those of maize and spinach LS. The positions in the gene corresponding to the 5' and the 3' ends of tobacco LS mRNA have been located on the DNA sequence by the S1 nuclease mapping procedure. The LS gene promoter sequence has homology with Escherichia coli promoter sequences; its terminator sequence is capable of forming a stem-and-loop structure. A sequence GGAGG, which is complementary to a sequence near the 3' end of tobacco chloroplast 16S rRNA and a putative ribosome binding site, occurs 6–10 bp upstream from the initiation codon.