The hypotrichous ciliate Euplotes octocarinatus has only one type of tRNACys with GCA anticodon encoded on a single macronuclear DNA molecule.
Deviations from the universal genetic code have evolved independently several times in ciliated protozoa. Thus, in some species UAA and UAG are no longer used as termination codons, but are read as glutamine, whereas in the genus Euplotes , UGA is translated as cysteine. We have investigated the nature of the tRNACys isoacceptor responsible for decoding UGA in Euplotes cells. Southern hybridization analyses indicated that a single DNA molecule of 630 bp encoding tRNACys exists in the macronucleus of Euplotes octocarinatus . Cloning and sequencing of this fragment revealed that it contains only one copy of a tRNACys gene, which codes for a normal tRNACys with GCA anticodon. This is the first report of the characterization of a tRNA gene in any hypotrichous ciliate. It contains putative signals for initiation and termination of transcription by RNA polymerase III and can be transcribed efficiently in vitro in HeLa cell nuclear extract. Intensive studies on the DNA and tRNA level involving PCR analyses have not disclosed the existence of any tRNA Cys isoacceptor with UCA or ICA anticodons. Translation of the UGA codon by tRNA sub GCA sup Cys necessitates a G:A mispairing in the first anticodon position. We discuss a number of aspects which might contribute to the finding that a near-cognate tRNA isoacceptor efficiently translates the UGA stop codon.
Grimm
M
M
Institut für Biochemie, Bayerische Julius-Maximilians-Universität, Biozentrum, Am Hubland, D-97074 Würzburg, Germany.
Brünen-Nieweler
C
C
Junker
V
V
Heckmann
K
K
Beier
H
H
D000818:Animals; D000926:Anticodon / Q000235:genetics*; D001483:Base Sequence; D015139:Blotting, Southern; D002467:Cell Nucleus / Q000235:genetics; D018388:Codon, Terminator; D016054:DNA, Protozoan / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics; D016812:Euplotes / Q000235:genetics*; D017125:Genes, Protozoan; D005815:Genetic Code; D015698:Genomic Library; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D012320:RNA Polymerase III / Q000378:metabolism; D016053:RNA, Protozoan / Q000235:genetics; D012350:RNA, Transfer, Cys / Q000235:genetics* / Q000302:isolation & purification; D015183:Restriction Mapping; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic
D000926:Anticodon; D018388:Codon, Terminator; D016054:DNA, Protozoan; D016053:RNA, Protozoan; D012350:RNA, Transfer, Cys; D012320:RNA Polymerase III