The signal for translational readthrough of a UGA codon in Sindbis virus RNA involves a single cytidine residue immediately downstream of the termination codon.
The nucleotide sequences surrounding termination codons influence the efficiency of translational readthrough. In this report, we examined the sequence requirement for efficient readthrough of the UGA codon in the Sindbis virus genomic RNA which regulates production of the putative viral RNA polymerase, nsP4. The UGA codon and its neighboring nucleotide sequences were subcloned into a heterologous coding context, and readthrough efficiency was measured by cell-free translation of RNA transcripts in rabbit reticulocyte lysates. The CUA codon immediately downstream of the UGA codon was found to be sufficient for efficient translational readthrough. Further mutagenesis of residues in the CUA triplet demonstrated that mutations at the second or third residues following the UGA codon (U and A, respectively) had little effect on readthrough efficiency. In contrast, replacement of the cytidine residue immediately downstream of the UGA codon with any of the other three nucleotides (U, A, or G) dramatically reduced the readthrough efficiency from approximately 10% to less than 1%. These results show that a simple sequence context can allow efficient readthrough of UGA codons in a mammalian translation system. Interestingly, compilation studies of nucleotide sequences surrounding eukaryotic termination codons indicate a strong bias against cytidine residues immediately 3' to UGA termination codons. Taken together with our results, this bias may reflect a selective pressure for efficient translation termination for most eukaryotic gene products.
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Department of Molecular Microbiology, Washington University School of Medicine, St. Louis, Missouri 63110.
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D000595:Amino Acid Sequence; D001483:Base Sequence; D002213:Capsid / Q000096:biosynthesis* / Q000302:isolation & purification; D002474:Cell-Free System; D003001:Cloning, Molecular; D003062:Codon / Q000378:metabolism*; D003562:Cytidine; D004591:Electrophoresis, Polyacrylamide Gel; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D014176:Protein Biosynthesis; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism*; D012045:Regulatory Sequences, Nucleic Acid; D015183:Restriction Mapping; D012845:Sindbis Virus / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins / Q000096:biosynthesis* / Q000302:isolation & purification
D003062:Codon; D012367:RNA, Viral; D014764:Viral Proteins; D003562:Cytidine