Mapping of a sequence essential for the nuclear transport of the Xenopus ribosomal transcription factor xUBF using a simple coupled translation-transport and acid extraction approach.
The amino acid sequences directing the nuclear transport of the RNA polymerase I transcription factor xUBF have been studied by a novel combination of in oocyte-coupled translation-nuclear transport and selective HCl extraction. Synthetic mRNA was used to direct the translation of labeled xUBF and its mutants in microinjected oocytes. After manual dissection of nuclei and cytoplasm, labeled xUBF and mutants were isolated essentially pure by HCl extraction. Using deletion mutations, a sequence essential, but not necessarily sufficient, for nuclear transport was mapped to a 29-amino-acid segment lying between the most carboxy-terminal putative HMG-box DNA-binding domain, HMG-box 5, and the highly acidic carboxy-terminal domain. It was shown that deletion of only 5 amino acids from this segment eliminated xUBF transport, and it could be deduced that at least 11 of the 29 amino acids were essential for nuclear transport. The segment of xUBF necessary for nuclear transport contains a sequence conforming to the bipartite nuclear transport motif consensus, but this sequence in itself was insufficient for nuclear transport.
Dimitrov
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Centre de Recherche en Cancérologie de l'Université Laval, Hôtel-Dieu de Québec, Canada.
Bachvarov
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Moss
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D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002467:Cell Nucleus / Q000378:metabolism*; D003593:Cytoplasm / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D009865:Oocytes / Q000378:metabolism; D035463:Pol1 Transcription Initiation Complex Proteins; D021382:Protein Sorting Signals / Q000737:chemistry; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000737:chemistry* / Q000378:metabolism; D014982:Xenopus laevis
D004268:DNA-Binding Proteins; D035463:Pol1 Transcription Initiation Complex Proteins; D021382:Protein Sorting Signals; D014157:Transcription Factors; C063844:transcription factor UBF