Restricted specificity of Xenopus TFIIIA for transcription of somatic 5S rRNA genes.
Xenopus transcription factor IIIA (TFIIIA) is phosphorylated on serine-16 by CK2. Replacements with alanine or glutamic acid were made at this position in order to address the question of whether phosphorylation possibly influences the function of this factor. Neither substitution has an effect on the DNA or RNA binding activity of TFIIIA. The wild-type factor and the alanine variant activate transcription of somatic- and oocyte-type 5S rRNA genes in nuclear extract immunodepleted of endogenous TFIIIA. The glutamic acid variant (S16E) supports the transcription of somatic-type genes at levels comparable to those of wild-type TFIIIA; however, there is no transcription of the oocyte-type genes. This differential behavior of the phosphomimetic mutant protein is also observed in vivo when using early-stage embryos, where this mutant failed to activate transcription of the endogenous oocyte-type genes. Template exclusion assays establish that the S16E mutant binds to the oocyte-type 5S rRNA genes and recruits at least one other polymerase III transcription factor into an inactive complex. Phosphorylation of TFIIIA by CK2 may allow the factor to continue to act as a positive activator of the somatic-type genes and simultaneously as a repressor of the oocyte-type 5S rRNA genes, indicating that there is a mechanism that actively promotes repression of the oocyte-type genes at the end of oogenesis.
Ghose
Romi
R
Department of Chemistry and Biochemistry, University of Notre Dame, Notre Dame, Indiana 46556, USA.
Malik
Mariam
M
Huber
Paul W
PW
D019943:Amino Acid Substitution; D000818:Animals; D001665:Binding Sites / Q000235:genetics; D047390:Casein Kinase II; D005260:Female; D066298:In Vitro Techniques; D016297:Mutagenesis, Site-Directed; D009865:Oocytes / Q000254:growth & development / Q000378:metabolism; D009866:Oogenesis / Q000235:genetics; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D012320:RNA Polymerase III / Q000235:genetics / Q000378:metabolism; D012341:RNA, Ribosomal, 5S / Q000235:genetics*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012694:Serine / Q000737:chemistry; D035561:Transcription Factor TFIIA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014981:Xenopus / Q000235:genetics* / Q000378:metabolism*
D012341:RNA, Ribosomal, 5S; D011994:Recombinant Proteins; D035561:Transcription Factor TFIIA; D012694:Serine; D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D012320:RNA Polymerase III