Fundamental cellular processes do not require vertebrate-specific sequences within the TATA-binding protein.
The 180-amino acid core of the TATA-binding protein (TBPcore) is conserved from Archae bacteria to man. Vertebrate TBPs contain, in addition, a large and highly conserved N-terminal region that is not found in other phyla. We have generated a line of mice in which the tbp allele is replaced with a version, tbp(Delta N), which lacks 111 of 135 N-terminal amino acid residues. Most tbp(Delta N/Delta N) fetuses die in midgestation. To test whether a disruption of general cellular processes contributed to this fetal loss, primary fibroblast cultures were established from +/+, Delta N/+, and Delta N/Delta N fetuses. The cultures exhibited no genotype-dependent differences in proliferation or in expression of the proliferative markers dihydrofolate reductase (DHFR) mRNA (S phase-specific) and cdc25B mRNA (G(2)-specific). The mutation had no effect on transcription initiation site fidelity by either RNA polymerase II (pol II) or pol III. Moreover, the mutation did not cause differences in levels of U6 RNA, a pol III-dependent component of the splicing machinery, in mRNA splicing efficiency, in expression of housekeeping genes from either TATA-containing or TATA-less promoters, or in global gene expression. Our results indicated that general eukaryotic cell functions are unaffected by deletion of these vertebrate-specific sequences from TBP. Thus, all activities of this polypeptide domain must either be compensated for by redundant activities or be restricted to situations that are not represented by primary fibroblasts.
Schmidt
Edward E
EE
Department of Veterinary Molecular Biology, Marsh Laboratories, Montana State University, Bozeman, Montana 59717, USA. eschmidt@montana.edu
Bondareva
Alla A
AA
Radke
Jay R
JR
Capecchi
Mario R
MR
D000818:Animals; D002478:Cells, Cultured; D017931:DNA Primers; D004622:Embryo, Mammalian; D004625:Embryo, Nonmammalian; D005347:Fibroblasts / Q000166:cytology / Q000502:physiology; D005786:Gene Expression Regulation / Q000502:physiology*; D051379:Mice; D018345:Mice, Knockout; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017384:Sequence Deletion; D013045:Species Specificity; D035181:TATA-Box Binding Protein / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014714:Vertebrates
D017931:DNA Primers; D012333:RNA, Messenger; D035181:TATA-Box Binding Protein