Functional characterization of elements in a human U6 small nuclear RNA gene distal control region.
The promoters of vertebrate U6 small nuclear RNA genes contain a distal control region whose presence results in at least an eightfold level of transcriptional activation in vivo. Previous transfection experiments have demonstrated that most of the distal control region of a human U6 gene resides in a restriction fragment located from -244 to -149 relative to the transcriptional start site. Three octamer-related motifs that bind recombinant Oct-1 transcription factor in vitro exist in this segment of DNA. However, transfection of human 293 cells with various plasmid templates in which these Oct-1 binding sites had been disrupted individually or in combination showed that only the consensus octamer motif located between positions -221 to -214 was functional. Even so, the consensus octamer motif mutant template was expressed at only a moderately reduced level relative to the wild-type promoter. When another octamer-related sequence located nearby, one that did not bind Oct-1 in vitro, was disrupted along with the perfect octamer site, expression was reduced fivefold in transfected cells. A factor that binds this functional, nonconsensus octamer site (NONOCT) was detected in crude cellular extracts. However, the NONOCT sequence was not essential for activation, since its disruption caused only a 40% reduction in U6 gene expression, and mutagenesis to convert the NONOCT sequence to a consensus octamer motif restored wild-type expression. Furthermore, in vitro transcription of a human U6 proximal promoter joined to a single copy of the octamer motif was stimulated by the addition of recombinant Oct-1 protein.
Danzeiser
D A
DA
Department of Biochemistry and Biophysics, Texas A&M University, College Station 77843-2128.
Urso
O
O
Kunkel
G R
GR
D001483:Base Sequence; D002460:Cell Line; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D005786:Gene Expression Regulation; D005796:Genes; D051863:Host Cell Factor C1; D006801:Humans; D066298:In Vitro Techniques; D008969:Molecular Sequence Data; D050811:Octamer Transcription Factor-1; D009838:Oligodeoxyribonucleotides / Q000737:chemistry; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D012342:RNA, Small Nuclear / Q000235:genetics*; D011994:Recombinant Proteins / Q000378:metabolism; D012045:Regulatory Sequences, Nucleic Acid; D014157:Transcription Factors / Q000235:genetics; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; C497692:HCFC1 protein, human; D051863:Host Cell Factor C1; D050811:Octamer Transcription Factor-1; D009838:Oligodeoxyribonucleotides; C494165:POU2F1 protein, human; D012342:RNA, Small Nuclear; D011994:Recombinant Proteins; D014157:Transcription Factors