Heterogeneous nuclear ribonucleoprotein K is a transcription factor.
The CT element is a positively acting homopyrimidine tract upstream of the c-myc gene to which the well-characterized transcription factor Spl and heterogeneous nuclear ribonucleoprotein (hnRNP) K, a less well-characterized protein associated with hnRNP complexes, have previously been shown to bind. The present work demonstrates that both of these molecules contribute to CT element-activated transcription in vitro. The pyrimidine-rich strand of the CT element both bound to hnRNP K and competitively inhibited transcription in vitro, suggesting a role for hnRNP K in activating transcription through this single-stranded sequence. Direct addition of recombinant hnRNP K to reaction mixtures programmed with templates bearing single-stranded CT elements increased specific RNA synthesis. If hnRNP K is a transcription factor, then interactions with the RNA polymerase II transcription apparatus are predicted. Affinity columns charged with recombinant hnRNP K specifically bind a component(s) necessary for transcription activation. The depleted factors were biochemically complemented by a crude TFIID phosphocellulose fraction, indicating that hnRNP K might interact with the TATA-binding protein (TBP)-TBP-associated factor complex. Coimmunoprecipitation of a complex formed in vivo between hnRNP K and epitope-tagged TBP as well as binding in vitro between recombinant proteins demonstrated a protein-protein interaction between TBP and hnRNP K. Furthermore, when the two proteins were overexpressed in vivo, transcription from a CT element-dependent reporter was synergistically activated. These data indicate that hnRNP K binds to a specific cis element, interacts with the RNA polymerase II transcription machinery, and stimulates transcription and thus has all of the properties of a transcription factor.
Michelotti
E F
EF
Laboratory of Pathology, National Cancer Institute, National Institute of Health, Bethesda, Maryland 20892, USA.
Michelotti
G A
GA
Aronsohn
A I
AI
Levens
D
D
D001483:Base Sequence; D001665:Binding Sites; D001667:Binding, Competitive; D015153:Blotting, Western; D002852:Chromatography, Ion Exchange; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D016259:Genes, myc; D006367:HeLa Cells; D034641:Heterogeneous-Nuclear Ribonucleoprotein K; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D012319:RNA Polymerase II / Q000378:metabolism; D012332:RNA, Heterogeneous Nuclear / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012261:Ribonucleoproteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D016329:Sp1 Transcription Factor / Q000378:metabolism; D035181:TATA-Box Binding Protein; D013698:Templates, Genetic; D035362:Transcription Factor TFIID; D014157:Transcription Factors / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014162:Transfection
D004268:DNA-Binding Proteins; D034641:Heterogeneous-Nuclear Ribonucleoprotein K; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D009838:Oligodeoxyribonucleotides; D012332:RNA, Heterogeneous Nuclear; D011994:Recombinant Proteins; D012261:Ribonucleoproteins; D016329:Sp1 Transcription Factor; D035181:TATA-Box Binding Protein; D035362:Transcription Factor TFIID; D014157:Transcription Factors; D012319:RNA Polymerase II