Specific binding of heterogeneous ribonucleoprotein particle protein K to the human c-myc promoter, in vitro.
A homopurine/homopyrimidine-like sequence is found 100-150 base pairs upstream of the human c-myc promoter P1. This element, termed the CT-element, has been shown to augment expression from P1, and it serves as a positive transcriptional element when coupled to a heterologous promoter in vivo and in vitro. Synthetic oligonucleotides comprising this element were used to form DNA-protein complexes in electrophoretic mobility shift assays. By using conventional and affinity methods, 61- and 34-kDa proteins were shown to be associated with these complexes. Amino acid sequence analysis and immunological methods have identified these proteins as heterogeneous ribonucleoprotein particle (hnRNP) proteins K and A1. Surprisingly, hnRNP protein K binds to the pyrimidine-rich strand of the CT-element in a sequence-specific manner as well as to the double-stranded molecule. Cotransfection of vectors encoding hnRNP protein K in the sense or anti-sense orientations with reporter plasmids driven by wild-type or mutant CT-elements demonstrates that hnRNP protein K augments gene expression in a cis-element-dependent manner. Taken together, these results suggest that hnRNP protein K may play a role in the transcriptional regulation of the human c-myc gene.
Takimoto
M
M
Laboratory of Pathology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Tomonaga
T
T
Matunis
M
M
Avigan
M
M
Krutzsch
H
H
Dreyfuss
G
G
Levens
D
D
D000595:Amino Acid Sequence; D001483:Base Sequence; D002467:Cell Nucleus / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D016259:Genes, myc; D005822:Genetic Vectors; D006367:HeLa Cells; D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D006801:Humans; D015151:Immunoblotting; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D016376:Oligonucleotides, Antisense / Q000378:metabolism; D010446:Peptide Fragments / Q000302:isolation & purification; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D016601:RNA-Binding Proteins / Q000378:metabolism*; D012261:Ribonucleoproteins / Q000302:isolation & purification / Q000378:metabolism*; D014162:Transfection
D000076245:Heterogeneous Nuclear Ribonucleoprotein A1; D034481:Heterogeneous-Nuclear Ribonucleoprotein Group A-B; D034441:Heterogeneous-Nuclear Ribonucleoproteins; D009838:Oligodeoxyribonucleotides; D016376:Oligonucleotides, Antisense; D010446:Peptide Fragments; D016601:RNA-Binding Proteins; D012261:Ribonucleoproteins