Identification of a nucleic acid-binding region within the largest subunit of Drosophila melanogaster RNA polymerase II.
The largest and the second-largest subunit of the multisubunit eukaryotic RNA polymerases are involved in interaction with the DNA template and the nascent RNA chain. Using Southwestern DNA-binding techniques and nitrocellulose filter binding assays of bacterially expressed fusion proteins, we have identified a region of the largest, 215-kDa, subunit of Drosophila RNA polymerase II that has the potential to bind nucleic acids nonspecifically. This nucleic acid-binding region is located between amino acid residues 309-384 and is highly conserved within the largest subunits of eukaryotic and bacterial RNA polymerases. A homology to a region of the DNA-binding cleft of Escherichia coli DNA polymerase I involved in binding of the newly synthesized DNA duplex provides indirect evidence that the nucleic acid-binding region of the largest subunit participates in interaction with double-stranded nucleic acids during transcription. The nonspecific DNA-binding behavior of the region is similar to that observed for the native enzyme in nitrocellulose filter binding assays and that of the separated largest subunit in Southwestern assays. A high content of basic amino acid residues is consistent with the electrostatic nature of nonspecific DNA binding by RNA polymerases.
Kontermann
R E
RE
Institute of Molecular Genetics, University of Heidelberg, Germany.
Kobor
M
M
Bautz
E K
EK
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D017124:Conserved Sequence; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D004331:Drosophila melanogaster / Q000201:enzymology*; D004926:Escherichia coli / Q000235:genetics; D006493:Heparin / Q000494:pharmacology; D046911:Macromolecular Substances; D015636:Magnesium Chloride / Q000494:pharmacology; D008969:Molecular Sequence Data; D009696:Nucleic Acids / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D012319:RNA Polymerase II / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D012965:Sodium Chloride / Q000494:pharmacology; D013329:Structure-Activity Relationship
D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D009696:Nucleic Acids; D011993:Recombinant Fusion Proteins; D015636:Magnesium Chloride; D012965:Sodium Chloride; D006493:Heparin; D012319:RNA Polymerase II