Identification of an RNA-binding Site in the ATP binding domain of Escherichia coli Rho by H2O2/Fe-EDTA cleavage protection studies.
Transcription factor Rho is a ring-shaped, homohexameric protein that causes transcript termination through actions on nascent RNAs that are coupled to ATP hydrolysis. The Rho polypeptide has a distinct RNA binding domain of known structure as well as an ATP binding domain for which a structure has been proposed based on homology modeling. Treatment of Rho with H2O2 in the presence of Fe-EDTA caused single-cut cleavage at a number of points that coincide with solvent-exposed loops in both the known and predicted structures, thereby providing support for the validity of the tertiary and quaternary structural models of Rho. The binding of ATP caused one distinct change in the cleavage pattern, a strong protection at a cleavage point in the P-loop of the ATP binding domain. Binding of RNA and single-stranded DNA (poly(dC)) caused strong protection at several accessible parts of the oligosaccharide/oligonucleotide binding (OB) fold in the RNA binding domain. RNA molecules but not DNA molecules also caused a strong, ATP-dependent protection at a cleavage site in the predicted Q-loop of the ATP binding domain. These results suggest that Rho has two distinct binding sites for RNA. Besides the site composed of multiples of the RNA binding domain, to which single-stranded DNA as well as RNA can bind, it has a separate, RNA-specific site on the Q-loop in the ATP binding domain. In the proposed quaternary structure of Rho, the Q-loops from the six subunits form the upper entrance to the hole in the ring-shaped hexamer through which the nascent transcript is translocated by actions coupled to ATP hydrolyses.
Wei
R R
RR
Department of Biology, Indiana University, Bloomington, Indiana 47405, USA.
Richardson
J P
JP
D000225:Adenine / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001665:Binding Sites; D015153:Blotting, Western; D004247:DNA / Q000378:metabolism; D004492:Edetic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism*; D006861:Hydrogen Peroxide / Q000494:pharmacology; D006868:Hydrolysis; D008024:Ligands; D008958:Models, Molecular; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D020836:Protein Structure, Quaternary; D017434:Protein Structure, Tertiary; D012313:RNA / Q000378:metabolism*; D013997:Time Factors; D014158:Transcription, Genetic; D020741:rho GTP-Binding Proteins / Q000737:chemistry* / Q000378:metabolism
D008024:Ligands; D012313:RNA; D000255:Adenosine Triphosphate; D004247:DNA; D004492:Edetic Acid; D006861:Hydrogen Peroxide; D000251:Adenosine Triphosphatases; D020741:rho GTP-Binding Proteins; D000225:Adenine