Site-directed alterations in the ATP-binding domain of rho protein affect its activities as a termination factor.
We have utilized oligonucleotide site-directed mutagenesis to test our prediction that Escherichia coli rho factor has an ATP-binding domain separate from its RNA-binding domain and similar to that of adenylate kinase. Single amino acid substitutions were generated in regions thought to be within the active site and catalytically important for the ATPase activity, changing lysine 181 and/or lysine 184 to glutamine, and aspartate 265 to valine and asparagine. The altered proteins were purified and characterized in vitro for RNA- and ATP-binding ability, ATPase activity, helicase activity, and ability to catalyze transcription termination. Our results indicate that 1) these amino acid alterations in the proposed ATP-binding domain do not interfere with RNA binding; 2) substitution of lysine 184 by glutamine actually improves the ATPase and related activities while the same substitution at lysine 181 reduces but does not eliminate activity; 3) the double mutation changing both lysine 181 and lysine 184 to glutamine eliminates ATPase activity; and 4) the aspartate at 265 is also required for ATP hydrolysis but not for ATP binding. These results are consistent with our proposal that the general tertiary structure of rho's ATP-binding domain is similar to that of adenylate kinase.
Dombroski
A J
AJ
Department of Biochemistry, University of Rochester Medical Center, New York 14642.
Brennan
C A
CA
Spear
P
P
Platt
T
T
D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism*; D001665:Binding Sites; D004338:Drug Combinations; D005973:Glutamine / Q000378:metabolism; D008239:Lysine / Q000378:metabolism; D008958:Models, Molecular; D010449:Peptide Mapping; D011487:Protein Conformation; D012313:RNA / Q000378:metabolism; D012234:Rho Factor / Q000378:metabolism*; D013329:Structure-Activity Relationship; D014157:Transcription Factors / Q000378:metabolism*
D004338:Drug Combinations; D012234:Rho Factor; D014157:Transcription Factors; D005973:Glutamine; D012313:RNA; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D008239:Lysine