Poly(A) polymerase I of Escherichia coli: characterization of the catalytic domain, an RNA binding site and regions for the interaction with proteins involved in mRNA degradation.
Poly(A) polymerase I (PAP I) of Escherichia coli is a member of the nucleotidyltransferase (Ntr) superfamily that includes the eukaryotic PAPs and all the known tRNA CCA-adding enzymes. Five highly conserved aspartic acids in the putative catalytic site of PAP I were changed to either alanine or proline, demonstrating their importance for polymerase activity. A glycine that is absolutely conserved in all Ntrs was also changed yielding a novel mutant protein in which ATP was wastefully hydrolysed in a primer-independent reaction. This is the first work to characterize the catalytic site of a eubacterial PAP and, despite the conservation of certain sequences, we predict that the overall architecture of the eukaryotic and eubacterial active sites is likely to be different. Binding sites for RNase E, a component of the RNA degradosome, and RNA were mapped by North-western and Far-western blotting using truncated forms of PAP I. Additional protein-protein interactions were detected between PAP I and CsdA, RhlE and SrmB, suggesting an unexpected connection between PAP I and these E. coli DEAD box RNA helicases. These results show that the functional organization of PAP I is similar to the eukaryotic PAPs with an N-terminal catalytic domain, a C-terminal RNA binding domain and sites for the interaction with other protein factors.
Raynal
L C
LC
Laboratoire de Microbiologie et Génétique Moléculaire, Centre National de la Recherche Scientifique (CNRS), Toulouse, France.
Carpousis
A J
AJ
D000251:Adenosine Triphosphatases / Q000378:metabolism; D001665:Binding Sites; D004722:Endoribonucleases / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology*; D016296:Mutagenesis; D009154:Mutation; D011062:Polynucleotide Adenylyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020365:RNA Helicases / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism*; D016415:Sequence Alignment; D013379:Substrate Specificity
D012333:RNA, Messenger; D011062:Polynucleotide Adenylyltransferase; D004722:Endoribonucleases; C022537:ribonuclease E; D000251:Adenosine Triphosphatases; D020365:RNA Helicases