title: Characterization of the E.coli poly(A) polymerase: nucleotide specificity, RNA-binding affinities and RNA structure dependence.
abstract: Polyadenylation of RNA molecules in bacteria and chloroplasts has been implicated as part of the RNA degradation pathway. The polyadenylation reaction is performed in Escherichia coli mainly by the enzyme poly(A) polymerase I (PAP I). In order to understand the molecular mechanism of RNA poly-adenylation in bacteria, we characterized the biochemical properties of this reaction in vitro using the purified enzyme. Unlike the PAP from yeast nucleus, which is specific for ATP, E.coli PAP I can use all four nucleotide triphosphates as substrates for addition of long ribohomopolymers to RNA. PAP I displays a high binding activity to poly(U), poly(C) and poly(A) ribohomopolymers, but not to poly(G). The 3'-ends of most of the mRNA molecules in bacteria are characterized by a stem-loop structure. We show here that in vitro PAP I activity is inhibited by a stem-loop structure. A tail of two to six nucleo-tides located 3' to the stem-loop structure is sufficient to overcome this inhibition. These results suggest that the stem-loop structure located in most of the mRNA 3'-ends may function as an inhibitor of poly-adenylation and degradation of the corresponding RNA molecule. However, RNA 3'-ends produced by endonucleolytic cleavage by RNase E in single-strand regions of mRNA molecules may serve as efficient substrates for polyadenylation that direct these molecules for rapid exonucleolytic degradation.
authors.affiliation: Department of Biology, Technion-Israel Institute of Technology, Haifa 32000, Israel.
authors.forename: S
authors.initials: S
authors.lastname: Yehudai-Resheff
authors.forename: G
authors.initials: G
authors.lastname: Schuster
mesh_terms: D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology; D009690:Nucleic Acid Conformation; D011062:Polynucleotide Adenylyltransferase / Q000032:analysis* / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D012329:RNA, Bacterial / Q000378:metabolism*; D013329:Structure-Activity Relationship
chemical_list: D012329:RNA, Bacterial; D011062:Polynucleotide Adenylyltransferase