Characterization of the E.coli poly(A) polymerase: nucleotide specificity, RNA-binding affinities and RNA structure dependence - PubMed (original) (raw)
Characterization of the E.coli poly(A) polymerase: nucleotide specificity, RNA-binding affinities and RNA structure dependence
S Yehudai-Resheff et al. Nucleic Acids Res. 2000.
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.
Figures
Figure 1
Characterization of E.coli PAP I. (A) A silver stained SDS–polyacrylamide gel of the PAP I fraction (50 ng). Molecular mass markers are shown on the left. (B) Western blot analysis. An aliquot of 30 ng of PAP I was separated by SDS–PAGE, transferred to a nitrocellulose membrane and allowed to react with antibodies raised against E.coli PAP I (20). (C) Detection of PAP I activity. In vitro transcribed [32P]RNA was incubated for 35 min without protein (lane –), with the addition of yeast PAP I (lane yeast) and with the addition of E.coli PAP I (lane E.coli). Following incubation, the RNA was isolated and analyzed by denaturing PAGE and autoradiography. A schematic representation of the non-polyadenylated and polyadenylated RNA molecules is shown on the right.
Figure 2
The E.coli PAP I activity is not specific for ATP. (A) Synthetic transcribed [32P]RNA of 380 nt was incubated with PAP I and 1 mM corresponding nucleotide. At 0, 5, 20, 40 and 60 min time points, the reaction was terminated and the RNA was isolated and analyzed by denaturing PAGE and autoradiography. (B) [32P]RNA as in (A) was incubated with PAP I for 40 min without (–) or with ATP or GTP at the concentrations shown in the figure. Following incubation, the reaction was terminated and the RNA was isolated and analyzed by denaturing PAGE and autoradiography. (C) PAP I isolated from yeast was incubated with synthetic transcribed [32P]RNA for 35 min without (–) or with the addition of 1 mM ATP, GTP, UTP or CTP. Following incubation, the RNA was isolated and analyzed as described in (A).
Figure 3
When incubated together PAP I incorporates all the nucleotides. Non-radioactive RNA of 260 nt was incubated with PAP and [α-32P]ATP for 40 min. The reaction was stopped by incubation at 70°C for 5 min and RNase A and RNase T1 (digesting at G, C and U but not A) were added for 1 h at 37°C following isolation of the RNA and analysis by denatured 12% PAGE and autoradiography (lane 2). In lane 3, non-radioactive CTP, GTP and UTP were added to the incubation with PAP I and the reaction proceeded as described for lane 2. The 260 nt radioactively labeled RNA is shown in lane 1 as a size marker. The polyadenylated RNA that was resistant to the ribonucleases and the digestion products produced when all nucleotides were used are indicated.
Figure 4
Binding of PAP I to different ribohomopolymers. (A) [32P]RNA was mixed with increasing amounts of the competitors poly(G), poly(A), poly(U) and poly(C) and 16.5 ng of PAP I. The competitor:[32P]RNA ratios were as follows: none (–), 10-fold (x10), 25-fold (x25), 50-fold (x50), 100-fold (x100) and 250-fold excess (x250). The mixture was immediately UV crosslinked and digested with RNase A and the proteins were then analyzed by SDS–PAGE and autoradiogaphy. (B) A graphical representation of the results of at least three independent competition experiments. The amount of [32P]RNA crosslinked to PAP I was quantified using a Fuji imaging analyzer and plotted as a function of the competitor excess.
Figure 5
RNA terminating with a stem–loop structure is poorly polyadenylated. Synthetic transcribed [32P]RNA corresponding to the chloroplast gene petD (A) or the E.coli gene thrA (B) in either their 3′-end processed (lanes 1 and 2) or unprocessed precursor (lanes 3 and 4) forms were used in an in vitro polyadenylation assay. The RNAs were incubated for 35 min without (lanes 1 and 3) or with 1 mM ATP (lanes 2 and 4) and E.coli PAP I. Following incubation, the RNAs were isolated and analyzed by denaturing PAGE and autoradiography. A schematic representation of the RNA substrate is shown at the bottom.
Figure 6
The addition of several nucleotides 3′ to the stem–loop is sufficient for efficient polyadenylation. [32P]RNA corresponding to the 3′-end of the E.coli malE mRNA with the addition of several nucleotides, as shown in the figure, was incubated with PAP I and ATP for the times indicated. Following incubation, the RNA was isolated and analyzed by denaturing PAGE and autoradiography.
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