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PMID:10361280
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Raynal, LC and Carpousis, AJ (1999) 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.Mol. Microbiol. 32:765-75
| Abstract | 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. |
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Annotations
| Gene product | Qualifier | GO ID | GO term name | Evidence Code | with/from | Aspect | Notes | Status |
|---|---|---|---|---|---|---|---|---|
|
PcnB | GO:0004652 |
polynucleotide adenylyltransferase activity |
IDA: Inferred from Direct Assay |
F |
Figure 2 shows results for assays for release of pyropohosphate and incorporation of radiolabeled AMP. |
complete | ||
|
PcnB | GO:0009451 |
RNA modification |
IDA: Inferred from Direct Assay |
P |
Oligo-histidine tagged PcnB was cloned onto an overexpression plasmid and purified using immobilized metal affinity chromatography. This purified enzyme was assayed for poly(A) polymerase activity and was shown to catalyze polyadenylation of RNA while releasing pyrophosphate (Figure 2). |
complete | ||
|
PcnB | GO:0005737 |
cytoplasm |
IDA: Inferred from Direct Assay |
C |
PcnB was purified from the supernatant of a centrifugation performed at 100 000 x g (pp. 766). |
complete | ||
|
PcnB | GO:0006396 |
RNA processing |
IDA: Inferred from Direct Assay |
P |
PcnB was shown to interact with known components of the RNA degradosome (Figure 3) and was also shown to stimulate the activity of degradosome enzymes (Figure 6B). |
complete | ||
|
PcnB | GO:0003723 |
RNA binding |
IDA: Inferred from Direct Assay |
F |
PcnB was shown to bind RNA by NorthWestern blot using [a-32P]-ATP radiolabeled RNA (Figure 5C). |
complete | ||
|
PcnB | GO:0006397 |
mRNA processing |
IDA: Inferred from Direct Assay |
P |
SeeGO:0006396 in this table. |
complete | ||
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