Identification of the coding region for a second poly(A) polymerase in Escherichia coli.
We had earlier identified the pcnB locus as the gene for the major Escherichia coli poly(A) polymerase (PAP I). In this report, we describe the disruption and identification of a candidate gene for a second poly(A) polymerase (PAP II) by an experimental strategy which was based on the assumption that the viability of E. coli depends on the presence of either PAP I or PAP II. The coding region thus identified is the open reading frame f310, located at about 87 min on the E. coli chromosome. The following lines of evidence support f310 as the gene for PAP II: (i) the deduced peptide encoded by f310 has a molecular weight of 36,300, similar to the molecular weight of 35,000 estimated by gel filtration of PAP II; (ii) the deduced f310 product is a relatively hydrophobic polypeptide with a pI of 9.4, consistent with the properties of partially purified PAP II; (iii) overexpression of f310 leads to the formation of inclusion bodies whose solubilization and renaturation yields poly(A) polymerase activity that corresponds to a 35-kDa protein as shown by enzyme blotting; and (iv) expression of a f310 fusion construct with hexahistidine at the N-terminus of the coding region allowed purification of a poly(A) polymerase fraction whose major component is a 36-kDa protein. E. coli PAP II has no significant sequence homology either to PAP I or to the viral and eukaryotic poly(A) polymerases, suggesting that the bacterial poly(A) polymerases have evolved independently. An interesting feature of the PAP II sequence is the presence of sets of two paired cysteine and histidine residues that resemble the RNA binding motifs seen in some other proteins.
Cao
G J
GJ
Boston Biomedical Research Institute, MA 02114, USA.
Pogliano
J
J
Sarkar
N
N
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D002479:Inclusion Bodies / Q000201:enzymology; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry; D011062:Polynucleotide Adenylyltransferase / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D016324:Sequence Tagged Sites
D010446:Peptide Fragments; D011994:Recombinant Proteins; D011062:Polynucleotide Adenylyltransferase