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Residual polyadenylation in poly(A) polymerase I ( pcnB ) mutants of Escherichia coli does not result from the activity encoded by the f310 gene
Author(s) -
Mohanty Bijoy K.,
Kushner Sidney R.
Publication year - 1999
Publication title -
molecular microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.857
H-Index - 247
eISSN - 1365-2958
pISSN - 0950-382X
DOI - 10.1046/j.1365-2958.1999.01674.x
Subject(s) - biology , escherichia coli , polymerase , mutant , gene , microbiology and biotechnology , polyadenylation , rna polymerase , plasmid , mutagenesis , dna polymerase i , genetics , polymerase chain reaction , gene expression , reverse transcriptase
As extracts of poly(A) polymerase I (PAP I) deficient strains of Escherichia coli appeared to contain considerable residual polyadenylating activity, efforts were undertaken to identify a second poly(A) polymerase. Recently, a gene ( f310 ) encoding the putative second poly(A) polymerase was cloned and sequenced. Here we have tested the ability of the F310 protein to add poly(A) tails in vivo by measuring total poly(A) levels in both f310 mutants and strains that overproduce F310. In addition, we have visualized poly(A) tails and examined ColE1 plasmid copy number in various genetic backgrounds. We also carried out direct biochemical measurements of AMP incorporation, using cell extracts after amplification of F310. All the data obtained indicate that F310 is not a poly(A) polymerase. Although the presence of two potential ATP binding domains in the F310 protein may account for its apparent ATP binding activity, its true biochemical function remains to be identified. In addition, we show that the f310 gene is transcribed, almost exclusively, during stationary phase from a σ s promoter.