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In vitro, polynucleotide phosphorylase of Escherichia coli can both synthesize RNA by using nucleotide diphosphates as precursors and exonucleolytically degrade RNA in the presence of inorganic phosphate. However, because of the high in vivo concentration of inorganic phosphate in exponentially growing cells, it has been assumed that the enzyme works(More)
In Escherichia coli, the post-transcriptional addition of poly(A) tails by poly(A) polymerase I (PAP I, pcnB) plays a significant role in cellular RNA metabolism. However, many important features of this system, including its regulation and the selection of polyadenylation sites, are still poorly understood. Here we show that the inactivation of Hfq (hfq),(More)
To help understand the role of polyadenylation in Escherichia coli RNA metabolism, we constructed an IPTG-inducible pcnB [poly(A) polymerase I, PAP I] containing plasmid that permitted us to vary poly(A) levels without affecting cell growth or viability. Increased polyadenylation led to a decrease in the half-life of total pulse-labelled RNA along with(More)
In Escherichia coli and Bacillus subtilis replication fork arrest occurs in the terminus at sequence-specific sites by the binding of replication terminator proteins to the fork arrest sites. The protein-DNA complex causes polar arrest of the replication forks by inhibiting the activity of the replicative helicases in only one orientation of the terminus(More)
This chapter provides detailed methodologies for isolating total RNA and polyadenylated RNA from E. coli and other prokaryotes, along with the procedures necessary to analyze the processing and decay of specific transcripts and determine their 3'- and 5'-ends. The RNA isolation methods described here facilitate isolating good-quality RNA in a very(More)
Tiling microarrays have proven to be a valuable tool for gaining insights into the transcriptomes of microbial organisms grown under various nutritional or stress conditions. Here, we describe the use of such an array, constructed at the level of 20 nt resolution for the Escherichia coli MG1655 genome, to observe genome-wide changes in the steady-state RNA(More)
Although the first poly(A) polymerase (PAP) was discovered in Escherichia coli in 1962, the study of polyadenylation in bacteria was largely ignored for the next 30 years. However, with the identification of the structural gene for E. coli PAP I in 1992, it became possible to analyze polyadenylation using both biochemical and genetic approaches.(More)
Polyadenylation of RNAs by poly(A) polymerase I (PAP I) in Escherichia coli plays a significant role in mRNA decay and general RNA quality control. However, many important features of this system, including the prevalence of polyadenylated mRNAs in the bacterium, are still poorly understood. By comparing the transcriptomes of wild-type and pcnB deletion(More)
The primase DnaG of Escherichia coli requires the participation of the replicative helicase DnaB for optimal synthesis of primer RNA for lagging strand replication. However, previous studies had not determined whether the activation of the primase or its loading on the template was accomplished by a helicase-mediated structural alteration of the(More)
The first step in the current model for the processing and maturation of mono- and polycistronic tRNA precursors in Escherichia coli involves initial cleavages by RNase E 1-3 nt downstream of each chromosomally encoded CCA determinant. Subsequently, each mature 5' terminus is generated by single RNase P cleavage, while the 3' terminus undergoes(More)