RapA, Escherichia coli RNA polymerase SWI/SNF subunit-dependent polyadenylation of RNA

In this work, we describe RapA-dependent polyadenylation of model RNA substrates and endogenous, RNA polymerase-associated nucleic acid fragments. We demonstrate that the Escherichia coli RNA polymerase obtained through the classic purification procedure carries endogenous RNA oligonucleotides, which, in the presence of ATP, are polyriboadenylated in a RapA-dependent manner by an accessory poly(rA) polymerase. RNA polymerase isolated from poly(A) polymerase- (PAP-) and polynucleotide phosphorylase- (PNP-) deficient E. coli strain lacks accessory (rA)(n)-synthetic activity. Experiments with reconstituted RNA polymerase-PAP and RNA polymerase-PNP mixtures suggest that RapA enables the polyadenylation by PAP of RNA polymerase-associated RNA. Mutations disrupting RapA's ATP-hydrolytic function disrupt RapA-dependent polyadenylation, and the rapA(-)E. coli strain displays a measurable reduction in RNA polyadenylation. RapA-dependent polyadenylation can also be modulated by mutations in the section of RapA's SWI/SNF domain linked to interaction with single-stranded nucleic acid. We have developed enzymatic assays in which model, synthetic RNAs are polyriboadenylated in a RapA-dependent manner. Taken together, our results are consistent with RapA acting as an RNA polymerase-associated, ATP-dependent RNA translocase. Our work further links RapA to RNA remodeling and provides new mechanistic insights into the functional interaction between RNA polymerase and RapA.     

Specific polyadenylation and purification of total messenger RNA from Escherichia coli.

Obtaining pure mRNA preparations from prokaryotes has been difficult, if not impossible, for want of a poly(A) tail on these messages. We have used poly(A) polymerase from yeast to effect specific polyadenylation of Escherichia coli polysomal mRNA in the presence of magnesium and manganese. The polyadenylated total mRNA, which could be subsequently purified by binding to and elution from oligo(dT) beads, had a size range of 0.4-4.0 kb. We have used hybridization to a specific plasmid-encoded gene to further confirm that the polyadenylated species represented mRNA. Withdrawal of Mg2+ from the polyadenylation reaction resulted in addition of poly(A) to 16S rRNA despite the presence of Mn2+, indicating the vital role of Mg2+ in maintaining the native structure of polysomes. Complete dissociation of polysomes into ribosomal subunits resulted in quantitative polyadenylation of both 16S and 23S rRNA species. Chromosomal lacZ gene-derived messages were quantitatively recovered in the oligo(dT)-bound fraction, as demonstrated by RT-PCR analysis. Potential advantages that accrue from the availability of pure total mRNA from prokaryotes is discussed.     

RNA polyadenylation and degradation in cyanobacteria are similar to the chloroplast but different from Escherichia coli

The mechanism of RNA degradation in Escherichia coli involves endonucleolytic cleavage, polyadenylation of the cleavage product by poly(A) polymerase, and exonucleolytic degradation by the exoribonucleases, polynucleotide phosphorylase (PNPase) and RNase II. The poly(A) tails are homogenous, containing only adenosines in most of the growth conditions. In the chloroplast, however, the same enzyme, PNPase, polyadenylates and degrades the RNA molecule; there is no equivalent for the E. coli poly(A) polymerase enzyme. Because cyanobacteria is a prokaryote believed to be related to the evolutionary ancestor of the chloroplast, we asked whether the molecular mechanism of RNA polyadenylation in the Synechocystis PCC6803 cyanobacteria is similar to that in E. coli or the chloroplast. We found that RNA polyadenylation in Synechocystis is similar to that in the chloroplast but different from E. coli. No poly(A) polymerase enzyme exists, and polyadenylation is performed by PNPase, resulting in heterogeneous poly(A)-rich tails. These heterogeneous tails were found in the amino acid coding region, the 5' and 3' untranslated regions of mRNAs, as well as in rRNA and the single intron located at the tRNA(fmet). Furthermore, unlike E. coli, the inactivation of PNPase or RNase II genes caused lethality. Together, our results show that the RNA polyadenylation and degradation mechanisms in cyanobacteria and chloroplast are very similar to each other but different from E. coli.     

DEAD-box RNA helicases in Escherichia coli

In spite of their importance in RNA metabolism, the function of DExD/H-box proteins (including DEAD-box proteins) is poorly understood at the molecular level. Here, we present recent progress achieved with the five DEAD-box proteins from Escherichia coli, which have been particularly well studied. These proteins, which have orthologues in many bacteria, participate, in particular, in specific steps of mRNA decay and ribosome assembly. In vitro, they behave as poorly processive RNA helicases, presumably because they only unwind a few base pairs at each cycle so that stable duplexes can reanneal rather than dissociate. Except for one of them (DbpA), these proteins lack RNA specificity in vitro, and specificity in vivo is likely conferred by partners that target them to defined substrates. Interestingly, at least one of them is multifunctional, presumably because it can interact with different partners. Altogether, several aspects of the information gathered with these proteins have become paradigms for our understanding of DEAD-box proteins in general.     

Decay of RNA in RNA processing mutants of Escherichia coli

Summary Stability of RNA was tested in strains of Escherichia, coli carrying single, double, or triple mutations in the RNA processing enzymes RNase III, RNase E and RNase P. Tests were carried out for total pulse labeled RNA, -galactosidase mRNA and for the decay of preexisting RNA during carbon starvation. Decay of RNA was measured at premissive and nonpermissive temperatures and in no case were significant differences between mutants and non-mutant strains found. Therefore, we conclude that the three processing enzymes; RNase III, E and P do not contribute significantly to turnover of RNA in Escherichia coli.     

A spectinomycin dependent mutant of Escherichia coli.

Summary A mutant of Escherichia coli B has been isolated which shows a novel phenotype of spectinomycin dependence. The mutant, termed RD, needs spectinomycin to grow at temperatures of 37° or below; it is unable to grow at 42° in either the presence or absence of spectinomycin. Secondary mutants which grow well in the absence of spectinomycin can be isolated spontaneously at a frequency of about 10^-6. Two-dimensional gel electrophoresis of ribosomal proteins from 25 of these revertants showed that two revertants had an alteration in S4; one other showed an alteration in L5, and one showed an apparent absence of L1. Mutant RD itself had an altered less basic S5, which was maintained in all the revertants that were checked.Genetic analysis indicated that RD was a double mutant: one mutation, which alone conferred a spectinomycin resistant phenotype on the strain, was located in the strA region of the E. coli chromosome and was represented by the mutation in S5. The other mutation, which conferred the dependence on spectinomycin, mapped close to the rif locus.     

Identifying Hfq-binding small RNA targets in Escherichia coli

The Hfq-binding small RNAs (sRNAs) have recently drawn much attention as regulators of translation in Escherichia coli. We attempt to identify the targets of this class of sRNAs in genome scale and gain further insight into the complexity of translational regulation induced by Hfq-binding sRNAs. Using a new alignment algorithm, most known negatively regulated targets of Hfq-binding sRNAs were identified. The results also show several interesting aspects of the regulatory function of Hfq-binding sRNAs.