Autogenous regulation of Escherichia coli polynucleotide phosphorylase expression revisited

The Escherichia coli polynucleotide phosphorylase (PNPase, encoded by pnp), a phosphorolytic exoribonuclease, post-transcriptionally regulates its own expression at the level of mRNA stability and translation. Its primary transcript is very efficiently processed by RNase III, an endonuclease that makes a staggered double strand cleavage about in the middle of a long stem-loop in the 5'-untranslated region. The processed pnp mRNA is then rapidly degraded in a PNPase-dependent manner. Two non-mutually exclusive models have been proposed to explain PNPase autogenous regulation. The earlier one suggested that PNPase impedes translation of the RNase III processed pnp mRNA thus exposing the transcript to degradative pathways. More recently this has been replaced by the current model, which maintains that PNPase would simply degrade the promoter proximal small RNA generated by the RNase III endonucleolytic cleavage thus destroying the double stranded structure at the 5'-end that otherwise stabilizes the pnp mRNA. In our opinion, however, the first model was not completely ruled out. Moreover, the RNA decay pathway acting upon the pnp mRNA after disruption of the 5' double stranded structure remained to be determined. Here we provide additional support to the current model and show that the RNase III-processed pnp mRNA devoid of the double stranded structure at its 5'-end is not translatable and is degraded by RNase E in a PNPase-independent manner. Thus the role of PNPase in autoregulation is simply to remove, in concert with RNase III, the 5'-fragment of the cleaved structure that both allows translation and prevents the RNase E-mediated PNPase-independent degradation of the pnp transcript

A proteomic approach to the analysis of RNA degradosome composition in Escherichia coli

The RNA degradosome is a bacterial protein machine devoted to RNA degradation and processing. In Escherichia coli it is typically composed of the endoribonuclease RNase E, which also serves as a scaffold for the other components, the exoribonuclease PNPase, the RNA helicase RhlB, and enolase. The variable presence of additional proteins, however, suggests that the degradosome is a flexible machine that may vary its composition in response to different conditions. Direct analysis of large protein complexes, together with simplified purification procedures, can facilitate qualitative and quantitative identification of RNA degradosome components under different physiological and genetic conditions and can help elucidating the role in the bacterial cell and the mechanisms of action of this macromolecular assembly. Herewith we describe the application of Multidimensional Protein Identification Technology (MudPIT) to the rapid and quantitative identification of the RNA degradosome components. RNA degradosome preparations obtained from specific conditions are enzymatically digested and the peptides are first fractionated by two dimensional (ion exchange and reversed phase) chromatography and analyzed by tandem mass spectrometry. Bioinformatic analysis by means of the SEQUEST algorithm, which correlates the experimental mass spectra with those predicted by peptide sequences in proteomic and translated genomic databases, allows for the identification of the corresponding proteins that compose the complex. The output protein lists of two or more degradosome samples is then compared so as to obtain a rapid evaluation of qualitative and quantitative differences in protein composition. Quantitative analysis is based on the observation that changes of relative protein abundance in different samples are reflected by the score values assigned to each protein component of the RNA degradosome identified by the MudPIT approach. This correlation can be validated by means of orthogonal independent methods. This general fully automated procedure may be applied to the characterization of any complex protein mixture

Polynucleotide phosphorylase-deficient mutants of Pseudomonas putida

In bacteria, polynucleotide phosphorylase (PNPase) is one of the main exonucleolytic activities involved in RNA turnover and is widely conserved. In spite of this, PNPase does not seem to be essential for growth if the organisms are not subjected to special conditions, such as low temperature. We identified the PNPase-encoding gene (pnp) of Pseudomonas putida and constructed deletion mutants that did not exhibit cold sensitivity. In addition, we found that the transcription pattern of pnp upon cold shock in P. putida was markedly different from that in Escherichia coli. It thus appears that pnp expression control and the physiological roles in the cold may be different in different bacterial species

S1 ribosomal protein over-expression inhibits RNase E-dependent decay in Escherichia coli

It is commonly accepted that in bacteria transcription, translation and degradation of RNA are tightly coordinated processes; however, the molecular bases of this interconnection are poorly understood. S1 is the largest E. coli ribosomal protein; it is very abundant and weakly associated with the 30S ribosomal subunit. Both its over-expression and depletion impair bacterial growth. In a previous work we showed that S1 over-expression leads to a general increase in mRNA stability. To further characterize this phenomenon, we tested the role of 5'-UTR in S1-dependent stabilization. As a model transcript, we chose the cspE mRNA, which is sensitive to S1 stabilization. We showed by EMSA that S1 still binds a cspE RNA lacking the 5'-UTR (cspE-Del), albeit with lower affinity relative to the complete mRNA (cspE-wt), suggesting that for this mRNA different S1 binding sites may exist. In vivo, cspE-Del was less stable than cspE-wt RNA. Upon S1 induction, both the transcripts were stabilized; moreover, different RNA decay intermediates, deriving from cspE-Del degradation at the 5'-end, were detected, whereas no cspE-wt degradation products were present. In an rne thermosensitive mutant expressing cspE-Del, these decay intermediates were produced and stabilized, suggesting that RNase E may be involved in their degradation but not in their production. On the whole, our data suggest that S1 may interact both with the 5'-UTR and/or the 3'-UTR and prevent RNase E-dependent mRNA degradation but not the RNase E-independent endonucleolytic pathway operating on the leaderless cspE mRNA

S1 ribosomal protein and the interplay between translation and mRNA decay

In bacteria, transcription, translation and mRNA decay are tightly interconnected processes; however, little is known about specific factors and molecular mechanisms involved in their co-ordination. The ribosomal protein S1, an \u201catypical\u201d ribosomal protein weakly associated with the 30S subunit of Escherichia coli ribosome, has been implicated in translation, transcription and control of RNA stability. It is thus a good candidate for playing a role in the interplay among these processes. We have addressed S1 function by assaying translation and decay of model full-length and leaderless mRNAs upon modulation of S1 intracellular concentration (from depletion to overexpression). We have shown that S1 over-expression leads to polysome disappearance and translation inhibition. Moreover, in the same condition, RNase E-dependent decay of both the cspE+ and leaderless \u394L-cspE mRNAs is prevented. Conversely, cleavage of \u394L-cspE mRNA by an unidentified endonuclease is not affected. Overall, our data suggest that ribosome-unbound S1 may inhibit translation and stabilize mRNA through the specific inhibition of RNase E-dependent decay

The lipopolysaccharide transport system of Gram-negative bacteria

The cell envelope of Gram-negative bacteria consists of two distinct membranes, the inner (IM) and the outer membrane (OM) separated by the periplasm. The OM contains in the outer leaflet the lipopolysaccharide (LPS), a complex lipid with important biological activities. In the host it elicits the innate immune response whereas in the bacterium it is responsible for the peculiar permeability barrier properties exhibited by the OM. The chemical structure of LPS and its biosynthetic pathways have been fully elucidated. By contrast only recently details of the transport and assembly of LPS into the OM have emerged. LPS is synthesized in the cytoplasm and at the inner leaflet of the IM and needs to cross two different compartments, the IM and the periplasm, to reach its final destination at the OM. This review focuses on recent studies that led to our present understanding of the protein machine implicated in LPS transport and in assembly at the cell surface