RNA processing by RNase III is involved in the synthesis of Escherichia coli polynucleotide phosphorylase

1987 ◽  
Vol 209 (1) ◽  
pp. 28-32 ◽  
Author(s):  
Renkichi Takata ◽  
Tsunehiro Mukai ◽  
Katsuji Hori
Keyword(s):  
Escherichia Coli ◽  
Rna Processing ◽  
Polynucleotide Phosphorylase ◽  
Rnase Iii

scholarly journals The Escherichia coli major exoribonuclease RNase II is a component of the RNA degradosome

2014 ◽  
Vol 34 (6) ◽  
Author(s):  
Feng Lu ◽  
Aziz Taghbalout
Keyword(s):  
Escherichia Coli ◽  
Rna Processing ◽  
Binding Sites ◽  
Rna Degradation ◽  
The Other ◽  
Polynucleotide Phosphorylase ◽  
Cell Extracts ◽  
Domains Of Life ◽  
Rna Degradosome ◽  
Rnase Ii

Multiprotein complexes that carry out RNA degradation and processing functions are found in cells from all domains of life. In Escherichia coli, the RNA degradosome, a four-protein complex, is required for normal RNA degradation and processing. In addition to the degradosome complex, the cell contains other ribonucleases that also play important roles in RNA processing and/or degradation. Whether the other ribonucleases are associated with the degradosome or function independently is not known. In the present work, IP (immunoprecipitation) studies from cell extracts showed that the major hydrolytic exoribonuclease RNase II is associated with the known degradosome components RNaseE (endoribonuclease E), RhlB (RNA helicase B), PNPase (polynucleotide phosphorylase) and Eno (enolase). Further evidence for the RNase II-degradosome association came from the binding of RNase II to purified RNaseE in far western affinity blot experiments. Formation of the RNase II–degradosome complex required the degradosomal proteins RhlB and PNPase as well as a C-terminal domain of RNaseE that contains binding sites for the other degradosomal proteins. This shows that the RNase II is a component of the RNA degradosome complex, a previously unrecognized association that is likely to play a role in coupling and coordinating the multiple elements of the RNA degradation pathways.

scholarly journals Autogenous Regulation of Escherichia coli Polynucleotide Phosphorylase Expression Revisited

2009 ◽  
Vol 191 (6) ◽  
pp. 1738-1748 ◽  
Author(s):  
Thomas Carzaniga ◽  
Federica Briani ◽  
Sandro Zangrossi ◽  
Giuseppe Merlino ◽  
Paolo Marchi ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Current Model ◽  
Dependent Manner ◽  
Rnase E ◽  
Polynucleotide Phosphorylase ◽  
Rnase Iii ◽  
Independent Manner ◽  
Stem Loop ◽  
Autogenous Regulation ◽  
Additional Support

ABSTRACT The Escherichia coli polynucleotide phosphorylase (PNPase; encoded by pnp), a phosphorolytic exoribonuclease, posttranscriptionally 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.

scholarly journals RNase III-Independent Autogenous Regulation of Escherichia coli Polynucleotide Phosphorylase via Translational Repression

2015 ◽  
Vol 197 (11) ◽  
pp. 1931-1938 ◽  
Author(s):  
Thomas Carzaniga ◽  
Gianni Dehò ◽  
Federica Briani
Keyword(s):  
Escherichia Coli ◽  
Posttranscriptional Regulation ◽  
Mrna Degradation ◽  
Translational Repression ◽  
Rnase E ◽  
Polynucleotide Phosphorylase ◽  
Rnase Iii ◽  
Primary Transcript ◽  
Content Type ◽  
Autogenous Regulation

ABSTRACTThe complex posttranscriptional regulation mechanism of theEscherichia colipnpgene, which encodes the phosphorolytic exoribonuclease polynucleotide phosphorylase (PNPase), involves two endoribonucleases, namely, RNase III and RNase E, and PNPase itself, which thus autoregulates its own expression. The models proposed forpnpautoregulation posit that the target of PNPase is a maturepnpmRNA previously processed at its 5′ end by RNase III, rather than the primarypnptranscript (RNase III-dependent models), and that PNPase activity eventually leads topnpmRNA degradation by RNase E. However, some published data suggest thatpnpexpression may also be regulated through a PNPase-dependent, RNase III-independent mechanism. To address this issue, we constructed isogenic Δpnp rnc+and ΔpnpΔrncstrains with a chromosomalpnp-lacZtranslational fusion and measured β-galactosidase activity in the absence and presence of PNPase expressed by a plasmid. Our results show that PNPase also regulates its own expression via a reversible RNase III-independent pathway acting upstream from the RNase III-dependent branch. This pathway requires the PNPase RNA binding domains KH and S1 but not its phosphorolytic activity. We suggest that the RNase III-independent autoregulation of PNPase occurs at the level of translational repression, possibly by competition forpnpprimary transcript between PNPase and the ribosomal protein S1.IMPORTANCEInEscherichia coli, polynucleotide phosphorylase (PNPase, encoded bypnp) posttranscriptionally regulates its own expression. The two models proposed so far posit a two-step mechanism in which RNase III, by cutting the leader region of thepnpprimary transcript, creates the substrate for PNPase regulatory activity, eventually leading topnpmRNA degradation by RNase E. In this work, we provide evidence supporting an additional pathway for PNPase autogenous regulation in which PNPase acts as a translational repressor independently of RNase III cleavage. Our data make a new contribution to the understanding of the regulatory mechanism ofpnpmRNA, a process long since considered a paradigmatic example of posttranscriptional regulation at the level of mRNA stability.

Heterodimer-based analysis of subunit and domain contributions to double-stranded RNA processing by Escherichia coli RNase III in vitro

2008 ◽  
Vol 410 (1) ◽  
pp. 39-48 ◽  
Author(s):  
Wenzhao Meng ◽  
Allen W. Nicholson
Keyword(s):  
Escherichia Coli ◽  
Rna Processing ◽  
Metal Ion ◽  
Catalytic Site ◽  
Ion Concentration ◽  
Hill Coefficient ◽  
Rnase Iii ◽  
Double Stranded Rna ◽  
Catalytic Sites ◽  
Scissile Bond

Members of the RNase III family are the primary cellular agents of dsRNA (double-stranded RNA) processing. Bacterial RNases III function as homodimers and contain two dsRBDs (dsRNA-binding domains) and two catalytic sites. The potential for functional cross-talk between the catalytic sites and the requirement for both dsRBDs for processing activity are not known. It is shown that an Escherichia coli RNase III heterodimer that contains a single functional wt (wild-type) catalytic site and an inactive catalytic site (RNase III[E117A/wt]) cleaves a substrate with a single scissile bond with a kcat value that is one-half that of wt RNase III, but exhibits an unaltered Km. Moreover, RNase III[E117A/wt] cleavage of a substrate containing two scissile bonds generates singly cleaved intermediates that are only slowly cleaved at the remaining phosphodiester linkage, and in a manner that is sensitive to excess unlabelled substrate. These results demonstrate the equal probability, during a single binding event, of placement of a scissile bond in a functional or nonfunctional catalytic site of the heterodimer and reveal a requirement for substrate dissociation and rebinding for cleavage of both phosphodiester linkages by the mutant heterodimer. The rate of phosphodiester hydrolysis by RNase III[E117A/wt] has the same dependence on Mg2+ ion concentration as that of the wt enzyme, and exhibits a Hill coefficient (h) of 2.0±0.1, indicating that the metal ion dependence essentially reflects a single catalytic site that employs a two-Mg2+-ion mechanism. Whereas an E. coli RNase III mutant that lacks both dsRBDs is inactive, a heterodimer that contains a single dsRBD exhibits significant catalytic activity. These findings support a reaction pathway involving the largely independent action of the dsRBDs and the catalytic sites in substrate recognition and cleavage respectively.

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