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 More recently, this has been replaced by the current model, which maintains that PNPase would simply degrade the promoter proximal small RNA generated by the RNase III endonucleolytic cleavage, thus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.

scholarly journals Modes of Overinitiation, dnaA Gene Expression, and Inhibition of Cell Division in a Novel Cold-Sensitive hda Mutant of Escherichia coli

2008 ◽  
Vol 190 (15) ◽  
pp. 5368-5381 ◽  
Author(s):  
Kazuyuki Fujimitsu ◽  
Masayuki Su'etsugu ◽  
Yoko Yamaguchi ◽  
Kensaku Mazda ◽  
Nisi Fu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Cell Division ◽  
Cell Cycle Progression ◽  
Synthetic Lethality ◽  
Active Form ◽  
Dependent Manner ◽  
Chromosomal Replication ◽  
Independent Manner ◽  
Multiple Copies ◽  
Cold Sensitive

ABSTRACT The chromosomal replication cycle is strictly coordinated with cell cycle progression in Escherichia coli. ATP-DnaA initiates replication, leading to loading of the DNA polymerase III holoenzyme. The DNA-loaded form of the β clamp subunit of the polymerase binds the Hda protein, which promotes ATP-DnaA hydrolysis, yielding inactive ADP-DnaA. This regulation is required to repress overinitiation. In this study, we have isolated a novel cold-sensitive hda mutant, the hda-185 mutant. The hda-185 mutant caused overinitiation of chromosomal replication at 25°C, which most likely led to blockage of replication fork progress. Consistently, the inhibition of colony formation at 25°C was suppressed by disruption of the diaA gene, an initiation stimulator. Disruption of the seqA gene, an initiation inhibitor, showed synthetic lethality with hda-185 even at 42°C. The cellular ATP-DnaA level was increased in an hda-185-dependent manner. The cellular concentrations of DnaA protein and dnaA mRNA were comparable at 25°C to those in a wild-type hda strain. We also found that multiple copies of the ribonucleotide reductase genes (nrdAB or nrdEF) or dnaB gene repressed overinitiation. The cellular levels of dATP and dCTP were elevated in cells bearing multiple copies of nrdAB. The catalytic site within NrdA was required for multicopy suppression, suggesting the importance of an active form of NrdA or elevated levels of deoxyribonucleotides in inhibition of overinitiation in the hda-185 cells. Cell division in the hda-185 mutant was inhibited at 25°C in a LexA regulon-independent manner, suggesting that overinitiation in the hda-185 mutant induced a unique division inhibition pathway.

scholarly journals Regulation of RraA, a Protein Inhibitor of RNase E-Mediated RNA Decay

2006 ◽  
Vol 188 (9) ◽  
pp. 3257-3263 ◽  
Author(s):  
Meng Zhao ◽  
Li Zhou ◽  
Yasuaki Kawarasaki ◽  
George Georgiou
Keyword(s):  
Escherichia Coli ◽  
Intergenic Region ◽  
Ectopic Expression ◽  
Dependent Manner ◽  
Feedback Circuit ◽  
Rnase E ◽  
Protein Inhibitor ◽  
E Coli ◽  
The Stability ◽  
Fusion Analysis

ABSTRACT The recently discovered RraA protein acts as an inhibitor of the essential endoribonuclease RNase E, and we demonstrated that ectopic expression of RraA affects the abundance of more than 700 transcripts in Escherichia coli (K. Lee, X. Zhan, J. Gao, J. Qiu, Y. Feng, R. Meganathan, S. N. Cohen, and G. Georgiou, Cell 114:623-634, 2003). We show that rraA is expressed from its own promoter, P rraA , located in the menA-rraA intergenic region. Primer extension and lacZ fusion analysis revealed that transcription from P rraA is elevated upon entry into stationary phase in a σs-dependent manner. In addition, the stability of the rraA transcript is dependent on RNase E activity, suggesting the involvement of a feedback circuit in the regulation of the RraA level in E. coli.

scholarly journals Regulation of Expression of the adhE Gene, Encoding Ethanol Oxidoreductase in Escherichia coli: Transcription from a Downstream Promoter and Regulation by Fnr and RpoS

1999 ◽  
Vol 181 (24) ◽  
pp. 7571-7579 ◽  
Author(s):  
Jorge Membrillo-Hernández ◽  
E. C. C. Lin
Keyword(s):  
Escherichia Coli ◽  
Transcriptional Start Site ◽  
Start Codon ◽  
Start Site ◽  
Rnase Iii ◽  
Regulation Of Expression ◽  
Experimental Conditions ◽  
Stem Loop ◽  
Translational Start ◽  
Transcriptional Start Sites

ABSTRACT The adhE gene of Escherichia coli, located at min 27 on the chromosome, encodes the bifunctional NAD-linked oxidoreductase responsible for the conversion of acetyl-coenzyme A to ethanol during fermentative growth. The expression of adhEis dependent on both transcriptional and posttranscriptional controls and is about 10-fold higher during anaerobic than during aerobic growth. Two putative transcriptional start sites have been reported: one at position −292 and the other at −188 from the translational start codon ATG. In this study we show, by using several different transcriptional and translational fusions to the lacZ gene, that both putative transcriptional start sites can be functional and each site can be redox regulated. Although both start sites are NarL repressible in the presence of nitrate, Fnr activates only the −188 start site and Fis is required for the transcription of only the −292 start site. In addition, it was discovered that RpoS activatesadhE transcription at both start sites. Under all experimental conditions tested, however, only the upstream start site is active. Available evidence indicates that under those conditions, the upstream promoter region acts as a silencer of the downstream transcriptional start site. Translation of the mRNA starting at −292, but not the one starting at −188, requires RNase III. The results support the previously postulated ribosomal binding site (RBS) occlusion model, according to which RNase III cleavage is required to release the RBS from a stem-loop structure in the long transcript.

scholarly journals Organization and Expression of the Polynucleotide Phosphorylase Gene (pnp) of Streptomyces: Processing of pnp Transcripts in Streptomyces antibioticus

2004 ◽  
Vol 186 (10) ◽  
pp. 3160-3172 ◽  
Author(s):  
Patricia Bralley ◽  
George H. Jones
Keyword(s):  
Intergenic Region ◽  
Transcription Initiation ◽  
Streptomyces Coelicolor ◽  
Open Reading Frames ◽  
Streptomyces Avermitilis ◽  
Polynucleotide Phosphorylase ◽  
Rnase Iii ◽  
Stem Loop ◽  
Streptomyces Antibioticus ◽  
E Coli

ABSTRACT We have examined the expression of pnp encoding the 3′-5′-exoribonuclease, polynucleotide phosphorylase, in Streptomyces antibioticus. We show that the rpsO-pnp operon is transcribed from at least two promoters, the first producing a readthrough transcript that includes both pnp and the gene for ribosomal protein S15 (rpsO) and a second, Ppnp, located in the rpsO-pnp intergenic region. Unlike the situation in Escherichia coli, where observation of the readthrough transcript requires mutants lacking RNase III, we detect readthrough transcripts in wild-type S. antibioticus mycelia. The Ppnp transcriptional start point was mapped by primer extension and confirmed by RNA ligase-mediated reverse transcription-PCR, a technique which discriminates between 5′ ends created by transcription initiation and those produced by posttranscriptional processing. Promoter probe analysis demonstrated the presence of a functional promoter in the intergenic region. The Ppnp sequence is similar to a group of promoters recognized by the extracytoplasmic function sigma factors, sigma-R and sigma-E. We note a number of other differences in rspO-pnp structure and function between S. antibioticus and E. coli. In E. coli, pnp autoregulation and cold shock adaptation are dependent upon RNase III cleavage of an rpsO-pnp intergenic hairpin. Computer modeling of the secondary structure of the S. antibioticus readthrough transcript predicts a stem-loop structure analogous to that in E. coli. However, our analysis suggests that while the readthrough transcript observed in S. antibioticus may be processed by an RNase III-like activity, transcripts originating from Ppnp are not. Furthermore, the S. antibioticus rpsO-pnp intergenic region contains two open reading frames. The larger of these, orfA, may be a pseudogene. The smaller open reading frame, orfX, also observed in Streptomyces coelicolor and Streptomyces avermitilis, may be translationally coupled to pnp and the gene downstream from pnp, a putative protease.

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