Species: Escherichia coli K-12 substr. MG1655
Subunit composition of
degradosome = [(Ppk)2][(Rne)4][(RhlB)2][(Pnp)3][(Eno)2]
polyphosphate kinase = (Ppk)2 (extended summary available)
ribonuclease E = (Rne)4 (extended summary available)
RNase E = Rne
RhlB, ATP-dependent RNA helicase of the RNA degradosome = (RhlB)2 (extended summary available)
polynucleotide phosphorylase = (Pnp)3 (extended summary available)
polynucleotide phosphorylase monomer = Pnp
enolase = (Eno)2 (extended summary available)
The degradosome is a large, multiprotein complex involved in RNA degradation. It consists of the RNA degradation enzymes RNase E and PNPase, as well as the ATP-dependent RNA helicase RhlB and the metabolic enzyme enolase [Py94, Carpousis94, Py96]. Polyphosphate kinase and the chaperone protein DnaK are also associated with and may be components of the degradosome [Blum97, Miczak96]. A "minimal" degradosome composed of only RNase E, PNPase and RhlB degrades malEF REP RNA in an ATP-dependent manner in vitro, with activity equivalent to purified whole degradosomes. RNase E enzymatic function is dispensible for this test case, whereas PNPase must be catalytically active and incorporated into the degradosome for degradation to occur [Coburn99]. Based on immunogold labeling studies, RhlB and RNase E are present in equimolar quantities in the degradosome, which is tethered to the cytoplasmic membrane via the amino-terminus of RNase E [Liou01].
RNase E provides the organizational structure for the degradosome. Its carboxy-terminal half binds PNPase, RhlB and enolase, and the loss of this portion of the protein prevents degradation of a number of degradosome substrates, including the ptsG and mukB mRNAs and RNA I [Kido96, Vanzo98, Morita04]. This scaffold region is flexible, with isolated segments of increased structure that may be involved in binding other degradosome constituents [Callaghan04]. RNase E binding to partner proteins can be selectively disrupted. Loss of RhlB and enolase binding results in reduced degradosome activity. Conversely, disrupted PNPase binding yields increased activity. Strains any alteration in RNase E binding do not grow as well as wild type [Leroy02]. The amino-terminal half of RNase E contains sequences involved in oligomerization [Vanzo98].
In vitro purified degradosome generates 147-nucleotide RNase E cleavage intermediates from rpsT mRNA. Continuous cycles of polyadenylation and PNPase cleavage are necessary and sufficient to break down these intermediates, though RNase II can block this second degradation step [Coburn98]. RNAs with 3' REP stabilizers or stem loops must be polyadenylated to allow breakdown by the degradosome [Khemici04, Blum99]. Poly(G) and poly(U) tails do not allow degradation, though addition of a stretch of mixed nucleotides copied from within a coding region has stimulated degradation of a test substrate [Blum99].
The DEAD-box helicases SrmB, RhlE and CsdA bind RNase E in vitro at a different site than RhlB. RhlE and CsdA can both replace RhlB in promoting PNPase activity in vitro [Khemici04a]. CsdA is induced by cold shock, and following a shift to 15 degrees C it copurifies with the degradosome [PrudhommeGenere04].
At least two poly(A)-binding proteins interact with the degradosome. The cold-shock protein CspE inhibits internal cleavage and breakdown of polyadenylated RNA by RNase E and PNPase by blocking digestion through the poly(A) tail. S1, a component of the 30S ribosome, binds to RNase E and PNPase without apparent effect on their activities [Feng01].
The global effects of mutations in degradeosome constituents on mRNA levels have been evaluated using microarrays [Bernstein04].
Locations: inner membrane
|Gene:||ppk||Accession Numbers: EG11510 (MetaCyc), b2501, ECK2497|
Locations: outer membrane, inner membrane, cytosol
Subunit composition of polyphosphate kinase = [Ppk]2
|Map Position: [2,621,066 -> 2,623,132]|
Molecular Weight of Polypeptide: 80.431 kD (from nucleotide sequence), 69.0 kD (experimental) [Ahn90]
Molecular Weight of Multimer: 270.0 kD (experimental) [Ahn90]
|MultiFun Terms:||metabolism → metabolism of other compounds → phosphorous metabolism|
Relationship Links: InterPro:IN-FAMILY:IPR001736, InterPro:IN-FAMILY:IPR003414, InterPro:IN-FAMILY:IPR024953, InterPro:IN-FAMILY:IPR025198, InterPro:IN-FAMILY:IPR025200, PDB:Structure:1XDO, PDB:Structure:1XDP, Pfam:IN-FAMILY:PF02503, Pfam:IN-FAMILY:PF13089, Pfam:IN-FAMILY:PF13090, Prosite:IN-FAMILY:PS50035
Polyphosphate kinase (PPK) catalyses several reactions in E. coli K-12.
PPK transfers the γ phosphate of ATP processively to generate inorganic polyphosphate (polyP or polyPi) and ADP. Phosphorylated enzyme (PPK-P) is an intermediate in this reaction. Purified PPK is a tetramer and requires Mg2+ for activity [Ahn90]. PPK in which histidine 435 or histidine 454 have been altered to glutamine or alanine results in enzyme that is unable to autophosphorylate and lacks polyphosphate kinase activity. Purified PPK catalyses the synthesis of polyP chains with a uniform length of 750 +/- 50 phosphate groups. No intermediate chain lengths were visualised and polyP chains ranging from 2 - 40 residues in length failed to act as primers for the synthesis reaction in vitro [Kumble96].
PPK also catalyses the reverse reaction which synthesizes ATP from inorganic polyphosphate and ADP [Ahn90]. Partially purified PPK from E. coli B is most active in ATP synthesis using polyphosphate molecules with a chain length greater than 132 and its activity decreases with decreasing chain length [Haeusler92].
Purified PPK can transfer a phosphate from inorganic polyphosphate to nucleotide diphosphates including ADP, GDP, CDP, UDP, dADP, dGDP, dCDP and TDP. It can also transfer a pyrophosphate group to GDP to form guanosine 5' tetraphosphate (ppGpp) [Kuroda97].
Overproduced PPK results in increased polyPi:AMP phosphotransferase (PAP) activity in E. coli K-12. PPK requires adenylate kinase (ADK) for PAP activity. PPK and ADK from a complex in the presence of polyPi in vitro [Ishige00].
Overproduced PPK is associated with the outer membrane [Akiyama92].
Cells overexpressing PPK have increased polyphosphate levels (estimated at 220 µg/1011 cells) compared to wild type (2 µg/1011 cells) while ppk::kan cells have decreased polyphosphate levels (0.16 µg/1011 cells). ppk::kan cells are more sensitive to hydrogen peroxide stress and to heat stress and show reduced survival in stationary phase [Crooke94, Rao96]. PPK is required to stimulate protein degradation upon nutritional downshift [Kuroda99].
ppk forms an operon with ppx, encoding an exopolyphosphatase [Akiyama93]
PPK has been identified as a component of the E. coli RNA degradosome [Blum97].
Native PPK purifies as a tetramer however different subunit organizations may be associated with the different functions of the enzyme. Experiments using radiation inactivation of enzyme activity suggest that the functional unit for PPK activity (both forward and reverse) is a dimer, the functional unit for autophosphorylation is a tetramer (at 5mM ATP) or dimer (at 1mM ATP) and the functional unit for ppGpp synthesis is a trimer [Tzeng00]
Crystal structures have been determined for polyphosphate kinase on its own and binding the non-hydrolysable ATP analogue AMP-PNP. The structure is an interlocked dimer. Each monomer has 4 structural domains: an amino terminal or N domain; a head domain and two carboxy terminal domains C1 and C2. A tunnel structure penetrates the centre of each monomer and contains the sites of catalysis. Histidine residue 435 directly interacts with the AMP-PNP γ phosphate group and probably represents the site of autophosphorylation [Zhu03a, Zhu05a].
Synonyms: smbB, ams, hmp1, RNase E
|Gene:||rne||Accession Numbers: EG10859 (MetaCyc), b1084, ECK1069|
Locations: cytosol, inner membrane
|Map Position: [1,140,405 <- 1,143,590]|
Molecular Weight of Polypeptide: 118.2 kD (from nucleotide sequence)
|MultiFun Terms:||information transfer → RNA related → RNA degradation|
|metabolism → degradation of macromolecules → RNA|
Unification Links: DIP:DIP-10727N, DisProt:DP00207, EcoliWiki:b1084, Mint:MINT-1220086, ModBase:P21513, PR:PRO_000023789, Pride:P21513, Protein Model Portal:P21513, RefSeq:NP_415602, SMR:P21513, UniProt:P21513
Relationship Links: InterPro:IN-FAMILY:IPR003029, InterPro:IN-FAMILY:IPR004659, InterPro:IN-FAMILY:IPR012340, InterPro:IN-FAMILY:IPR019307, InterPro:IN-FAMILY:IPR021968, InterPro:IN-FAMILY:IPR022967, InterPro:IN-FAMILY:IPR028878, PDB:Structure:1SLJ, PDB:Structure:1SMX, PDB:Structure:1SN8, PDB:Structure:2BX2, PDB:Structure:2C0B, PDB:Structure:2C4R, PDB:Structure:2FYM, PDB:Structure:2VMK, PDB:Structure:2VRT, PDB:Structure:3GCM, PDB:Structure:3GME, PDB:Structure:3H1C, PDB:Structure:3H8A, Pfam:IN-FAMILY:PF00575, Pfam:IN-FAMILY:PF10150, Pfam:IN-FAMILY:PF12111, Prosite:IN-FAMILY:PS50126, Smart:IN-FAMILY:SM00316
9S rRNA + 2 H2O → 5S rRNA + 2 a single-stranded RNA,
RNase E mRNA processing substrate + n H2O → RNase E processing product mRNA + n a single-stranded RNA,
a polycistronic tRNA precursor + H2O → a tRNA precursor with a 5' extension and a short 3' extension + a partially processed polycistronic tRNA precursor,
a polycistronic tRNA precursor + H2O → a tRNA precursor with a 5' extension and a long 3' trailer + a partially processed polycistronic tRNA precursor,
RNase E degradation substrate mRNA + n H2O → n a single-stranded RNA
Ribonuclease E (RNase E) is a single-strand-specific endonuclease that is essential for viability. It processes rRNA, tRNA and other RNAs, is involved in plasmid and phage stability and is part of the degradosome, a multienzyme complex involved in mRNA degradation.
RNase E is involved in processing and cleavage of several rRNAs. It processes the 9S rRNA precursor to yield the mature 5S rRNA by cleaving quite near the 5' end and downstream from the 3' end of the final product [Ghora78, Roy83, Apirion78]. RNase E also participates in the 5' maturation of 16S rRNA from its 17S precursor, as well as being able to cleave single-stranded regions within mature 16S and 23S rRNAs [Li99b, Bessarab98].
RNase E initiates the processing of both poly- and monicistronic tRNA transcripts, including those within rRNA transcripts, by cleaving within a few nucleotides of the mature 3' CCA terminus, thus allowing RNase P and other 3' to 5' exonucleases to complete tRNA maturation [Ow02, Li02]. RNase E similarly cleaves at the 3' CCA terminus of the ssrA precursor to yield its final form [LinChao99]. RNase E may also be involved in processing of the 5' leader of precursor tRNAs [Soderbom05].
RNase E carries out the 3' processing of M1 mRNA, which codes for the catalytic subunit of RNase P [Lundberg95, Sim01]. Other mRNA processing substrates include the cell division inhibitor DicF, the RNA polymerase sigma70 activity modulator 6S RNA, the polycistronic histidine operon mRNA and the papAB primary transcript, which is cleaved to yield stable papA and unstable papB mRNA [Faubladier90, Kim04, Alifano94, Nilsson91].
The stability of plasmids R1 and Colicin E1 is influenced by RNase E. It initiates degradation of CopA, the R1 copy regulator RNA [Soderbom98]. RNase E also cleaves near the 5' end of the sok component of the hok/sok sense/antisense RNA plasmid stabilization mechanism from R1, allowing subsequent degradation by another degradeosome component, PNPase [Dam97]. The Colicin E1 DNA synthesis inhibitor RNA, RNAI, is also cleaved at its 5' end by RNase E [Tomcsanyi85, LinChao91]. Finally, RNase E cleaves FinP, which binds to the 5'-untranslated region of the positive F-plasmid transfer regulator traJ [Jerome99].
RNase E processing maintains the balance between phage f1 proteins pII and PX by cleaving the mRNA coding for pII, thus maintaining a normal replication cycle [Kokoska98]. RNase E is required more generally for production of certain phage f1 mRNAs as well [Stump96]. RNase E processes T4 gene 32 mRNA, cleaves T4 soc mRNA and is involved generally in the destabilizing of T4 mRNA [Mudd88, Otsuka03, Mudd90].
A number of cellular mRNAs are degraded by RNase E. mRNA decay slows 2-3 fold in an rne mutant, and RNase E is the rate-limiting enzyme in the degradation of many of its substrates [Babitzke91, Jain02]. RNase E cleaves both sodB mRNA and its antisense RNA RyhB, though cleavage of the latter can be blocked by Hfq binding to the cleavage site [Afonyushkin05, Masse03, Moll03, Folichon03]. Hfq also overlaps cleavage sites in the dsrA and ompA mRNAs [Moll03]. The rpsO-pnp transcript is cleaved near the beginning of the rpsO coding sequence and on both sides of the rspO 3' stem-loop terminator, after which it is rapidly degraded by PNPase [Hajnsdorf99, Regnier91, Braun96, Hajnsdorf94]. Ribosome binding blocks this cleavage [Braun98a]. RNase E is responsible for a number of cleavages within the unc transcripts, which code for subunits of the F1/F0-ATPase [Patel92, Patel95]. It destabilizes the secE, nusG, L11-L1, L10 and beta cistrons from transcripts from the secEnusG and rplKAJLrpoBC operons, though this is not reflected in a change in mRNA abundance [Chow94]. Other transcripts that are degraded by RNase E include ftsA-ftsZ, thrS, pstG, pnp and rnb [Cam96, Nogueira01, Kimata01, Hajnsdorf94a, Zilhao95]. RNase E is also involved in limiting the abundance of mRNAs from rspT, dsbC, pth and tetR [Le02, Zhan04, CruzVera02, Baumeister91]. Finally, although overexpressed RNase G can partially complement a lack of RNase E, about a hundred RNAs are only degraded by RNase E, including many mRNAs coding for proteins involved in energy generation and macromolecule synthesis and degradation [Lee02].
RNase E regulates its own abundance by cleaving within the 5' untranslated region of rne mRNA. As RNase E activity can be titrated by other substrates, this acts to modulate its expression to match cellular needs [Mudd93, Diwa02, Sousa01]. Appending the 5' region of rne to heterologous RNA confers RNase E regulation [Jain95].
In a pnp/rnb/rne triple mutant, RNA polyadenylation is longer and more abundant [OHara95]. Conversely, RNase E indirectly increases polyadenylation by generating new 3' ends on which PAP I, which has a binding region for RNase E, can act [Mohanty00, Raynal99]. Increased polyadenylation stabilizes the rne transcript [Mohanty99, Mohanty02].
RNase E cleaves at regions that are single stranded and rich in A/U sequences [Kim04a, Mackie92, Bessarab98, Babitzke91]. Though RNase E has no canonical target sequence, the effects of local sequence on cleavage placement and effectiveness have been thoroughly characterized [Kaberdin03, McDowall94]. Secondary structure in the form of adjacent stem-loops has been shown to be necessary for RNase E cleavage for a number of substrates, and it has been suggested that these structures maintain a stretch of single-stranded RNA for the enzyme to cleave [Ehretsmann92, Cormack92, Diwa00]. In other cases, however, secondary structures play no definite role in susceptibility or actually impede RNase E cleavage [Mackie93, McDowall95, Lopez96].
RNase E binds to the 5'-monophosphate end of its substrate but then cleaves farther in moving 3' to 5', suggesting a scanning mechanism [Feng02]. In the absence of a 5'-monophosphate, cleavage is slowed [Jiang04]. Blocking the 5' end, either by circularizing the RNA or by adding a 5'-triphosphate also inhibits cleavage [Mackie00, Mackie98].
The catalytic parameters of RNase E have been thoroughly evaluated [Redko03].
RNase E's enzymatic and RNA-binding functions are split between its amino-terminal and carboxy-terminal portions, respectively [McDowall96, Taraseviciene95]. The carboxy-terminal section of the protein and its arginine-rich RNA-binding domain (ARRBD) is required for mRNA degradation and enhances RNase E autoregulatory cleavage of rne mRNA, but is dispensible for rRNA processing [Ow00, Lopez99, Jiang00, Kaberdin00]. Contradicting this observation, it has been reported that the RNA-binding domain of RNase E is not required for feedback regulation [Diwa02a]. Mutations within the RNA-binding do lead to defective binding, but have no effect on RNA cleavage activity [Shin08].
RNase E is catalytically active only as a tetramer, with its RNA-binding domains facing outward [Callaghan03]. Crystallographic and NMR analysis of the isolated RNA-binding domain indicates that it forms a homodimer, possibly contributing to overall tetramer formation [Schubert04]. A crystal structure of the amino-terminal catalytic domain to 2.9 Å resolution shows that the tetramer consists of a dimer of dimers and contains divalent magnesium ion [Callaghan05]. The tetrameric structure is maintained by cysteine-zinc-cysteine linkages between adjacent Rne monomers [Callaghan05a].
The previously reported RNase K appears to be a proteolytic fragment of RNase E [Mudd93].
Synonyms: mmrA?, b3780, G704, mmrA
|Gene:||rhlB||Accession Number: EG10844 (MetaCyc)|
Subunit composition of RhlB, ATP-dependent RNA helicase of the RNA degradosome = [RhlB]2
|Map Position: [3,962,388 <- 3,963,653]|
Molecular Weight of Polypeptide: 47.126 kD (from nucleotide sequence)
|MultiFun Terms:||information transfer → RNA related → RNA degradation|
|metabolism → degradation of macromolecules → RNA|
Relationship Links: InterPro:IN-FAMILY:IPR000629, InterPro:IN-FAMILY:IPR001650, InterPro:IN-FAMILY:IPR011545, InterPro:IN-FAMILY:IPR014001, InterPro:IN-FAMILY:IPR014014, InterPro:IN-FAMILY:IPR023554, InterPro:IN-FAMILY:IPR027417, Pfam:IN-FAMILY:PF00270, Pfam:IN-FAMILY:PF00271, Prosite:IN-FAMILY:PS00039, Prosite:IN-FAMILY:PS51192, Prosite:IN-FAMILY:PS51194, Prosite:IN-FAMILY:PS51195, Smart:IN-FAMILY:SM00487, Smart:IN-FAMILY:SM00490
RhlB is an ATP-dependent helicase that is a component of the degradosome, where it aids degradation of structured RNA by PNPase and is required for full degradosome activity [Py96, Coburn99]. RhlB is a member of the family of DEAD-box helicases, though rhlb conditional lethality is not complemented by another DEAD-box helicase, SrmB [Kalman91a]. RhlB binds to the degradosome scaffold RNase E, an interaction that both makes it part of the complex and enhances its ATPase activity fifteen fold [Vanzo98]. In at least one case, rpsT mRNA, substrate polyadenylation is required following cleavage by RNase E to allow RhlB and PNPase to complete degradation [Coburn99].
RhlB forms homodimers and can bind PNPase even in the absence of RNase E. In vitro, RhlB unwinding of dsRNA allows PNPase degradation to occur [Liou02].
Whereas uvr mutant strains show better recovery from irradiation in minimal versus rich medium, uvr mutants that also lack RhlB function recover equally well on either medium. This may be due to a delay in DNA synthesis that occurs during growth on minimal medium or on any medium when RhlB activity is absent [Sharma83, Sharma85].
RhlB has a role independent of PNPase in the RNase E-dependent cleavage of the products of T7 phage RNA polymerase. In an rhlb null, lacZ and other RNAs transcribed by T7 RNAP are stabilized. RhlB is not generally required for RNase E cleavage of non-T7 substrates, however [Khemici05]. The T7 gene 0.7 protein kinase phosphorylates RhlB, stabilizing lac and cat RNAs transcribed by T7 RNAP [Marchand01].
Synonyms: bfl, PNPase
|Gene:||pnp||Accession Numbers: EG10743 (MetaCyc), b3164, ECK3152|
Locations: cytosol, membrane
|Map Position: [3,307,055 <- 3,309,190]|
Molecular Weight of Polypeptide: 77.101 kD (from nucleotide sequence)
|MultiFun Terms:||information transfer → RNA related → RNA degradation|
|metabolism → degradation of macromolecules → RNA|
Relationship Links: InterPro:IN-FAMILY:IPR001247, InterPro:IN-FAMILY:IPR003029, InterPro:IN-FAMILY:IPR004087, InterPro:IN-FAMILY:IPR004088, InterPro:IN-FAMILY:IPR012162, InterPro:IN-FAMILY:IPR012340, InterPro:IN-FAMILY:IPR015847, InterPro:IN-FAMILY:IPR015848, InterPro:IN-FAMILY:IPR020568, InterPro:IN-FAMILY:IPR022967, InterPro:IN-FAMILY:IPR027408, Panther:IN-FAMILY:PTHR11252, PDB:Structure:1SRO, PDB:Structure:3CDI, PDB:Structure:3CDJ, PDB:Structure:3GCM, PDB:Structure:3GLL, PDB:Structure:3GME, PDB:Structure:3H1C, Pfam:IN-FAMILY:PF00013, Pfam:IN-FAMILY:PF00575, Pfam:IN-FAMILY:PF01138, Pfam:IN-FAMILY:PF03725, Pfam:IN-FAMILY:PF03726, Prosite:IN-FAMILY:PS50084, Prosite:IN-FAMILY:PS50126, Smart:IN-FAMILY:SM00316, Smart:IN-FAMILY:SM00322
a tRNA precursor with a 5' extension and a long 3' trailer + n H2O → a tRNA precursor with a 5' extension and a short 3' extension + n a nucleoside 5'-monophosphate,
(ribonucleotides)(n) + phosphate ↔ (ribonucleotides)(n-1) + a nucleoside diphosphate
Polynucleotide phosphorylase (PNPase) is a 3' to 5' exonuclease and a 3'-terminal oligonucleotide polymerase. It degrades various mRNAs, is involved in cold shock regulation, is a part of tRNA maturation and degradation, adds heteropolymeric tails to some RNAs and is a component of the degradosome, a multienzyme complex that carries out RNA degradation.
PNPase is involved in general mRNA degradation. Loss of PNPase leads to an increase in steady-state levels of mRNA, as well as increasing mRNA half lives in the absence of the 3' exonuclease RNase II [Mohanty03, Kinscherf75]. PNPase also has a role in mRNA degradation during carbon starvation, where it may be required for breakdown of small rRNA fragments produced by other RNases [Kaplan74, Kaplan75].
A number of specific PNPase substrates have been identified. PNPase is involved in degradation of lac mRNA, rnb mRNA, mRNA coding for ribosomal protein S20, and the RNA-OUT antisense RNA [HarEl79, Pepe94, Mackie89, Zilhao96]. It also degrades sok antisense RNA and thrS and rpsO mRNA following cleavage by RNase E [Dam97, Nogueira01, Braun96, Hajnsdorf94]. PNPase binds to but does not degrade RNA containing 8-oxoguanine [Hayakawa01].
PNPase-mediated degradation is required for regulation of the cold shock response. PNPase degrades a number of mRNAs induced by cold shock, including those coding for CspA, RbfA, CsdA, RpoE, RseA, Rnr and many others [Yamanaka01a, Cairrao03, Polissi03]. The isolated PNPase S1 RNA-binding domain can complement a deletion in four cold-shock genes [Xia01].
The 3' to 5' processive cleavage of RNA by PNPase depends on the composition and structure of the 3' end of the substrate [Plamann90, Cisneros96]. RhlB and poly(A) polymerase I (PAP I) in concert with the degradosome are required for PNPase-mediated degradation of cistrons with 3' REP-stabilizers [Khemici04].
Binding of the protein Hfq to poly(A) tracts prevents PNPase degradation of these tails in vitro [Folichon03]. RNA with 3' stem-loops are resistant to degradation by pure PNPase or whole degradosome in vitro, but addition of even a short poly(A) or mixed nucleotide tail overcomes this block [Causton94, Blum99, Lisitsky99]. Polyadenylation similarly destabilizes rpsO mRNA against degradation by RNase E, RNase II and PNPase, and is required for sok RNA degradation [Hajnsdorf95, Hajnsdorf96, Dam97]. Both 3' adenylation and 5' phosphorylation affect the rate of degradation of RNA I [Xu95d]. PNPase itself modulates polyadenylation of several RNAs [Mohanty00].
PNPase is involved in tRNA processing and maintenance. Though purified PNPase is incapable of completely processing tRNA in vitro, it is effective, along with RNase II, in trimming long 3' trailing sequences to yield 2-4 nucleotide intermediates which will be trimmed by RNases T and PH [Deutscher88, Li94a]. PNPase is also partially required for repair of 3'-terminal CCA sequences in tRNAs in the absence of tRNA nucleotidyltransferase [Reuven97]. PNPase is also involved in the degradation of mutant tRNA, in a process that is enhanced by polyadenylation by PAP I [Li02b].
PNPase also catalyzes the "reverse" reaction, converting nucleoside diphosphates into polyribonucleotides [Littauer57, Gillam78, Gillam80]. PNPase generates heteropolymeric tails on RNA and is responsible for residual polyadenylation detected in PAP I deficient strains [Mohanty00a]. Hfq, which binds to the 3' end of RNA and prevents PNPase-mediated degradation, also prevents PNPase-mediated addition of nucleosides to bound RNA, while promoting PAP I activity [Folichon05].
PNPase is a trimer of Pnp monomers [Portier75, Soreq77]. Each Pnp monomer has two RNA-binding sites, KH and S1, that are dispensible for strict catalytic function but are required for Pnp autoregulation, growth at low temperature, and the generation of oligonucleotides [Jarrige02, MatusOrtega07, Guissani76]. The S1 domain is a five-stranded antiparallel β barrel with conserved residues on one face forming the RNA binding site [Bycroft97].
PNPase binds the signaling molecule c-di-GMP; binding enhances several PNPase activities, including ADP/Pi phosphoryl exchange and poly(A) synthesis [Tuckerman11].
PNPase is subject to autoregulation at the mRNA level. RNase III cleaves a stem-loop in the pnp mRNA leader sequence, following which PNPase binds and degrades the 5' half of the cleaved duplex [Portier87, Takata89, Jarrige01, RobertLe92, Takata87, Carzaniga09]. PNPase autoregulation also decreases as general RNA polyadenylation increases and following a shift to cold temperatures [Mohanty02, Mathy01, Zangrossi00, Beran01].
Strains lacking both PNPase and RNase II activity are inviable and collect mRNA fragments 100-1,500 nucleotides long [Donovan86]. In a triple mutant in pnp, rnb and rne, mRNA degradation slows three- to fourfold and the length and number of poly(A) tails increases [Arraiano88, OHara95]. In a pnp mutant lacking RNase PH function, the 50S ribosomal subunit and 23S rRNA is degraded [Zhou97a].
Even in the absence of the degradosome scaffold RNase E, PNPase and the helicase RhlB interact. In vitro, RhlB unwinding of dsRNA allows PNPase degradation to occur [Liou02].
PNPase is required to prevent phage P4 superinfection. This prevention requires binding of CI antisense RNA to sequences on nascent P4 transcripts; PNPase processes CI RNA [Piazza96].
pnp shows differential codon adaptation, resulting in differential translation efficiency signatures, in thermophilic microbes. It was therefore predicted to play a role in the heat shock response. A pnp deletion mutant was shown to be more sensitive than wild-type specifically to heat shock, but not other stresses [Kri14].
|Gene:||eno||Accession Numbers: EG10258 (MetaCyc), b2779, ECK2773|
Locations: cytosol, membrane, extracellular space, cytoskeleton
Subunit composition of enolase = [Eno]2
|Map Position: [2,904,665 <- 2,905,963]|
Molecular Weight of Polypeptide: 45.655 kD (from nucleotide sequence), 46.0 kD (experimental) [Spring71]
Molecular Weight of Multimer: 90.0 kD (experimental) [Spring71]
pI: 5.64, 5.7
|MultiFun Terms:||metabolism → central intermediary metabolism|
|metabolism → energy metabolism, carbon → glycolysis|
Relationship Links: InterPro:IN-FAMILY:IPR000941, InterPro:IN-FAMILY:IPR020809, InterPro:IN-FAMILY:IPR020810, InterPro:IN-FAMILY:IPR020811, InterPro:IN-FAMILY:IPR029017, InterPro:IN-FAMILY:IPR029065, Panther:IN-FAMILY:PTHR11902, PDB:Structure:1E9I, PDB:Structure:2FYM, PDB:Structure:3H8A, Pfam:IN-FAMILY:PF00113, Pfam:IN-FAMILY:PF03952, Prints:IN-FAMILY:PR00148, Prosite:IN-FAMILY:PS00164
Enolase catalyzes the interconversion of 2-phosphoglycerate and phosphoenolpyruvate during glycolysis and gluconeogenesis in E. coli. It is also a component of the E. coli degradosome complex that degrades RNA. In the degradosome enolase has been shown to bind to the C-terminal scaffold region of ribonuclease E. However, the role of enolase in RNA metabolism has not been fully defined [Miczak96, Kuhnel01, Liou01, Morita04, Callaghan04, Chandran06, Nurmohamed10, DominguezMalfav13, Lu14a].
Enolase has been purified from cell extracts of E. coli B and characterized. It was shown to be a dimer in solution and is dependent upon Mg2+ for its structure [Spring71]. The E. coli K-12 eno gene was later cloned, sequenced and functionally expressed in a temperature-sensitive eno mutant strain [Klein96].
The crystal structure of recombinant enolase from E. coli K-12 has been determined at 2.5 Å resolution, revealing that its dimer interface is enriched in charged residues relative to typical homodimer interfaces [Kuhnel01]. A later 1.6 Å structure shows enolase bound to its recognition site on RNase E [Chandran06].
E. coli enolase is functionally similar to enolases in other organisms, notably in its dependence on Mg2+, inhibition by fluoride in the presence of phosphate, and in its catalytic parameters. Its pH optimum is significantly higher than vertebrate enolases and is somewhat above those of yeast and plant enolases [Spring71].
E. coli K-12 enolase mutants were shown to grow on glycerate and succinate. They accumulated glycolytic pathway intermediates above the blocked enzyme upon addition of glucose or glycerol to resting cultures [Irani77].
Enolase is required for the rapid, degradosome-mediated degradation of ptsG mRNA in response to high levels of glucose 6-phosphate or fructose 6-phosphate [Morita04].
Immunofluorescence microscopy studies showed that enolase and other components of the degradosome are associated with the E. coli cytoskeleton and are organized into extended helical structures [Taghbalout07].
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