Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.8 (polynucleotide phosphorylase)
 723 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The reaction of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole [NBD-Cl] with purified eel electrophax Na+ and K+ stimulated adenosine triphosphatase [(Na-K)ATPase] has been monitored by changes in the (Na-K)ATPase activity, the K+ stimulated p-nitrophenyl phosphatase [PNPase] activity, and the protein ultraviolet absorption spectrum. The NBD-Cl reacts with two tyrosine residues per mol of enzyme (approximately 6-7 nmol/mg of protein), as judged by changes in protein absorption spectra and incorporation of [14C]NBD-Cl. The modified tyrosine groups are located on the Mr = 95 000 polypeptide chain and react at different rates. Only one tyrosine modification is necessary for complete inhibition of (Na-K)ATPase activity, although both must be modified for complete inhibition of PNPase activity. Reversal of these modifications by 2-mercaptoethanol restores 65% of both activities. Na+ increases the rate of tyrosine modification, K+ decreases the rate, and ATP affords the more reactive tyrosine group complete protection. NBD-Cl modification of approximately 6-7 nmol of tyrosine groups/mg of protein results in a large decrease in ATP affinity as judged by equilibrium binding. These results are compared with similar results obtained from NBD-Cl modification of the coupling factors of oxidative phosphorylation and photophosphorylation. A model is presented suggesting an asymmetric arrangement of two 95 000 polypeptide chains with a single tyrosine residue at the ATP site.
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PMID:Reaction of (Na-K)ATPase with 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole: evidence for an essential tyrosine at the active site. 14 73

A thermophilic polynucleotide phosphorylase lacking polynucleotide phosphoryltic activity was purified from Thermus thermophilus HB-8 strain. The enzyme is an altered form of the native polynucleotide phosphorylase, probably attacked by the proteinase(s) of this extreme thermophile during the purification process. This modified enzyme lacks phosphorolytic activity to poly(A) while retaining weak activity to phosphorolyse tetranucleotides or hexanucleotides. The purified enzyme was shown to be homogenous by electrophoretic analysis in polyacrylamide gel. This enzyme had a molecular weight of 190 000 as calculated both from electrophoresis on polyacrylamide gel and from the Stoke's radius derived from the gel filtration pattern and the sedimentation coefficient. The enzyme was separated into three polypeptide chains by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate; their molecular weights were calculated to be 92000, 73000 and 35000. The enzyme was thermophilic and thermotolerant, exhibiting its maximal activity at 70 degrees C. The four ribonucleoside diphosphates (ADP, GDP, UDP and CDP) were polymerized to the extent of 7-S size.
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PMID:Thermophilic polynucleotide phosphorylase from Thermus thermophilus. Purification and properties of an altered form of enzyme which lacks phosphorolytic activity to polynycleotide. 89 51

Benzimidazoles are weak mutagens acting through base substitutions; they are incorporated into nucleic acids. Experiments with deoxyribohomopolymers as templates demonstrated that benzimidazole nucleoside triphosphate is polymerized by RNA polymerase only in the presence of poly dC, i.e., instead of guanine. In plasmolyzed Escherichia coli cells, benzimidazole ribonucleoside diphosphate is polymerized by polynucleotide phosphorylase and can, after blocking of the normal mRNA synthesis with actinomycin D, be used as a messenger for polypeptide formation. The addition of radioactive amino acids to this system showed that benzimidazole is not read preferentially as guanine, as would have been expected from the RNA polymerase results. Instead, the reading was position dependent and brnzimidazole is recognized (1) in the first codon position as adenine, (2) in the second as purine, and (3) in the third possibly only as base. Benzimidazole mutagenicity is thus explained as a G in equilibrium A transition.
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PMID:The molecular mechanism of benzimidazole mutagenicity: in vitro studies on transcription and translation. 110 1

As a first step to approach the structural and functional analysis of DNA-dependent RNA polymerase II (EC 2.7.7.8), we have isolated genomic sequences for the large subunit of the human enzyme. The sequences homologous to Drosophila RNA polymerase II large subunit sequences are present in the genome as single copy genes, when assayed at high stringency. The polypeptide information is encoded in a mRNA of 7.35 kilobases, as determined by Northern blot analysis. In vitro translation reveals a polypeptide of 220 kDa, similar in electrophoretic mobility to the largest subunit of the enzyme. A fusion-polypeptide synthesized in bacteria contains a region that cross-reacts with anti-RNA polymerase II antiserum. Antiserum directed against the purified fusion protein reacts with the large subunit of RNA polymerase II, whether in the intact IIA (220 kDa) or in the degraded IIB (180 kDa) forms. Moreover, the antifusion protein antibody inhibits not only the purified calf thymus RNA polymerase II activity but also specific RNA polymerase II transcription in a HeLa cell extract. Thus, the DNA fragment isolated contains structural and functional domains of the human RNA polymerase II large subunit.
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PMID:The gene encoding the large subunit of human RNA polymerase II. 299 7

We have isolated suppressor mutants that suppress temperature-sensitive colony formation and anucleate cell production of a mukB mutation. A linkage group (smbB) of the suppressor mutations is located in the rne/ams/hmp gene encoding the processing endoribonuclease RNase E. All of the rne (smbB) mutants code for truncated RNase E polypeptides lacking a carboxyl-terminal half. The amount of MukB protein was higher in these rne mutants than that in the rne+ strain. These rne mutants grew nearly normally in the mukB+ genetic background. The copy number of plasmid pBR322 in these rne mutants was lower than that in the rne+ isogenic strain. The results suggest that these rne mutations increase the half-lives of mukB mRNA and RNAI of pBR322, the antisense RNA regulating ColE1-type plasmid replication. We have demonstrated that the wild-type RNase E protein bound to polynucleotide phosphorylase (PNPase) but a truncated RNase E polypeptide lacking the C-terminal half did not. We conclude that the C-terminal half of RNase E is not essential for viability but plays an important role for binding with PNPase. RNase E and PNPase of the multiprotein complex presumably cooperate for effective processing and turnover of specific substrates, such as mRNAs and other RNAs in vivo.
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PMID:RNase E polypeptides lacking a carboxyl-terminal half suppress a mukB mutation in Escherichia coli. 868 98

The Escherichia coli RNA degradosome is the prototype of a recently discovered family of multiprotein machines involved in the processing and degradation of RNA. The interactions between the various protein components of the RNA degradosome were investigated by Far Western blotting, the yeast two-hybrid assay, and coimmunopurification experiments. Our results demonstrate that the carboxy-terminal half (CTH) of ribonuclease E (RNase E) contains the binding sites for the three other major degradosomal components, the DEAD-box RNA helicase RhlB, enolase, and polynucleotide phosphorylase (PNPase). The CTH of RNase E acts as the scaffold of the complex upon which the other degradosomal components are assembled. Regions for oligomerization were detected in the amino-terminal and central regions of RNase E. Furthermore, polypeptides derived from the highly charged region of RNase E, containing the RhlB binding site, stimulate RhlB activity at least 15-fold, saturating at one polypeptide per RhlB molecule. A model for the regulation of the RhlB RNA helicase activity is presented. The description of RNase E now emerging is that of a remarkably complex multidomain protein containing an amino-terminal catalytic domain, a central RNA-binding domain, and carboxy-terminal binding sites for the other major components of the RNA degradosome.
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PMID:Ribonuclease E organizes the protein interactions in the Escherichia coli RNA degradosome. 973 74

We report here the identification of a new lipoprotein, NlpI, in Escherichia coli K-12. The NlpI structural gene (nlpI) is located between the genes pnp (polynucleotide phosphorylase) and deaD (RNA helicase) at 71 min on the E. coli chromosome. The nlpI gene encodes a putative polypeptide of approximately 34 kDa, and multiple lines of evidence clearly demonstrate that NlpI is indeed a lipoprotein. An nlpI::cm mutation rendered growth of the cells osmotically sensitive, and incubation of the insertion mutant at an elevated temperature resulted in the formation of filaments. The altered phenotype of the mutant was a direct consequence of the mutation in nlpI, since it was complemented by the wild-type nlpI gene alone. Overexpression of the unaltered nlpI gene in wild-type cells resulted in the loss of the rod morphology and the formation of single prolate ellipsoids and pairs of prolate ellipsoids joined by partial constrictions. NlpI may be important for an as-yet-undefined step in the overall process of cell division.
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PMID:Identification and characterization of a new lipoprotein, NlpI, in Escherichia coli K-12. 1040 May 90

RNase E is an essential Escherichia coli endonuclease, which controls both 5S rRNA maturation and bulk mRNA decay. While the C-terminal half of this 1061-residue protein associates with polynucleotide phosphorylase (PNPase) and several other enzymes into a 'degradosome', only the N-terminal half, which carries the catalytic activity, is required for growth. We characterize here a mutation (rne131 ) that yields a metabolically stable polypeptide lacking the last 477 residues of RNAse E. This mutation resembles the N-terminal conditional mutation rne1 in stabilizing mRNAs, both in bulk and individually, but differs from it in leaving rRNA processing and cell growth unaffected. Another mutation (rne105 ) removing the last 469 residues behaves similarly. Thus, the C-terminal half of RNase E is instrumental in degrading mRNAs, but dispensable for processing rRNA. A plausible interpretation is that the former activity requires that RNase E associates with other degradosome proteins; however, PNPase is not essential, as RNase E remains fully active towards mRNAs in rne+pnp mutants. All mRNAs are not stabilized equally by the rne131 mutation: the greater their susceptibility to RNase E, the larger the stabilization. Artificial mRNAs generated by E. coli expression systems based on T7 RNA polymerase can be genuinely unstable, and we show that the mutation can improve the yield of such systems without compromising cell growth.
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PMID:The C-terminal half of RNase E, which organizes the Escherichia coli degradosome, participates in mRNA degradation but not rRNA processing in vivo. 1041 35

Different representatives of bacteria have different number of amino acid residues in the ribosomal proteins S1. This number varies from 111 (Spiroplasma kunkelii) to 863 a.a. (Treponema pallidum). Traditionally and for lack of this protein three-dimensional structure, its architecture is represented as repeating S1 domains. Number of these domains depends on the protein's length. Domain's quantity and its boundaries data are contained in the specialized databases, such as SMART, Pfam and PROSITE. However, for the same object these data may be very different. For search of domain's quantity and its boundaries, new approach, based on the analysis of dicted secondary structure (PsiPred), was used. This approach allowed us to reveal structural domains in amino acid sequences of S1 proteins and at that number varied from one to six. Alignment of S1 proteins, containing different domain's number, with the S1 RNAbinding domain of Escherichia coli PNPase elicited a fact that in family of ribosomal proteins SI one domain has maximal homology with S1 domain from PNPase. This conservative domain migrates along polypeptide chain and locates in proteins, containing different domain's number, according to specified pattern. In this domain as well in the S1 domain from PNPase, residues Phe-19, Phe-22, His-34, Asp-64 and Arg-68 are clustered on the surface and formed RNA binding site.
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PMID:[Family of ribosomal proteins S1 contains unique conservative domain]. 2087 33