3.1.13.1 A815F degrades RNA duplexes with 7 or 14 nucleotides of ssRNA overhang significantly slower (about 4fold) than the wild-type enzyme 700205 3.1.13.1 A815W degrades RNA duplexes with 7 or 14 nucleotides of ssRNA overhang significantly slower (about 3fold) than the wild-type enzyme 700205 3.1.13.1 C284Y RNase II thermolability of the rnb500 phenotype is due to the Cys284Tyr mutation within the RNB domain, which abolishes activity by increasing protein kinetic instability at the nonpermissive temperature. Expression of RNase II C284Y and the double mutant (D126N and C284Y) does not allow growth at the nonpermissive temperature of 44°C. Structural mapping and partial multiple sequence alignment of RNase II thermosensitive phenotype mutations, overview -, 750548 3.1.13.1 C425A cannot be classified as polymorphic in the Japanese population. In the Korean, Mongolian, Ovambo, Turkish, and German DNA no genotype other than homozygotic 425C allele in RNASE2 at each single nucleotide polymorphism site is found 697158 3.1.13.1 D126N site-directed mutagenesis, the RNase II mutant exhibits a slightly decreased growth at 44°C suggesting some thermosensitivity which does not account for a major phenotype. This individual mutation is not detrimental for the function of RNase II in vivo. the RNase II D126N variant exhibits substantial catalytic activity -, 750548 3.1.13.1 D126N/C284Y site-directed mutagenesis, expression of RNase II C284Y and the double mutant (D126N and C284Y) does not allow growth at the nonpermissive temperature of 44°C -, 750548 3.1.13.1 D155M truncated RNase II protein pETIIDELTACSD1DELTAS1 consisting of the nuclease domain alone, but lacking any part of CSD2. Removal of the RNA-binding domains does allow RNase II to proceed further 698974 3.1.13.1 D201N activity is highly impaired, 0.2% of the specific activity of wild-type enzyme 709032 3.1.13.1 D201N significant loss of activity in degradation of poly(A) (0.2% of that of the wild-type enzyme). Generates a 10-11-nt fragment as a major degradation product, although longer reaction times result in the usual 4-nt fragment as a secondary product 693048 3.1.13.1 D201N site-directed mutagenesis, kinetic constants for enzyme-RNA interaction compared to the wild-type enzyme 716841 3.1.13.1 D201N/E390A site-directed mutagenesis, kinetic constants for enzyme-RNA interaction compared to the wild-type enzyme 716841 3.1.13.1 D201N/E390A very similar specific specific activity to the wild-type 709032 3.1.13.1 D201N/Y313F shows less than 0.1% of the specific activity present in the wild-type 709032 3.1.13.1 D201N/Y313F site-directed mutagenesis, kinetic constants for enzyme-RNA interaction compared to the wild-type enzyme 716841 3.1.13.1 D201N/Y313F/E390A shows less than 0.1% of the specific activity present in the wild-type 709032 3.1.13.1 D201N/Y313F/E390A site-directed mutagenesis, kinetic constants for enzyme-RNA interaction compared to the wild-type enzyme 716841 3.1.13.1 D207N site-directed mutagenesis, kinetic constants for enzyme-RNA interaction compared to the wild-type enzyme 716841 3.1.13.1 D207N still retains 12% activity 693048 3.1.13.1 D209N catalytically inactive, is unable to complement RNase II deletion 709993 3.1.13.1 D209N has less than 1% of the wild-type RNase activity, has similar affinities for the RNA substrate as the wild-type enzyme -, 700000 3.1.13.1 D209N site-directed mutagenesis, kinetic constants for enzyme-RNA interaction compared to the wild-type enzyme 716841 3.1.13.1 D210N significant loss of activity in degradation of poly(A) (0.3% of that of the wild-type enzyme). Generates a 10-11-nt fragment as a major degradation product, although longer reaction times result in the usual 4-nt fragment as a secondary product 693048 3.1.13.1 D210N site-directed mutagenesis, kinetic constants for enzyme-RNA interaction compared to the wild-type enzyme 716841 3.1.13.1 D275A is stable and produced in amounts similar to those seen for the wild-type enzyme, but it cannot repress competence -, 698601 3.1.13.1 D278N mutation at the catalytic center of RNase R, is inactive on A(4), but retains 4% activity of wild-type RNase R on poly(A) and A(17) 698974 3.1.13.1 D283R is stable and produced in amounts similar to those seen for the wild-type enzyme, but it cannot repress competence -, 698601 3.1.13.1 D551N abolishes the exonucleolytic activity 700205 3.1.13.1 D551N mutation in Rrp44-cat abolishes the exonucleolytic activity of Rrp44 without affecting its ability to bind RNA 681884 3.1.13.1 E390A site-directed mutagenesis, kinetic constants for enzyme-RNA interaction compared to the wild-type enzyme 716841 3.1.13.1 E390A specific activity is very similar to that of the wild-type 709032 3.1.13.1 E542A extraordinary catalysis and binding abilities that turns RNase II into a super-enzyme. More than a 100fold increase in the specific exoribonucleolytic activity, significantly increases affinity for the poly(A) substrate 709032 3.1.13.1 E542A site-directed mutagenesis, kinetic constants for enzyme-RNA interaction compared to the wild-type enzyme 716841 3.1.13.1 F358A site-directed mutagenesis, kinetic constants for enzyme-RNA interaction compared to the wild-type enzyme 716841 3.1.13.1 F358A the protein is 2fold more active than the wild-type 693048 3.1.13.1 K501Q site-directed mutagenesis, mutation of Lys501 results in up to 80% reduction in acetylation of RNase II 751728 3.1.13.1 K501R site-directed mutagenesis, mutation of Lys501 results in up to 80% reduction in acetylation of RNase II 751728 3.1.13.1 additional information construction of a large set of RNase II truncated proteins and comparison of them to the wild-type regarding their exoribonucleolytic activity and RNA-binding ability. The dissociation constants are determined using different single- or double-stranded substrates. The results obtained reveal that S1 is the most important domain in the establishment of stable RNA–protein complexes, and its elimination results in a drastic reduction on RNA-binding ability. The N-terminal CSD plays a very specific role in RNase II, preventing a tight binding of the enzyme to single-stranded poly(A) chains. The biochemical results obtained with a mutant that lacks both putative RNA-binding domains, reveals the presence of an additional region involved in RNA binding. Such region, is identified by sequence analysis and secondary structure prediction as a third putative RNA-binding domain located at the N-terminal part of RNB catalytic domain 681386 3.1.13.1 additional information construction of deletion mutant DELTArnb. Comparison of the mutant transcriptome with the wild-type, to determine the global effects of the deletion of the exoribonucleases in exponential phase, reveals that the deletion of RNase II significantly affects 187 transcripts. Many of the transcripts are actually down-regulated in the exoribonuclease mutants when compared to the wild-type control. The exoribonuclease also affects some stable RNAs. The RNase II mutant is shown to produce more biofilm than the wild-type control. Differential expression analysis of the transcriptome of exoribonucleases mutants and phenotypes, overview. For example, nirB (Nitrite reductase [NAD(P)H] large subunit) is down-regulated in DELTArnb with a fold-change of 0.36 while in the DELTArnr mutant nirB is up-regulated with a fold-change of 9.11 750198 3.1.13.1 additional information construction of deletion mutant DELTArnb. Comparison of the mutant transcriptome with the wild-type, to determine the global effects of the deletion of the exoribonucleases in exponential phase, reveals that the deletion of RNase R significantly affects 202 transcripts. Many of the transcripts are actually down-regulated in the exoribonuclease mutants when compared to the wild-type control. The exoribonuclease also affects some stable RNAs. The RNase R mutant is shown to produce more biofilm than the wild-type control. Differential expression analysis of the transcriptome of exoribonucleases mutants and phenotypes, overview 750198 3.1.13.1 additional information DELTACSDb and DELTAS1b mutants, are more than 90% soluble. Similarly, the solubility of the RNB derivative, which lacks both putative RNA-binding domains, is also greater than 90%. The DELTACSDa mutant is only 60% soluble. Elimination of the whole CSD domain (DELTACSDb) or part of it (DELTACSDa) does not affect the exonucleolytic activity of RNase II, and even improves its activity significantly -, 700000 3.1.13.1 additional information hybrid proteins are constructed by replacing the S1 domain of RNase II for the S1 from RNase R and PNPase, and their exonucleolytic activity and RNA-binding ability are examined. Both the S1 domains of RNase R and PNPase are able to partially reverse the drop of RNA-binding ability and exonucleolytic activity resulting from removal of the S1 domain of RNase II. The S1 domains investigated are not equivalent 677105 3.1.13.1 additional information RNase RDELTACSDs is missing the first 221 amino acids of RNase R, which include CSD1 and CSD2. RNase RDELTABasic lacks the 83 amino acids from the C terminus, which comprise the low complexity, highly basic region. RNase RDELTAS1 is truncated 170 amino acids from the C-terminus to remove both the S1 domain and the low complexity, highly basic region. RNase RDELTACSDsDELTAS1 consists of the nuclease domain alone, and, therefore, lacks all of the putative RNA-binding domains. Decrease in affinity upon deletion of either the CSDs or the S1 domain. RNase RDELTABasic displays 2fold higher activity than full-length wild-type RNase R. RNase RDELTACSDs looses 30% of the activity of full-length RNase R on poly(A) 90% on the shorter A(17) substrate. The RNase RDELTACSDsDELTAS1 truncated protein retains only 0.5% activity of the full-length protein on poly(A), and only 0.02% activity on A(17). All of the RNase R-truncated proteins have comparable activity on A(4) 698974 3.1.13.1 additional information Rrp44 242-1001 (Rrp44DELTAN) lacks the predicted N-terminal PIN domain, shows no detectable difference in activity toward ssRNA substrates as compared to recombinant Rrp44 700205 3.1.13.1 R500A shows more than a 40000fold reduction in specific activity when compared with the wild-type 709032 3.1.13.1 R500A site-directed mutagenesis, kinetic constants for enzyme-RNA interaction compared to the wild-type enzyme 716841 3.1.13.1 R500K shows less than 0.1% of the specific activity present in the wild-type 709032 3.1.13.1 Y253A 26% of the activity of the enzyme persists, significantly impairs RNA binding 693048 3.1.13.1 Y253A site-directed mutagenesis, kinetic constants for enzyme-RNA interaction compared to the wild-type enzyme 716841 3.1.13.1 Y253A/F358A 12% of the activity of the enzyme persists, whereas RNA binding affinity is not significantly affected 693048 3.1.13.1 Y253A/F358A site-directed mutagenesis, kinetic constants for enzyme-RNA interaction compared to the wild-type enzyme 716841 3.1.13.1 Y313A 100fold reduction of specific activity 709032 3.1.13.1 Y313A site-directed mutagenesis, kinetic constants for enzyme-RNA interaction compared to the wild-type enzyme 716841 3.1.13.1 Y313F site-directed mutagenesis, kinetic constants for enzyme-RNA interaction compared to the wild-type enzyme 716841 3.1.13.1 Y313F specific activity is very similar to that of the wild-type 709032 3.1.13.1 Y313F/E390A site-directed mutagenesis, kinetic constants for enzyme-RNA interaction compared to the wild-type enzyme 716841 3.1.13.1 Y313F/E390A specific activity is not affected 709032