Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.2.1.23 (beta-galactosidase)
 14,648 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The properties of the enzyme ribonuclease N were investigated. By comparing the distribution in the cell of RNase N with the bonafide intracellular beta-galactosidase, and the periplasmic alkaline phosphatase enzymes, we showed that RNase N is an intracellular enzyme. Since previous studies suggested that it is an endoribonuclease, it was compared to RNase III, the only other known intracellular endoribonuclease in Escherichia coli. Using homopolymers and co-polymers we found that, while RNase III could digest double-stranded RNA only, RNase N digested single-stranded and double-stranded RNA with similar efficiency. Furthermore, all RNAs used, natural as well as synthetic, were substrates for the enzyme. Using 5 S rRNA as a substrate it was confirmed that the enzyme is an endonuclease. The final products of the reaction of this enzyme are 5'-mononucleotides. The molecular weight of the enzyme is about 120,000 and it seems to contain two subunits which are similar in size. These properties thus differentiate this enzyme from all other known ribonucleases in E. coli.
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PMID:Characterization of an endoribonuclease, RNase N, from Escherichia coli. 9

The metabolism of mRNA from the lactose (lac) operon of Escherichia coli has been studied in ribonuclease (RNase) III-deficient strains (rnc-105). The induction lag for beta-galactosidase from the first gene was twice as long, and enzyme synthesis was reduced 10-fold in one such mutant compared with its isogenic rnc+ sister; in the original mutant strain AB301-105, synthesis of beta-galactosidase was not even detectable, although transduction analysis revealed the presence of a normal lac operon. This defect does not reflect a loss of all lac operon activity galactoside acetyltransferase from the last gene was synthesized even in strain AB301-105 but at a rate several times lower than normal. Hybridization analyses suggested that both the frequency of transcription initiation and the time to transcribe the entire operon are normal in rnc-105 strains. The long induction lag was caused by a longer translation time. This defect led to translational polarity with reduced amounts of distal mRNA to give a population of smaller-sized lac mRNA molecules. All these pleiotropic effects seem to result from RNase III deficiency, since it was possible to select revertants to rnc+ that grew and expressed the lac operon at normal rates. However, the rnc-105 isogenic strains (but not AB301-105) also changed very easily to give a more normal rate of beta-galactosidase synthesis without regaining RNase III activity or a faster growth rate. The basis for this reversion is not known; it may represent a "phenotypic suppression" rather than result from a stable genetic change. Such suppressor effects could account for earlier reports of a noninvolvement of RNase III in mRNA metabolism in deliberately selected lac+ rnc-105 strains. The ribosomes from rnc-105 strains were as competent as ribosomes from rnc+ strains to form translation initiation complexes in vitro. However, per mass, beta-galactosidase mRNA from AB301-105 was at least three times less competent to form initiation complexes than was A19 beta-galactosidase mRNA. RNase III may be important in the normal cell to prepare lac mRNA for translation initiation. A defect at this step could account for all the observed changes in lac expression. A potential target within a secondary structure at the start of the lac mRNA is considered. Expression of many operons may be affected by RNase III activity; gal and trp operon expressions were also abnormal in RNase III- strains.
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PMID:Altered mRNA metabolism in ribonuclease III-deficient strains of Escherichia coli. 9 20

An isogenic pair of Escherichia coli strains, one carrying an rnc+ and the other an rnc- allele (a mutation which reduces the level of ribonuclease III), was compared. The rnc- strain fails to grow at very elevated temperatures (for E. coli) while the rnc+ strain does grow exponentially. Assaying the residual RNase III like activity in extracts of the rnc- strain at different pHs and at different temperatures suggested that this residual RNase III like activity is not due to RNase III. This raised the possibility that the rnc- strain is devoid of any RNase III activity in the cell. Comparing the decay of newly synthesized RNA and functional decay of beta-galactosidase mRNA in such strains revealed that in both strains these parameters proceed in similar rates, which suggests that RNase III is not involved in the metabolism of mRNA. During carbon starvation preexisting total RNA, as well as 23S and 16S rRNA, decay faster in the rnc- strain, thus eliminating the possibility that RNase III is the endoribonuclease which initiates the decay of rRNA during starvation (Kaplan and Apirion, 1975a).
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PMID:Consequences of losing ribonuclease III on the Escherichia coli cell. 77 91

It has been previously shown that the pnp messenger RNAs are cleaved by RNase III at the 5' end and that these cleavages induce a rapid decay of these messengers. A translational fusion between pnp and lacZ was introduced into the chromosome of a delta lac strain to study the expression of pnp. In the presence of increased cellular concentrations of polynucleotide phosphorylase, the level of the hybrid beta-galactosidase is repressed, whereas the synthesis rate of the corresponding message is not significantly affected. In the absence of pnp, the level of the hybrid protein increases strongly. Thus, polynucleotide phosphorylase is post-transcriptionally autocontrolled. However, autocontrol is totally abolished in strains where the RNase III site on the pnp message has been deleted or in strains devoid of RNase III. These results suggest that polynucleotide phosphorylase requires RNase III cleavages to autoregulate the translation of its message. Other mutations in the ribosome binding site region support the hypothesis that this 3' to 5' processive enzyme could recognize a specific repressor binding site at the 5' end of pnp mRNA. Implications of these results on the mechanism of regulation and on messenger degradation are discussed.
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PMID:E.coli polynucleotide phosphorylase expression is autoregulated through an RNase III-dependent mechanism. 162 24

Stability of RNA was tested in strains of Escherichia coli carrying single, double, or triple mutations in the RNA processing enzymes RNase III, RNase E and RNase P. Tests were carried out for total pulse labeled RNA, beta-galactosidase mRNA and for the decay of preexisting RNA during carbon starvation. Decay of RNA was measured at permissive and nonpermissive temperatures, and in no case were significant differences between mutants and non-mutant strains found. Therefore, we conclude that the three processing enzymes; RNase III, E and P do not contribute significantly to turnover of RNA IN Escherichia coli.
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PMID:Decay of RNA in RNA processing mutants of Escherichia coli. 615 28

Using RNA-directed synthesis of the alpha-peptide of beta-galactosidase as an assay, a factor was purified that inactivated further function of the mRNA. In the presence of Ca2+ ions to inhibit most nuclease activity, inactivation of mRNA occurred during incubation with ribosomes or with a 1 M KCl wash of ribosomes. The inactivation activity required Mg2+ ions, and purified as a single factor which did not bind to DEAE-cellulose, but bound reversibly to phosphocellulose. The factor eluted from Sephadex G-150 with an apparent molecular weight of about 43,000. Purified 700-fold, it showed no detectable exonuclease activity, and little or no cleavage of a variety of single-stranded substrates, including full length lac operon mRNA; but repurified inactivated mRNA was still inactive for protein synthesis. The factor did not inhibit poly(U)-directed polyphenylalanine synthesis. When proteins isolated from the ribosomal wash were individually tested, highly purified RNase III, which purifies in the same way and has the same size, also inactivated lac mRNA. The ribosomal wash from an RNase III- strain showed little if any activity compared to that from an isogenic RNase III+ strain. The possibility of a site-specific inactivating cleavage of mRNA by RNase III at or near the 5' end is considered.
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PMID:Functional inactivation of lac alpha-peptide mRNA by a factor that purifies that Escherichia coli RNase III. 625 91

Since the speF-potE operon (pPT71 clone) encoding inducible ornithine decarboxylase (iODC) and polyamine transport potE protein is inducible at acidic pH, a gene encoding a protein involved in the enhancement of expression of the operon was searched for. Using the fused gene containing the upstream sequence of the speF-potE operon and the open reading frame of beta-galactosidase as a reporter gene, a clone (pPTS23) which causes the increase of beta-galactosidase activity at acidic pH was isolated. The clone also increased iODC activity at acidic pH and was identified as a gene encoding RNase III. This is the first example that RNase III increases the translational efficiency of mRNA derived from Escherichia coli gene by cutting the 5'-untranslated region of mRNA.
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PMID:Involvement of ribonuclease III in the enhancement of expression of the speF-potE operon encoding inducible ornithine decarboxylase and polyamine transport protein. 816 35

New single-copy vectors based on lambda phage have been developed for creating either transcriptional (operon) or translational (gene) fusions to the lacZ gene. The improvements of these vectors over the previous lambda TL61 vector include: (i) incorporation of a tetracycline-resistance-encoding gene (TcR) to permit direct selection of lysogens, (ii) low-background beta-galactosidase activity, (iii) the ability to accept DNA inserts up to 8 kb in size, and (iv) an expanded multiple cloning site (MCS). The new transcriptional fusion vector retains the RNase III processing site downstream from the MCS which ensures independent translation of lacZ. The set of three translational fusion vectors allow for convenient subcloning in any of the three translational reading frames.
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PMID:A refined vector system for the in vitro construction of single-copy transcriptional or translational fusions to lacZ. 863 51

Pairs of very closely related Escherichia coli strains were prepared, one having the wild-type allele for ribonuclease III, an enzyme which specifically degrades double-stranded RNA, and the other having a mutant RNase III allele. Growth and phage plating efficiency were compared in these strains. The RNase III+ strains grow better than the RNase III- strains and plate T7 and lambda phage better, but T4 plates with the same efficiency on both strains. On the other hand, the half lives of newly synthesized RNA as well as of functional beta-galactosidase mRNA are similar in both kind of strains. These two parameters, however, are significantly longer in both strains as compared to the original strain from which they were derived. Also, no difference in the differential induction of beta-galactosidase was observed between such strains. Thus, we have to conclude that either ribonuclease III does not play a significant role in the functioning and stability of newly synthesized mRNA, or that enough enzymatic activity was left, residual RNase III or some other enzyme to deal with double-stranded regions in the message.
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PMID:Unaltered stability of newly synthesized RNA in strains of Escherichia coli missing a ribonuclease specific for double-stranded RNA. 1609 99