scholarly journals Comparative Study of Cyanobacterial and E. coli RNA Polymerases: Misincorporation, Abortive Transcription, and Dependence on Divalent Cations

2011 ◽  
Vol 2011 ◽  
pp. 1-11 ◽  
Author(s):  
Masahiko Imashimizu ◽  
Kan Tanaka ◽  
Nobuo Shimamoto
Keyword(s):  
Rna Polymerase ◽  
Divalent Cations ◽  
Physiological Role ◽  
Rna Polymerases ◽  
E Coli ◽  
Order Of Magnitude ◽  
Photosynthetic Machinery ◽  
The Difference ◽  
The Cost

If Mg2+ ion is replaced by Mn2+ ion, RNA polymerase tends to misincorporate noncognate nucleotide, which is thought to be one of the reasons for the toxicity of Mn2+ ion. Therefore, most cells have Mn2+ ion at low intracellular concentrations, but cyanobacteria need the ion at a millimolar concentration to maintain photosynthetic machinery. To analyse the mechanism for resistance against the abundant Mn2+ ion, we compared the properties of cyanobacterial and E. coli RNA polymerases. The cyanobacterial enzyme showed a lower level of abortive transcription and less misincorporation than the E. coli enzyme. Moreover, the cyanobacterial enzyme showed a slower rate of the whole elongation by an order of magnitude, paused more frequently, and cleaved its transcript faster in the absence of NTPs. In conclusion, cyanobacterial RNA polymerase maintains the fidelity of transcription against Mn2+ ion by deliberate incorporation of a nucleotide at the cost of the elongation rate. The cyanobacterial and the E. coli enzymes showed different sensitivities to Mg2+ ion, and the physiological role of the difference is also discussed.

scholarly journals The Difference δ-Cells Make in Glucose Control

Physiology ◽  
2018 ◽  
Vol 33 (6) ◽  
pp. 403-411 ◽  
Author(s):  
Mark O. Huising ◽  
Talitha van der Meulen ◽  
Jessica L. Huang ◽  
Mohammad S. Pourhosseinzadeh ◽  
Glyn M. Noguchi
Keyword(s):  
Glucose Control ◽  
Feedback Loop ◽  
Physiological Role ◽  
Glucagon Secretion ◽  
Negative Feedback Loop ◽  
Β Cells ◽  
Insulin And Glucagon Secretion ◽  
The Difference ◽  
Delta Cells

The role of beta and α-cells to glucose control are established, but the physiological role of δ-cells is poorly understood. Delta-cells are ideally positioned within pancreatic islets to modulate insulin and glucagon secretion at their source. We review the evidence for a negative feedback loop between delta and β-cells that determines the blood glucose set point and suggest that local δ-cell-mediated feedback stabilizes glycemic control.

scholarly journals Deletion of the rpoZ gene, encoding the ω subunit of RNA polymerase, results in pleiotropic surface-related phenotypes in Mycobacterium smegmatis

Microbiology ◽  
2006 ◽  
Vol 152 (6) ◽  
pp. 1741-1750 ◽  
Author(s):  
Renjith Mathew ◽  
Raju Mukherjee ◽  
Radhakrishnan Balachandar ◽  
Dipankar Chatterji
Keyword(s):  
Rna Polymerase ◽  
Mycobacterium Smegmatis ◽  
Biofilm Formation ◽  
Physiological Role ◽  
Wild Type ◽  
Gene Encoding ◽  
Biofilm Growth ◽  
Mutant Bacterium ◽  
Sliding Motility

The ω subunit, the smallest subunit of bacterial RNA polymerase, is known to be involved in maintaining the conformation of the β′ subunit and aiding its recruitment to the rest of the core enzyme assembly in Escherichia coli. It has recently been shown in Mycobacterium smegmatis, by creating a deletion mutation of the rpoZ gene encoding ω, that the physiological role of the ω subunit also includes providing physical protection to β′. Interestingly, the mutant had altered colony morphology. This paper demonstrates that the mutant mycobacterium has pleiotropic phenotypes including reduced sliding motility and defective biofilm formation. Analysis of the spatial arrangement of biofilms by electron microscopy suggests that the altered phenotype of the mutant arises from a deficiency in generation of extracellular matrix. Complementation of the mutant strain with a copy of the wild-type rpoZ gene integrated in the bacterial chromosome restored both sliding motility and biofilm formation to the wild-type state, unequivocally proving the role of ω in the characteristics observed for the mutant bacterium. Analysis of the cell wall composition demonstrated that the mutant bacterium had an identical glycopeptidolipid profile to the wild-type, but failed to synthesize the short-chain mycolic acids characteristic of biofilm growth in M. smegmatis.

scholarly journals Escherichia coli NsrR Regulates a Pathway for the Oxidation of 3-Nitrotyramine to 4-Hydroxy-3-Nitrophenylacetate

2008 ◽  
Vol 190 (18) ◽  
pp. 6170-6177 ◽  
Author(s):  
Linda D. Rankin ◽  
Diane M. Bodenmiller ◽  
Jonathan D. Partridge ◽  
Shirley F. Nishino ◽  
Jim C. Spain ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Physiological Role ◽  
Growth Conditions ◽  
Growth Defect ◽  
E Coli ◽  
Mutant Phenotypes ◽  
Culture Supernatants ◽  
Chip Chip

ABSTRACT Chromatin immunoprecipitation and microarray (ChIP-chip) analysis showed that the nitric oxide (NO)-sensitive repressor NsrR from Escherichia coli binds in vivo to the promoters of the tynA and feaB genes. These genes encode the first two enzymes of a pathway that is required for the catabolism of phenylethylamine (PEA) and its hydroxylated derivatives tyramine and dopamine. Deletion of nsrR caused small increases in the activities of the tynA and feaB promoters in cultures grown on PEA. Overexpression of nsrR severely retarded growth on PEA and caused a marked repression of the tynA and feaB promoters. Both the growth defect and the promoter repression were reversed in the presence of a source of NO. These results are consistent with NsrR mediating repression of the tynA and feaB genes by binding (in an NO-sensitive fashion) to the sites identified by ChIP-chip. E. coli was shown to use 3-nitrotyramine as a nitrogen source for growth, conditions which partially induce the tynA and feaB promoters. Mutation of tynA (but not feaB) prevented growth on 3-nitrotyramine. Growth yields, mutant phenotypes, and analyses of culture supernatants suggested that 3-nitrotyramine is oxidized to 4-hydroxy-3-nitrophenylacetate, with growth occurring at the expense of the amino group of 3-nitrotyramine. Accordingly, enzyme assays showed that 3-nitrotyramine and its oxidation product (4-hydroxy-3-nitrophenylacetaldehyde) could be oxidized by the enzymes encoded by tynA and feaB, respectively. The results suggest that an additional physiological role of the PEA catabolic pathway is to metabolize nitroaromatic compounds that may accumulate in cells exposed to NO.

scholarly journals Activities and some properties of 5′-nucleotidase, adenosine kinase and adenosine deaminase in tissues from vertebrates and invertebrates in relation to the control of the concentration and the physiological role of adenosine

1978 ◽  
Vol 174 (3) ◽  
pp. 965-977 ◽  
Author(s):  
J R S Arch ◽  
E A Newsholme
Keyword(s):  
Adenosine Deaminase ◽  
Physiological Role ◽  
Adenosine Kinase ◽  
British Library ◽  
Adenosine Concentration ◽  
Effects Of Ph ◽  
The Difference ◽  
Low Activity

1. The maximal activities of 5′-nucleotidase, adenosine kinase and adenosine deaminase together with the Km values for their respective substrates were measured in muscle, nervous tissue and liver from a large range of animals to provide information on the mechanism of control of adenosine concentration in the tissues. 2. Detailed evidence that the methods used were optimal for the extraction and assay of these enzymes has been deposited as Supplementary Publication SUP 50088 (16pages) at the British Library Lending Division, Boston Spa, Wetherby, West Yorkshire LS23 7BQ, U.K.,from whom copies can be obtained on the terms indicated in Biochem. J. (1978), 169, 5. This evidence includes the effects of pH and temperature on the activities of the enzymes. 3. In many tissues, the activities of 5′-nucleotidase were considerably higher than the sum of the activities of adenosine kinase and deaminase, which suggests that the activity of the nucleotidase must be markedly inhibited in vivo so that adenosine does not accumulate. In the tissues in which comparison is possible, the Km of the nucleotidase is higher than the AMP content of the tissue, and since some of the latter may be bound within the cell, the low concentration of substrate may, in part, be responsible for a low activity in vivo. 4. In most tissues and animals investigated, the values of the Km of adenosine kinase for adenosine are between one and two orders of magnitude lower than those for the deaminase. It is suggested that 5′-nucleotidase and adenosine kinase are simultaneously active so that a substrate cycle between AMP and adenosine is produced: the difference in Km values between kinase and deaminase indicates that, via the cycle, small changes in activity of kinase or nucleotidase produce large changes in adenosine concentration. 5. The activities of adenosine kinase or deaminase from vertebrate muscles are inversely correlated with the activities of phosphorylase in these muscles. Since the magnitude of the latter activities are indicative of the anaerobic nature of muscles, this negative correlation supports the hypothesis that an important role of adenosine is the regulation of blood flow in the aerobic muscles.

scholarly journals Association of ω with the C-Terminal Region of the β′ Subunit Is Essential for Assembly of RNA Polymerase in Mycobacterium tuberculosis

2018 ◽  
Vol 200 (12) ◽  
Author(s):  
Chunyou Mao ◽  
Yan Zhu ◽  
Pei Lu ◽  
Lipeng Feng ◽  
Shiyun Chen ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Mycobacterium Tuberculosis ◽  
Rna Polymerase ◽  
Essential Role ◽  
Β Subunit ◽  
Content Type ◽  
E Coli ◽  
Core Enzyme ◽  
Loop Region

ABSTRACT The ω subunit is the smallest subunit of bacterial RNA polymerase (RNAP). Although homologs of ω are essential in both eukaryotes and archaea, this subunit has been known to be dispensable for RNAP in Escherichia coli and in other bacteria. In this study, we characterized an indispensable role of the ω subunit in Mycobacterium tuberculosis . Unlike the well-studied E. coli RNAP, the M. tuberculosis RNAP core enzyme cannot be functionally assembled in the absence of the ω subunit. Importantly, substitution of M. tuberculosis ω with ω subunits from E. coli or Thermus thermophilus cannot restore the assembly of M. tuberculosis RNAP. Furthermore, by replacing different regions in M. tuberculosis ω with the corresponding regions from E. coli ω, we found a nonconserved loop region in M. tuberculosis ω essential for its function in RNAP assembly. From RNAP structures, we noticed that the location of the C-terminal region of the β′ subunit (β′CTD) in M. tuberculosis RNAP but not in E. coli or T. thermophilus RNAP is close to the ω loop region. Deletion of this β′CTD in M. tuberculosis RNAP destabilized the binding of M. tuberculosis ω on RNAP and compromised M. tuberculosis core assembly, suggesting that these two regions may function together to play a role in ω-dependent RNAP assembly in M. tuberculosis . Sequence alignment of the ω loop and the β′CTD regions suggests that the essential role of ω is probably restricted to mycobacteria. Together, our study characterized an essential role of M. tuberculosis ω and highlighted the importance of the ω loop region in M. tuberculosis RNAP assembly. IMPORTANCE DNA-dependent RNA polymerase (RNAP), which consists of a multisubunit core enzyme (α 2 ββ′ω) and a dissociable σ subunit, is the only enzyme in charge of transcription in bacteria. As the smallest subunit, the roles of ω remain the least well studied. In Escherichia coli and some other bacteria, the ω subunit is known to be nonessential for RNAP. In this study, we revealed an essential role of the ω subunit for RNAP assembly in the human pathogen Mycobacterium tuberculosis , and a mycobacterium-specific ω loop that plays a role in this function was also characterized. Our study provides fresh insights for further characterizing the roles of bacterial ω subunit.

scholarly journals Mutational Analysis of the Chlamydia trachomatis rRNA P1 Promoter Defines Four Regions Important for Transcription In Vitro

1998 ◽  
Vol 180 (9) ◽  
pp. 2359-2366 ◽  
Author(s):  
Ming Tan ◽  
Tamas Gaal ◽  
Richard L. Gourse ◽  
Joanne N. Engel
Keyword(s):  
Escherichia Coli ◽  
Chlamydia Trachomatis ◽  
Rna Polymerase ◽  
Mutational Analysis ◽  
In Vitro Transcription ◽  
Rna Polymerases ◽  
Rich Region ◽  
E Coli ◽  
Transcription In Vitro

ABSTRACT We have characterized the Chlamydia trachomatisribosomal promoter, rRNA P1, by measuring the effect of substitutions and deletions on in vitro transcription with partially purifiedC. trachomatis RNA polymerase. Our analyses indicate that rRNA P1 contains potential −10 and −35 elements, analogous toEscherichia coli promoters recognized by E-ς70. We identified a novel AT-rich region immediately downstream of the −35 region. The effect of this region was specific for C. trachomatis RNA polymerase and strongly attenuated by single G or C substitutions. Upstream of the −35 region was an AT-rich sequence that enhanced transcription by C. trachomatis and E. coli RNA polymerases. We propose that this region functions as an UP element.

The role of divalent cations in the activation of the NADP+-specific isocitrate dehydrogenase from Pisum sativum L.

1977 ◽  
Vol 55 (9) ◽  
pp. 928-934 ◽  
Author(s):  
Robert J. Maloney ◽  
David T. Dennis
Keyword(s):  
Ionic Strength ◽  
Stability Constants ◽  
Isocitrate Dehydrogenase ◽  
Divalent Cations ◽  
Free Form ◽  
Saturation Kinetics ◽  
The Difference ◽  
The Stability ◽  
Kinetics Of

A divalent cation electrode was used to measure the stability constants (association constants) for the magnesium and manganese complexes of the substrates for the NADP+-specific isocitrate dehydrogenase (EC 1.1.1.42) from pea stems. At an ionic strength of 26.5 mM and at pH 7.4 the stability constants for the Mg2+–isocitrate and Mg2+–NADP+ complexes were 0.85 ± 0.2 and 0.43 ± 0.04 mM−1 respectively and for the Mn2+–isocitrate and Mn2+–NADP+ complexes they were 1.25 ± 0.07 and 0.75 ± 0.09 mM−1 respectively. At the same ionic strength but at pH 6.0 the Mg2+–NADPH and Mn2+–NADPH complexes had stability constants of 0.95 ± 0.23 and 1.79 ± 0.34 mM−1 respectively. Oxalosuccinate and α-ketoglutarate do not form measureable complexes under these conditions. Saturation kinetics of the enzyme with respect to isocitrate and metal ions are consistent with the metal–isocitrate complex being the substrate for the enzyme. NADP+ binds to the enzyme in the free form. Saturation kinetics of NADPH and Mn2+ indicate that the metal–NADPH complex is the substrate in the reverse reaction. In contrast the pig heart enzyme appears to bind free NADPH and Mn2+. A scheme for the reaction mechanism is presented and the difference between the reversibility of the NAD+ and NADP+ enzyme is discussed in relation to the stability of the NADH and NADPH metal complexes.

scholarly journals Crystal structures of γ-glutamyltranspeptidase in complex with inhibitors

2014 ◽  
Vol 70 (a1) ◽  
pp. C847-C847
Author(s):  
Kei Hirabayashi ◽  
Tomoyo Ida ◽  
Chunjie Li ◽  
Hideyuki Suzuki ◽  
Keiichi Fukuyama ◽  
...  
Keyword(s):  
Crystal Structures ◽  
Cellular Response ◽  
Physiological Role ◽  
Time Lapse ◽  
Binding Mode ◽  
E Coli ◽  
Glutathione Biosynthesis ◽  
Intracellular Glutathione ◽  
H Pylori

γ-Glutamyltranspeptidase (GGT; EC 2.3.2.2) is involved in the degradation of γ-glutamyl compounds such as glutathione (GSH; γ-glutamyl-cysteinyl-glycine) . A major physiological role of this enzyme is to cleave the extracellular GSH as a source of cysteine for intracellular glutathione biosynthesis. Another crucial role of GGT is to cleave glutathione-S-conjugates as a key step in detoxification of xenobiotics and drug metabolism. In mammals, GGT has been implicated in physiological disorders such as Parkinson's disease, other neurodegenerative diseases including Alzheimer's disease and cardiovascular disease. The indispensable roles played by GGT in GSH-mediated detoxification and cellular response to oxidative stress suggest that GGT is an attractive pharmaceutical target. We here report the binding mode of acivicin, a well-known glutamine antagonist, to B. subtilis GGT at 1.8 Å resolution showing that acivicin is bound to the Oγ atom of Thr403, the catalytic nucleophile of the enzyme, through its C3 atom [1]. The observed electron density around the C3 atom was best fitted to the planar and sp2 hybridized carbon, consistent with a simple nucleophilic substitution of Cl at the imino carbon by Oγ atom of Thr403. Furthermore, comparison of three bacterial enzymes, the GGTs from E. coli, H. pylori and B. subtilis in complex with acivicin, showed significant diversity in the orientation of the dihydroisoxazole ring among three GGTs. The differences are discussed in terms of the recognition of the α-amino and α-carboxy groups in preference to the dihydroisoxazole ring as observed in time-lapse soaking crystal structures of B. subtilis GGT with acivicin.

scholarly journals Role of Second-Largest RNA Polymerase I Subunit Zn-Binding Domain in Enzyme Assembly

2003 ◽  
Vol 2 (5) ◽  
pp. 1046-1052 ◽  
Author(s):  
Tatyana Naryshkina ◽  
Adrian Bruning ◽  
Olivier Gadal ◽  
Konstantin Severinov
Keyword(s):  
Rna Polymerase ◽  
Binding Domain ◽  
Rna Polymerases ◽  
Rna Polymerase I ◽  
Cysteine Residues ◽  
Per Se ◽  
Essential Function ◽  
Polymerase I

ABSTRACT The second-largest subunits of eukaryal RNA polymerases are similar to the β subunits of prokaryal RNA polymerases throughout much of their lengths. The second-largest subunits from eukaryal RNA polymerases contain a four-cysteine Zn-binding domain at their C termini. The domain is also present in archaeal homologs but is absent from prokaryal homologs. Here, we investigated the role of the C-terminal Zn-binding domain of Rpa135, the second-largest subunit of yeast RNA polymerase I. Analysis of nonfunctional Rpa135 mutants indicated that the Zn-binding domain is required for recruitment of the largest subunit, Rpa190, into the RNA polymerase I complex. Curiously, the essential function of the Rpa135 Zn-binding domain is not related to Zn2+ binding per se, since replacement of only one of the four cysteine residues with alanine led to the loss of Rpa135 function. Even more strikingly, replacement of all four cysteines with alanines resulted in functional Rpa135.

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