scholarly journals ISOLATION, GENETIC MAPPING AND SOME CHARACTERIZATION OF A MUTATION IN ESCHERICHIA COLI THAT AFFECTS THE PROCESSING OF RIBONUCLEIC ACID

Genetics ◽  
1978 ◽  
Vol 90 (4) ◽  
pp. 659-671
Author(s):  
David Apirion
Keyword(s):  
Escherichia Coli ◽  
Temperature Sensitivity ◽  
Single Point ◽  
5S Rrna ◽  
23S Rrna ◽  
Single Point Mutation ◽  
Temperature Sensitive ◽  
Rnase E ◽  
Nonpermissive Temperature ◽  
Ribonuclease E

ABSTRACT Temperature-sensitive mutants were isolated from an rnc (RNase III-) strain of Escherichia coli, and their rRNA metabolism was analyzed on 3% polyacrylamide gels. One of these mutants was unable to produce 23S and 5S rRNAs at the nonpermissive temperature. When an rnc  + allele was introduced to this strain, it remained temperature sensitive. At the nonpermissive temperature, this strain could then produce 23S rRNA but was unable to make normal levels of 5S rRNA. In matings and transduction experiments, the defect in rRNA metabolism and temperature sensitivity behaved as a syndrome caused by a single point mutation, which was mapped at min 23.5 on the E. coli chromosome. This mutation probably affects an enzyme, ribonuclease E (RNase E), which introduces a cut in the nascent rRNA transcript between the 23S and the 5S rRNA cistrons. The mutation rne is recessive with respect to temperature sensitivity and the pattern of rRNA. Revertants able to grow at 43° and with normal metabolism of rRNA were isolated; genetic analysis showed that they do not contain the original rne mutation, suggesting that they were true revertants. By combining the rne mutation with an rnc mutation, double rnc rne strains were synthesized, which behaved very similarly to the original rnc strain from which the rne mutation was isolated. Such strains have RNA metabolism that is similar to that of rnc strains at permissive temperatures, but at the nonpermissive temperature they fail to synthesize p23, m23 and 5S rRNAs. Thus, the experiments reported here, together with previous studies, suggest the existence of a new processing ribonuclease activity in Escherichia coli, which is called ribonuclease E.

scholarly journals A Single Point Mutation, Asn16→Lys, Dictates the Temperature-Sensitivity of the Reovirus tsG453 Mutant

Viruses ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 289
Author(s):  
Kathleen K. M. Glover ◽  
Danica M. Sutherland ◽  
Terence S. Dermody ◽  
Kevin M. Coombs
Keyword(s):  
Amino Acid ◽  
Temperature Sensitivity ◽  
Reverse Genetics ◽  
Core Protein ◽  
Single Point ◽  
Single Amino Acid ◽  
Single Point Mutation ◽  
Temperature Sensitive ◽  
Amino Acid Substitutions ◽  
Gene Encoding

Studies of conditionally lethal mutants can help delineate the structure-function relationships of biomolecules. Temperature-sensitive (ts) mammalian reovirus (MRV) mutants were isolated and characterized many years ago. Two of the most well-defined MRV ts mutants are tsC447, which contains mutations in the S2 gene encoding viral core protein σ2, and tsG453, which contains mutations in the S4 gene encoding major outer-capsid protein σ3. Because many MRV ts mutants, including both tsC447 and tsG453, encode multiple amino acid substitutions, the specific amino acid substitutions responsible for the ts phenotype are unknown. We used reverse genetics to recover recombinant reoviruses containing the single amino acid polymorphisms present in ts mutants tsC447 and tsG453 and assessed the recombinant viruses for temperature-sensitivity by efficiency-of-plating assays. Of the three amino acid substitutions in the tsG453 S4 gene, Asn16-Lys was solely responsible for the tsG453ts phenotype. Additionally, the mutant tsC447 Ala188-Val mutation did not induce a temperature-sensitive phenotype. This study is the first to employ reverse genetics to identify the dominant amino acid substitutions responsible for the tsC447 and tsG453 mutations and relate these substitutions to respective phenotypes. Further studies of other MRV ts mutants are warranted to define the sequence polymorphisms responsible for temperature sensitivity.

scholarly journals Intragenic suppressors of temperature-sensitive rne mutations lead to the dissociation of RNase E activity on mRNA and tRNA substrates in Escherichia coli

2008 ◽  
Vol 36 (16) ◽  
pp. 5306-5318 ◽  
Author(s):  
T. Perwez ◽  
D. Hami ◽  
V. F. Maples ◽  
Z. Min ◽  
B.-C. Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Temperature Sensitive ◽  
Rnase E

scholarly journals A single-point mutation in HCF causes temperature-sensitive cell-cycle arrest and disrupts VP16 function.

1997 ◽  
Vol 11 (6) ◽  
pp. 726-737 ◽  
Author(s):  
H Goto ◽  
S Motomura ◽  
A C Wilson ◽  
R N Freiman ◽  
Y Nakabeppu ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Arrest ◽  
Point Mutation ◽  
Single Point ◽  
Single Point Mutation ◽  
Temperature Sensitive ◽  
Sensitive Cell ◽  
Cycle Arrest

scholarly journals The First Small-Molecule Inhibitors of Members of the Ribonuclease E Family

2015 ◽  
Vol 5 (1) ◽  
Author(s):  
Louise Kime ◽  
Helen A. Vincent ◽  
Deena M. A. Gendoo ◽  
Stefanie S. Jourdan ◽  
Colin W. G. Fishwick ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Small Molecules ◽  
Small Molecule ◽  
High Throughput Screening ◽  
Small Molecule Inhibitors ◽  
Rnase E ◽  
Ribonuclease E ◽  
New Antibiotics ◽  
Drug Leads ◽  
Number Of Bacteria

Abstract The Escherichia coli endoribonuclease RNase E is central to the processing and degradation of all types of RNA and as such is a pleotropic regulator of gene expression. It is essential for growth and was one of the first examples of an endonuclease that can recognise the 5′-monophosphorylated ends of RNA thereby increasing the efficiency of many cleavages. Homologues of RNase E can be found in many bacterial families including important pathogens, but no homologues have been identified in humans or animals. RNase E represents a potential target for the development of new antibiotics to combat the growing number of bacteria that are resistant to antibiotics in use currently. Potent small molecule inhibitors that bind the active site of essential enzymes are proving to be a source of potential drug leads and tools to dissect function through chemical genetics. Here we report the use of virtual high-throughput screening to obtain small molecules predicted to bind at sites in the N-terminal catalytic half of RNase E. We show that these compounds are able to bind with specificity and inhibit catalysis of Escherichia coli and Mycobacterium tuberculosis RNase E and also inhibit the activity of RNase G, a paralogue of RNase E.

scholarly journals Mutant Kinesin-2 Motor Subunits Increase Chromosome Loss

2005 ◽  
Vol 16 (8) ◽  
pp. 3810-3820 ◽  
Author(s):  
Mark S. Miller ◽  
Jessica M. Esparza ◽  
Andrew M. Lippa ◽  
Fordyce G. Lux ◽  
Douglas G. Cole ◽  
...  
Keyword(s):  
Chromosome Segregation ◽  
Single Point ◽  
Intraflagellar Transport ◽  
Single Amino Acid ◽  
Single Point Mutation ◽  
Chromosome Loss ◽  
Temperature Sensitive ◽  
Dominant Effect ◽  
Flagellar Assembly ◽  
Gene Maps

The Chlamydomonas anterograde intraflagellar transport motor, kinesin-2, is isolated as a heterotrimeric complex containing two motor subunits and a nonmotor subunit known as kinesin-associated polypeptide or KAP. One of the two motor subunits is encoded by the FLA10 gene. The sequence of the second motor subunit was obtained by mass spectrometry and sequencing. It shows 46.9% identity with the Fla10 motor subunit and the gene maps to linkage group XII/XIII near RPL9. The temperature-sensitive flagellar assembly mutants fla1 and fla8 are linked to this kinesin-2 motor subunit. In each strain, a unique single point mutation gives rise to a unique single amino acid substitution within the motor domain. The fla8 strain is named fla8-1 and the fla1 strain is named fla8-2. The fla8 and fla10 alleles show a chromosome loss phenotype. To analyze this chromosome loss phenotype, intragenic revertants of fla8-1, fla8-2, and fla10-14 were generated. The analysis of the mutants and the revertants demonstrates the importance of a pocket in the amino terminus of these motor subunits for both motor activity and for a novel, dominant effect on the fidelity of chromosome segregation.

scholarly journals Altered spectrum of multidrug resistance associated with a single point mutation in the Escherichia coli RND-type MDR efflux pump YhiV (MdtF)

2006 ◽  
Vol 59 (6) ◽  
pp. 1216-1222 ◽  
Author(s):  
Jürgen A. Bohnert ◽  
Sabine Schuster ◽  
Eva Fähnrich ◽  
Rainer Trittler ◽  
Winfried V. Kern
Keyword(s):  
Escherichia Coli ◽  
Multidrug Resistance ◽  
Point Mutation ◽  
Efflux Pump ◽  
Single Point ◽  
Single Point Mutation

scholarly journals Isolation and Characterization of β-Ketoacyl-Acyl Carrier Protein Reductase (fabG) Mutants of Escherichia coli and Salmonella enterica Serovar Typhimurium

2004 ◽  
Vol 186 (6) ◽  
pp. 1869-1878 ◽  
Author(s):  
Chiou-Yan Lai ◽  
John E. Cronan
Keyword(s):  
Escherichia Coli ◽  
Fatty Acid ◽  
Fatty Acid Synthesis ◽  
Salmonella Enterica ◽  
Acyl Carrier Protein ◽  
Acid Synthesis ◽  
Temperature Sensitive ◽  
Carrier Protein ◽  
Nonpermissive Temperature ◽  
Serovar Typhimurium

ABSTRACT FabG, β-ketoacyl-acyl carrier protein (ACP) reductase, performs the NADPH-dependent reduction of β-ketoacyl-ACP substrates to β-hydroxyacyl-ACP products, the first reductive step in the elongation cycle of fatty acid biosynthesis. We report the first documented fabG mutants and their characterization. By chemical mutagenesis followed by a tritium suicide procedure, we obtained three conditionally lethal temperature-sensitive fabG mutants. The Escherichia coli [fabG (Ts)] mutant contains two point mutations: A154T and E233K. The β-ketoacyl-ACP reductase activity of this mutant was extremely thermolabile, and the rate of fatty acid synthesis measured in vivo was inhibited upon shift to the nonpermissive temperature. Moreover, synthesis of the acyl-ACP intermediates of the pathway was inhibited upon shift of mutant cultures to the nonpermissive temperature, indicating blockage of the synthetic cycle. Similar results were observed for in vitro fatty acid synthesis. Complementation analysis revealed that only the E233K mutation was required to give the temperature-sensitive growth phenotype. In the two Salmonella enterica serovar Typhimurium fabG(Ts) mutants one strain had a single point mutation, S224F, whereas the second strain contained two mutations (M125I and A223T). All of the altered residues of the FabG mutant proteins are located on or near the twofold axes of symmetry at the dimer interfaces in this homotetrameric protein, suggesting that the quaternary structures of the mutant FabG proteins may be disrupted at the nonpermissive temperature.

Temperature Sensitivity of Cell Growth in Escherichia coli associated with the Temperature Sensitive R(KM) Factor

Nature ◽  
1968 ◽  
Vol 219 (5151) ◽  
pp. 284-285 ◽  
Author(s):  
YOSHIRO TERAWAKI ◽  
YOSHIHIRO KAKIZAWA ◽  
HISAO TAKAYASU ◽  
MASANOSUKE YOSHIKAWA
Keyword(s):  
Escherichia Coli ◽  
Cell Growth ◽  
Temperature Sensitivity ◽  
Temperature Sensitive

scholarly journals Gene-Specific Random Mutagenesis of Escherichia coli In Vivo: Isolation of Temperature-Sensitive Mutations in the Acyl Carrier Protein of Fatty Acid Synthesis

2006 ◽  
Vol 188 (1) ◽  
pp. 287-296 ◽  
Author(s):  
Nicholas R. De Lay ◽  
John E. Cronan
Keyword(s):  
Escherichia Coli ◽  
Fatty Acid ◽  
Fatty Acid Synthesis ◽  
Acyl Carrier Protein ◽  
Lipid Synthesis ◽  
Acid Synthesis ◽  
Temperature Sensitive ◽  
Carrier Protein ◽  
Nonpermissive Temperature ◽  
Overlap Extension

ABSTRACT Acyl carrier proteins (ACPs) are very small acidic proteins that play a key role in fatty acid and complex lipid synthesis. Moreover, recent data indicate that the acyl carrier protein of Escherichia coli has a large protein interaction network that extends beyond lipid synthesis. Despite extensive efforts over many years, no temperature-sensitive mutants with mutations in the structural gene (acpP) that encodes ACP have been isolated. We report the isolation of three such mutants by a new approach that utilizes error-prone PCR mutagenesis, overlap extension PCR, and phage λ Red-mediated homologous recombination and that should be generally applicable. These mutants plus other experiments demonstrate that ACP function is essential for the growth of E. coli. Each of the mutants was efficiently modified with the phosphopantetheinyl moiety essential for the function of ACP in lipid synthesis, and thus lack of function at the nonpermissive temperature cannot be attributed to a lack of prosthetic group attachment. All of the mutant proteins were largely stable at the nonpermissive temperature except the A68T/N73D mutant protein. Fatty acid synthesis in strains that carried the D38V or A68T/N73D mutations was inhibited upon a shift to the nonpermissive temperature and in the latter case declined to a small percentage of the rate of the wild-type strain.

scholarly journals Involvement of an Essential Gene, mviN, in Murein Synthesis in Escherichia coli

2008 ◽  
Vol 190 (21) ◽  
pp. 7298-7301 ◽  
Author(s):  
Azusa Inoue ◽  
Yoshimitsu Murata ◽  
Hiroshi Takahashi ◽  
Naoko Tsuji ◽  
Shingo Fujisaki ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Essential Gene ◽  
Temperature Sensitive ◽  
Nonpermissive Temperature ◽  
Sensitive Mutant ◽  
Temperature Sensitive Mutant ◽  
Mutant Cells

ABSTRACT We isolated a temperature-sensitive mutant with a mutation in mviN, an essential gene in Escherichia coli. At the nonpermissive temperature, mviN mutant cells swelled and burst. An intermediate in murein synthesis, polyprenyl diphosphate-N-acetylmuramic acid-(pentapeptide)-N-acetyl-glucosamine, accumulated in mutant cells. These results indicated that MviN is involved in murein synthesis.

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