scholarly journals Generation of pre-tRNAs from polycistronic operons is the essential function of RNase P in Escherichia coli

2020 ◽  
Vol 48 (5) ◽  
pp. 2564-2578 ◽  
Author(s):  
Bijoy K Mohanty ◽  
Ankit Agrawal ◽  
Sidney R Kushner
Keyword(s):  
Escherichia Coli ◽  
Rnase P ◽  
Ribonuclease P ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Essential Function ◽  
Sensitive Allele ◽  
Polymerase I ◽  
First Time

Abstract Ribonuclease P (RNase P) is essential for the 5′-end maturation of tRNAs in all kingdoms of life. In Escherichia coli, temperature sensitive mutations in either its protein (rnpA49) and or RNA (rnpB709) subunits lead to inviability at nonpermissive temperatures. Using the rnpA49 temperature sensitive allele, which encodes a partially defective RNase P at the permissive temperature, we show here for the first time that the processing of RNase P-dependent polycistronic tRNA operons to release pre-tRNAs is the essential function of the enzyme, since the majority of 5′-immature tRNAs can be aminoacylated unless their 5′-extensions ≥8 nt. Surprisingly, the failure of 5′-end maturation elicits increased polyadenylation of some pre-tRNAs by poly(A) polymerase I (PAP I), which exacerbates inviability. The absence of PAP I led to improved aminoacylation of 5′-immature tRNAs. Our data suggest a more dynamic role for PAP I in maintaining functional tRNA levels in the cell.

scholarly journals The effects of acridine orange on deoxyribonucleic acid in Escherichia coli

1978 ◽  
Vol 171 (3) ◽  
pp. 567-573 ◽  
Author(s):  
G R Barker ◽  
N Hardman
Keyword(s):  
Escherichia Coli ◽  
Acridine Orange ◽  
Dna Polymerase ◽  
Deoxyribonucleic Acid ◽  
Insoluble Fraction ◽  
Low Molecular Weight ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Dna Polymerase I ◽  
Polymerase I

1. Acridine Orange inhibits growth of Escherichia coli K12 when incubated at pH 7.9, but not at pH 7.4.2. At a non-permissive temperature for DNA polymerase I, Acridine Orange inhibits growth of a temperature-sensitive strain and also increases the rate of elimination of the F'-Lac plasmid. 3. DNA isolated from cells treated with Acridine Orange under conditions that inhibit growth contains material of low molecular weight, which is absent from DNA isolated from cells treated under conditions in which growth is not impaired. 4. Cells incubated with Acridine Orange at both pH 7.4 and 7.9 suffer degradation of DNA, as shown by loss of labelled DNA from the acid-insoluble fraction, which is not observed with untreated cells at either pH. 5. The results suggest that elimination of the F'-Lac plasmid by Acridine Orange requires inactivation of repair processes.

scholarly journals The mechanism of recA polA lethality: suppression by RecA-independent recombination repair activated by the lexA(Def) mutation in Escherichia coli.

Genetics ◽  
1995 ◽  
Vol 139 (4) ◽  
pp. 1483-1494 ◽  
Author(s):  
Y Cao ◽  
T Kogoma
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Dna Polymerase ◽  
Reca Protein ◽  
Minor Role ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Dna Polymerase I ◽  
Recombination Repair ◽  
Polymerase I

Abstract The mechanism of recA polA lethality in Escherichia coli has been studied. Complementation tests have indicated that both the 5'-->3' exonuclease and the polymerization activities of DNA polymerase I are essential for viability in the absence of RecA protein, whereas the viability and DNA replication of DNA polymerase I-defective cells depend on the recombinase activity of RecA. An alkaline sucrose gradient sedimentation analysis has indicated that RecA has only a minor role in Okazaki fragment processing. Double-strand break repair is proposed for the major role of RecA in the absence of DNA polymerase I. The lexA(Def)::Tn5 mutation has previously been shown to suppress the temperature-sensitive growth of recA200(Ts) polA25::spc mutants. The lexA(Def) mutation can alleviate impaired DNA synthesis in the recA200(Ts) polA25::spc mutant cells at the restrictive temperature. recF+ is essential for this suppression pathway. recJ and recQ mutations have minor but significant adverse effects on the suppression. The recA200(Ts) allele in the recA200(Ts) polA25::spc lexA(Def) mutant can be replaced by delta recA, indicating that the lexA(Def)-induced suppression is RecA independent. lexA(Def) reduces the sensitivity of delta recA polA25::spc cells to UV damage by approximately 10(4)-fold. lexA(Def) also restores P1 transduction proficiency to the delta recA polA25::spc mutant to a level that is 7.3% of the recA+ wild type. These results suggest that lexA(Def) activates a RecA-independent, RecF-dependent recombination repair pathway that suppresses the defect in DNA replication in recA polA double mutants.

scholarly journals Spt6 Is Essential for rRNA Synthesis by RNA Polymerase I

2015 ◽  
Vol 35 (13) ◽  
pp. 2321-2331 ◽  
Author(s):  
Krysta L. Engel ◽  
Sarah L. French ◽  
Olga V. Viktorovskaya ◽  
Ann L. Beyer ◽  
David A. Schneider
Keyword(s):  
Rna Polymerase ◽  
Transcription Initiation ◽  
Initiation Factor ◽  
Rrna Synthesis ◽  
Temperature Sensitive ◽  
Protein Levels ◽  
Pol I ◽  
Low Levels ◽  
Sensitive Allele ◽  
Polymerase I

Spt6 (suppressor ofTy6) has many roles in transcription initiation and elongation by RNA polymerase (Pol) II. These effects are mediated through interactions with histones, transcription factors, and the RNA polymerase. Two lines of evidence suggest that Spt6 also plays a role in rRNA synthesis. First, Spt6 physically associates with a Pol I subunit (Rpa43). Second, Spt6 interacts physically and genetically with Spt4/5, which directly affects Pol I transcription. Utilizing a temperature-sensitive allele,spt6-1004, we show that Spt6 is essential for Pol I occupancy of the ribosomal DNA (rDNA) and rRNA synthesis. Our data demonstrate that protein levels of an essential Pol I initiation factor, Rrn3, are reduced when Spt6 is inactivated, leading to low levels of Pol I-Rrn3 complex. Overexpression ofRRN3rescues Pol I-Rrn3 complex formation; however, rRNA synthesis is not restored. These data suggest that Spt6 is involved in either recruiting the Pol I-Rrn3 complex to the rDNA or stabilizing the preinitiation complex. The findings presented here identify an unexpected, essential role for Spt6 in synthesis of rRNA.

Eight UCA codons differentially affect the expression of the lacZ gene in the divE42 mutant of Escherichia coli

2000 ◽  
Vol 46 (6) ◽  
pp. 577-583 ◽  
Author(s):  
Takashi Kubo ◽  
Toshiko Aiso ◽  
Reiko Ohki
Keyword(s):  
Gene Expression ◽  
Escherichia Coli ◽  
Temperature Sensitive ◽  
Lacz Gene ◽  
Permissive Temperature ◽  
Wild Type ◽  
Double Mutation ◽  
Mutant Genes ◽  
Beta Galactosidase ◽  
Serine Codons

In the divE mutant, which has a temperature-sensitive mutation in the tRNA1Ser gene, the synthesis of beta-galactosidase is dramatically decreased at the non-permissive temperature. In Escherichia coli, the UCA codon is only recognized by tRNA1Ser. Several genes containing UCA codons are normally expressed at 42°C in the divE mutant. Therefore, it is unlikely that the defect is due to the general translational deficiency of the mutant tRNA1Ser. In this study, we constructed mutant lacZ genes, in which one or several UCA codons at eight positions were replaced with other serine codons such as UCU or UCC, and we examined the expression of these mutant genes in the divE mutant. We found that a single UCA codon at position 6 or 462 was sufficient to cause the same level of reduced beta-galactosidase synthesis as that of the wild-type lacZ gene, and that the defect in beta-galactosidase synthesis was accompanied by a low level of lacZ mRNA. It was also found that introduction of an rne-1 pnp-7 double mutation restored the expression of mutant lacZ genes with only UCA codons at position 6 or 462. A polarity suppressor mutation in the rho gene had no effect on the defect in lacZ gene expression in the divE mutant. We propose a model to explain these results.Key words: divE gene, tRNA1Ser, lacZ gene expression, UCA codon.

scholarly journals The role of ribonuclease II in the maturation of precursor 16S ribosomal ribonucleic acid in Escherichia coli

1974 ◽  
Vol 140 (3) ◽  
pp. 443-450 ◽  
Author(s):  
John R. Dean ◽  
John Sykes
Keyword(s):  
Escherichia Coli ◽  
Enzyme Activity ◽  
Maximum Activity ◽  
Exponential Phase ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Experimental Conditions ◽  
Inhibition Of Growth ◽  
Ribosomal Ribonucleic Acid

The suggested involvement of ribonuclease II in the maturation of rRNA has been examined directly by determining the activity of the enzyme and the amount of p16S rRNA in cell-free extracts from Escherichia coli A19 and its temperature-sensitive derivative N464 grown under experimental conditions designed to vary the amounts of enzyme and precursor independently. In strain A19 the enzyme showed maximum activity in circumstances where the amount of p16S rRNA was normal (e.g. exponential-phase cells) or raised eight times (e.g. during inhibition of growth by methionine starvation of the relaxed auxotroph or by chloramphenicol or puromycin treatment). In strain N464 at the non-permissive temperature the ribonuclease II activity may be decreased by 50% without effect upon the amount of p16S rRNA, whereas in methionine starvation of this strain the enzyme activity is at a maximum and the p16S rRNA is eight times that in exponential-phase cells. These observations are discussed in relation to the previously implied role of ribonuclease II in the maturation of rRNA within ribosome precursors.

fusB is an allele of nadD, encoding nicotinate mononucleotide adenylyltransferase in Escherichia coli

Microbiology ◽  
2003 ◽  
Vol 149 (9) ◽  
pp. 2427-2433 ◽  
Author(s):  
Martin Stancek ◽  
Leif A. Isaksson ◽  
Monica Rydén-Aulin
Keyword(s):  
Escherichia Coli ◽  
Amino Acids ◽  
Temperature Sensitivity ◽  
Minimal Medium ◽  
Synthetic Medium ◽  
Normal Growth ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Atp Binding Site ◽  
Pleiotropic Phenotype

Isolation of the temperature-sensitive Escherichia coli mutant 72c has been described previously. The mutant allele was named fusB and causes a pleiotropic phenotype, the most striking features of which, besides temperature sensitivity, are the inability to grow on synthetic medium and supersensitivity to trimethoprim, an antibiotic that inhibits the C1 metabolism. This work shows that the fusB mutation is a frameshift mutation in the nadD gene that encodes nicotinate mononucleotide adenylyltransferase. The frameshift leads to a change of the last 10 amino acids and an addition of 17 amino acids. This lesion, renamed nadD72, leads to very little NAD+ and NADPH synthesis at the permissive temperature and essentially no synthesis at the non-permissive temperature. As a comparison, a new mutation in the nadD gene, with an amino acid change in the ATP-binding site, has been isolated. Its NAD+ synthesis is decreased at 30 °C but the level is still sufficient to support normal growth. At 42 °C, NAD+ synthesis is reduced further, which leads to temperature sensitivity on minimal medium. This mutation was designated nadD74. Thus, a small decrease in NAD+ levels affects ability to grow on minimal medium at 42 °C, while a large decrease leads to a more pleiotropic phenotype.

scholarly journals MEIOTIC RECOMBINATION AND DNA SYNTHESIS IN A NEW CELL CYCLE MUTANT OF SACCHAROMYCES CEREVISIAE

Genetics ◽  
1978 ◽  
Vol 90 (1) ◽  
pp. 49-68
Author(s):  
Yona Kassir ◽  
Giora Simchen
Keyword(s):  
Cell Cycle ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Nuclear Division ◽  
Temperature Shift ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Permissive Temperature ◽  
Essential Function ◽  
Normal Meiosis

ABSTRACT Vegetative cells carrying the new temperature-sensitive mutation cdc40 arrest at the restrictive temperature with a medial nuclear division phenotype. DNA replication is observed under these conditions, but most cells remain sensitive to hydroxyurea and do not complete the ongoing cell cycle if the drug is present during release from the temperature block. It is suggested that the cdc40 lesion affects an essential function in DNA synthesis. Normal meiosis is observed at the permissive temperature in cdc40 homozygotes. At the restrictive temperature, a full round of premeiotic DNA replication is observed, but neither commitment to recombination nor later meiotic events occur. Meiotic cells that are already committed to the recombination process at the permissive temperature do not complete it if transferred to the restrictive temperature before recombination is realized. These temperature shift-up experiments demonstrate that the CDC40 function is required for the completion of recombination events, as well as for the earlier stage of recombination commitment. Temperature shift-down experiments with cdc40 homozygotes suggest that meiotic segregation depends on the final events of recombination rather than on commitment to recombination.

scholarly journals Role of Escherichia coli DNA Polymerase I in Conferring Viability upon the dnaN159 Mutant Strain

2007 ◽  
Vol 189 (13) ◽  
pp. 4688-4695 ◽  
Author(s):  
Robert W. Maul ◽  
Laurie H. Sanders ◽  
James B. Lim ◽  
Rosemary Benitez ◽  
Mark D. Sutton
Keyword(s):  
Escherichia Coli ◽  
Dna Polymerase ◽  
Temperature Sensitive ◽  
Mutant Form ◽  
Sliding Clamp ◽  
Pol I ◽  
Pol Iii ◽  
Polymerase I

ABSTRACT The Escherichia coli dnaN159 allele encodes a mutant form of the β-sliding clamp (β159) that is impaired for interaction with the replicative DNA polymerase (Pol), Pol III. In addition, strains bearing the dnaN159 allele require functional Pol I for viability. We have utilized a combination of genetic and biochemical approaches to characterize the role(s) played by Pol I in the dnaN159 strain. Our findings indicate that elevated levels of Pol I partially suppress the temperature-sensitive growth phenotype of the dnaN159 strain. In addition, we demonstrate that the β clamp stimulates the processivity of Pol I in vitro and that β159 is impaired for this activity. The reduced ability of β159 to stimulate Pol I in vitro correlates with our finding that single-stranded DNA (ssDNA) gap repair is impaired in the dnaN159 strain. Taken together, these results suggest that (i) the β clamp-Pol I interaction may be important for proper Pol I function in vivo and (ii) in the absence of Pol I, ssDNA gaps may persist in the dnaN159 strain, leading to lethality of the dnaN159 ΔpolA strain.

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