scholarly journals Suppression of Nonsense Mutations Induced by Expression of an RNA Complementary to a Conserved Segment of 23S rRNA

1999 ◽  
Vol 181 (17) ◽  
pp. 5257-5262 ◽  
Author(s):  
Natalya S. Chernyaeva ◽  
Emanuel J. Murgola ◽  
Alexander S. Mankin
Keyword(s):  
Escherichia Coli ◽  
Reporter Genes ◽  
Chain Termination ◽  
23S Rrna ◽  
Specific Interactions ◽  
Peptidyl Transferase ◽  
Nonsense Mutations ◽  
Peptidyl Transferase Center ◽  
Codon Positions

ABSTRACT We identified a short RNA fragment, complementary to theEscherichia coli 23S rRNA segment comprising nucleotides 735 to 766 (in domain II), which when expressed in vivo results in the suppression of UGA nonsense mutations in two reporter genes. Neither UAA nor UAG mutations, examined at the same codon positions, were suppressed by the expression of this antisense rRNA fragment. Our results suggest that a stable phylogenetically conserved hairpin at nucleotides 736 to 760 in 23S rRNA, which is situated close to the peptidyl transferase center, may participate in one or more specific interactions during peptide chain termination.

scholarly journals 23S rRNA Nucleotides in the Peptidyl Transferase Center Are Essential for Tryptophanase Operon Induction

2009 ◽  
Vol 191 (11) ◽  
pp. 3445-3450 ◽  
Author(s):  
Rui Yang ◽  
Luis R. Cruz-Vera ◽  
Charles Yanofsky
Keyword(s):  
23S Rrna ◽  
Tryptophan Residue ◽  
Transferase Activity ◽  
Wild Type ◽  
Peptidyl Transferase ◽  
Peptidyl Transferase Center ◽  
Free Tryptophan ◽  
Operon Expression

ABSTRACT Distinct features of the ribosomal peptide exit tunnel are known to be essential for recognition of specific amino acids of a nascent peptidyl-tRNA. Thus, a tryptophan residue at position 12 of the peptidyl-tRNA TnaC-tRNAPro leads to the creation of a free tryptophan binding site within the ribosome at which bound tryptophan inhibits normal ribosome functions. The ribosomal processes that are inhibited are hydrolysis of TnaC-tRNAPro by release factor 2 and peptidyl transfer of TnaC of TnaC-tRNAPro to puromycin. These events are normally performed in the ribosomal peptidyl transferase center. In the present study, changes of 23S rRNA nucleotides in the 2585 region of the peptidyl transferase center, G2583A and U2584C, were observed to reduce maximum induction of tna operon expression by tryptophan in vivo without affecting the concentration of tryptophan necessary to obtain 50% induction. The growth rate of strains with ribosomes with either of these changes was not altered appreciably. In vitro analyses with mutant ribosomes with these changes showed that tryptophan was not as efficient in protecting TnaC-tRNAPro from puromycin action as wild-type ribosomes. However, added tryptophan did prevent sparsomycin action as it normally does with wild-type ribosomes. These findings suggest that these two mutational changes act by reducing the ability of ribosome-bound tryptophan to inhibit peptidyl transferase activity rather than by reducing the ability of the ribosome to bind tryptophan. Thus, the present study identifies specific nucleotides within the ribosomal peptidyl transferase center that appear to be essential for effective tryptophan induction of tna operon expression.

Variety of nonsense suppressor phenotypes associated with mutational changes at conserved sites in Escherichia coli ribosomal RNA

1995 ◽  
Vol 73 (11-12) ◽  
pp. 925-931 ◽  
Author(s):  
Emanuel J. Murgola ◽  
Frances T. Pagel ◽  
Kathryn A. Hijazi ◽  
Alexey L. Arkov ◽  
Wenbing Xu ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
16S Rrna ◽  
Ribosomal Rna ◽  
Intramolecular Interaction ◽  
Large Subunit ◽  
Chain Termination ◽  
Peptide Chain ◽  
Nonsense Suppression ◽  
23S Rrna ◽  
Nonsense Mutations

To screen for ribosomal RNA mutants defective in peptide chain termination, we have been looking for rRNA mutants that exhibit different patterns of suppression of nonsense mutations and that do not suppress missense mutations at the same positions in the same reporter gene. The rRNA mutations were induced by segment-directed randomly mutagenic PCR treatment of a cloned rrnB operon, followed by subcloning of the mutagenesis products and transformation of strains containing different nonsense mutations in the Escherichia coli trpA gene. To date, we have repeatedly obtained only two small sets of mutations, one in the 3′ domain of 16S rRNA, at five nucleotides out of the 610 mutagenized (two in helix 34 and three in helix 44), and the other in 23S rRNA at only four neighboring nucleotide positions (in a highly conserved hexanucleotide loop) within me 1.4 kb mutagenized segment. There is variety, however, in the suppression patterns of the mutants, ranging from suppression of UAG or UGA, through suppression of UAG and UGA, but not UAA, to suppression of all three termination codons. The two helices in 16S rRNA have previously been associated both physically and functionally with the decoding center of the ribosome. The 23S region is part of the binding site for the large subunit protein L11 and the antibiotic thiostrepton, both of which have been shown to affect peptide chain termination. Finally, we have demonstrated that the 23S mutant A1093, which suppresses trpA UGA mutations very efficiently, is lethal at temperatures above 36 °C (when highly expressed). This lethality is overcome by secondary 23S rRNA mutations in domain V. Our results suggest that specific regions of 16S and 23S rRNA are involved in peptide chain termination, that the lethality of A1093 is caused by high-level UGA suppression, and that intramolecular interaction between domains II and V of 23S rRNA may play a role in peptide chain termination at the UGA stop codon.Key words: 16S and 23S rRNAs, PCR mutagenesis, nonsense suppression, peptide chain termination, intramolecular interaction.

scholarly journals The Cfr rRNA Methyltransferase Confers Resistance to Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A Antibiotics

2006 ◽  
Vol 50 (7) ◽  
pp. 2500-2505 ◽  
Author(s):  
Katherine S. Long ◽  
Jacob Poehlsgaard ◽  
Corinna Kehrenberg ◽  
Stefan Schwarz ◽  
Birte Vester
Keyword(s):  
Escherichia Coli ◽  
Antimicrobial Agents ◽  
Susceptibility Testing ◽  
Drug Binding ◽  
Animal Origin ◽  
23S Rrna ◽  
Peptidyl Transferase ◽  
E Coli ◽  
Peptidyl Transferase Center ◽  
Drug Classes

ABSTRACT A novel multidrug resistance phenotype mediated by the Cfr rRNA methyltransferase is observed in Staphylococcus aureus and Escherichia coli. The cfr gene has previously been identified as a phenicol and lincosamide resistance gene on plasmids isolated from Staphylococcus spp. of animal origin and recently shown to encode a methyltransferase that modifies 23S rRNA at A2503. Antimicrobial susceptibility testing shows that S. aureus and E. coli strains expressing the cfr gene exhibit elevated MICs to a number of chemically unrelated drugs. The phenotype is named PhLOPSA for resistance to the following drug classes: Phenicols, Lincosamides, Oxazolidinones, Pleuromutilins, and Streptogramin A antibiotics. Each of these five drug classes contains important antimicrobial agents that are currently used in human and/or veterinary medicine. We find that binding of the PhLOPSA drugs, which bind to overlapping sites at the peptidyl transferase center that abut nucleotide A2503, is perturbed upon Cfr-mediated methylation. Decreased drug binding to Cfr-methylated ribosomes has been confirmed by footprinting analysis. No other rRNA methyltransferase is known to confer resistance to five chemically distinct classes of antimicrobials. In addition, the findings described in this study represent the first report of a gene conferring transferable resistance to pleuromutilins and oxazolidinones.

scholarly journals Evidence that the supE44 Mutation of Escherichia coli Is an Amber Suppressor Allele of glnX and that It Also Suppresses Ochre and Opal Nonsense Mutations

2010 ◽  
Vol 192 (22) ◽  
pp. 6039-6044 ◽  
Author(s):  
B. Singaravelan ◽  
B. R. Roshini ◽  
M. Hussain Munavar
Keyword(s):  
Escherichia Coli ◽  
Growth Phase ◽  
Stationary Growth Phase ◽  
Luciferase Assay ◽  
Logarithmic Phase ◽  
Nonsense Mutations ◽  
Translational Readthrough ◽  
Amber Suppressor ◽  
Nonsense Codons

ABSTRACT Translational readthrough of nonsense codons is seen not only in organisms possessing one or more tRNA suppressors but also in strains lacking suppressors. Amber suppressor tRNAs have been reported to suppress only amber nonsense mutations, unlike ochre suppressors, which can suppress both amber and ochre mutations, essentially due to wobble base pairing. In an Escherichia coli strain carrying the lacZU118 episome (an ochre mutation in the lacZ gene) and harboring the supE44 allele, suppression of the ochre mutation was observed after 7 days of incubation. The presence of the supE44 lesion in the relevant strains was confirmed by sequencing, and it was found to be in the duplicate copy of the glnV tRNA gene, glnX. To investigate this further, an in vivo luciferase assay developed by D. W. Schultz and M. Yarus (J. Bacteriol. 172:595-602, 1990) was employed to evaluate the efficiency of suppression of amber (UAG), ochre (UAA), and opal (UGA) mutations by supE44. We have shown here that supE44 suppresses ochre as well as opal nonsense mutations, with comparable efficiencies. The readthrough of nonsense mutations in a wild-type E. coli strain was much lower than that in a supE44 strain when measured by the luciferase assay. Increased suppression of nonsense mutations, especially ochre and opal, by supE44 was found to be growth phase dependent, as this phenomenon was only observed in stationary phase and not in logarithmic phase. These results have implications for the decoding accuracy of the translational machinery, particularly in stationary growth phase.

scholarly journals SrmB, a DEAD-box helicase involved in Escherichia coli ribosome assembly, is specifically targeted to 23S rRNA in vivo

2009 ◽  
Vol 37 (19) ◽  
pp. 6540-6549 ◽  
Author(s):  
Dmitrii Trubetskoy ◽  
Florence Proux ◽  
Frédéric Allemand ◽  
Marc Dreyfus ◽  
Isabelle Iost
Keyword(s):  
Escherichia Coli ◽  
23S Rrna ◽  
Ribosome Assembly ◽  
Dead Box

scholarly journals An rRNA Fragment and Its Antisense Can Alter Decoding of Genetic Information

1998 ◽  
Vol 180 (10) ◽  
pp. 2744-2748 ◽  
Author(s):  
Alexey L. Arkov ◽  
Alexander Mankin ◽  
Emanuel J. Murgola
Keyword(s):  
Antisense Rna ◽  
Genetic Information ◽  
Translation Termination ◽  
23S Rrna ◽  
Release Factor ◽  
Expression Library ◽  
Nonsense Mutations ◽  
New Approach ◽  
E Coli

ABSTRACT rRNA plays a central role in protein synthesis and is intimately involved in the initiation, elongation, and termination stages of translation. However, the mode of its participation in these reactions, particularly as to the decoding of genetic information, remains elusive. In this paper, we describe a new approach that allowed us to identify an rRNA segment whose function is likely to be related to translation termination. By screening an expression library of random rRNA fragments, we identified a fragment of the Escherichia coli 23S rRNA (nucleotides 74 to 136) whose expression caused readthrough of UGA nonsense mutations in certain codon contexts in vivo. The antisense RNA fragment produced a similar effect, but in neither case was readthrough of UAA or UAG observed. Since termination at UGA in E. coli specifically requires release factor 2 (RF2), our data suggest that the fragments interfere with RF2-dependent termination.

scholarly journals Independent Regulation of Two Genes in Escherichia coli by Tetracyclines and Tet Repressor Variants

2004 ◽  
Vol 186 (13) ◽  
pp. 4399-4401 ◽  
Author(s):  
Annette Kamionka ◽  
Miriam Sehnal ◽  
Oliver Scholz ◽  
Wolfgang Hillen
Keyword(s):  
Escherichia Coli ◽  
Cross Talk ◽  
Reporter Genes ◽  
The Other ◽  
Regulation System ◽  
The Novel ◽  
Wild Type ◽  
Tet Repressor

ABSTRACT We report a regulation system in Escherichia coli for independent regulation of two distinct reporter genes by application of Tet repressors with different specificities. One Tet repressor variant comprises wild-type tet operator (tetO) recognition and exclusive induction with the novel inducer 4-dedimethylamino-anhydrotetracycline. The other Tet repressor variant shows tetO-4C recognition and induction with tetracycline. We demonstrate that both variants are independently active in vivo and allow selective regulation of two genes in the same cell without any cross talk.

scholarly journals Acfr-Like Gene from Clostridium difficile Confers Multiple Antibiotic Resistance by the Same Mechanism as thecfrGene

2015 ◽  
Vol 59 (9) ◽  
pp. 5841-5843 ◽  
Author(s):  
Lykke H. Hansen ◽  
Birte Vester
Keyword(s):  
Escherichia Coli ◽  
Antibiotic Resistance ◽  
Clostridium Difficile ◽  
Primer Extension ◽  
23S Rrna ◽  
Multiple Antibiotic Resistance ◽  
Gene Sequences ◽  
Peptidyl Transferase ◽  
Content Type

ABSTRACTThe Cfr RNA methyltransferase causes multiple resistances to peptidyl transferase inhibitors by methylation of A2503 23S rRNA. Manycfr-like gene sequences in the databases code for unknown functions. This study confirms that a Cfr-like protein from aPeptoclostridium difficile(formerlyClostridium difficile) strain does function as a Cfr protein. The enzyme is expressed inEscherichia coliand shows elevated MICs for five classes of antibiotics. A primer extension stop indicates a modification at A2503 in 23S rRNA.

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