UV-induced modifications in the peptidyl transferase loop of 23S rRNA dependent on binding of the streptogramin B antibiotic, pristinamycin IA

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.

Download Full-text

23S rRNA Similarity from Selection for Peptidyl Transferase Mimicry†

Biochemistry ◽

10.1021/bi963135j ◽

1997 ◽

Vol 36 (22) ◽

pp. 6614-6623 ◽

Cited By ~ 25

Author(s):

Mark Welch ◽

Irene Majerfeld ◽

Michael Yarus

Keyword(s):

23S Rrna ◽

Peptidyl Transferase ◽

Selection For

Download Full-text

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

Journal of Bacteriology ◽

10.1128/jb.00096-09 ◽

2009 ◽

Vol 191 (11) ◽

pp. 3445-3450 ◽

Cited By ~ 27

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.

Download Full-text

A ketolide resistance mutation in domain II of 23S rRNA reveals the proximity of hairpin 35 to the peptidyl transferase centre

Molecular Microbiology ◽

10.1046/j.1365-2958.1999.01203.x ◽

1999 ◽

Vol 31 (2) ◽

pp. 633-639 ◽

Cited By ~ 128

Author(s):

Liqun Xiong ◽

Sunita Shah ◽

Pascale Mauvais ◽

Alexander S. Mankin

Keyword(s):

Resistance Mutation ◽

23S Rrna ◽

Peptidyl Transferase ◽

Peptidyl Transferase Centre

Download Full-text

Linezolid and Tiamulin Cross-Resistance in Staphylococcus aureus Mediated by Point Mutations in the Peptidyl Transferase Center

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01015-07 ◽

2008 ◽

Vol 52 (5) ◽

pp. 1737-1742 ◽

Cited By ~ 50

Author(s):

Keith Miller ◽

Colin J. Dunsmore ◽

Colin W. G. Fishwick ◽

Ian Chopra

Keyword(s):

Staphylococcus Aureus ◽

Point Mutations ◽

Single Copy ◽

23S Rrna ◽

Cross Resistance ◽

Limiting Factor ◽

Peptidyl Transferase ◽

Inhibit Protein Synthesis ◽

Resistant Mutants ◽

Selection Of

ABSTRACT Oxazolidinone and pleuromutilin antibiotics are currently used in the treatment of staphylococcal infections. Although both antibiotics inhibit protein synthesis and have overlapping binding regions on 23S rRNA, the potential for cross-resistance between the two classes through target site mutations has not been thoroughly examined. Mutants of Staphylococcus aureus resistant to linezolid were selected and found to exhibit cross-resistance to tiamulin, a member of the pleuromutilin class of antibiotics. However, resistance was unidirectional because mutants of S. aureus selected for resistance to tiamulin did not exhibit cross-resistance to linezolid. This contrasts with the recently described PhLOPSA phenotype, which confers resistance to both oxazolidinones and pleuromutilins. The genotypes responsible for the phenotypes we observed were examined. Selection with tiamulin resulted in recovery of mutants with changes in the single-copy rplC gene (Gly155Arg, Ser158Leu, or Arg149Ser), whereas selection with linezolid led to recovery of mutants with changes (G2576U in 23S rRNA) in all five copies of the multicopy operon rrn. In contrast, cross-resistance to linezolid was exhibited by tiamulin-resistant mutants generated in a single-copy rrn knockout strains of Escherichia coli, illustrating that the copy number of 23S rRNA is the limiting factor in the selection of 23S rRNA tiamulin-resistant mutants. The interactions of linezolid and tiamulin with the ribosome were modeled to seek explanations for resistance to both classes in the 23S rRNA mutants and the lack of cross-resistance between tiamulin and linezolid following mutation in rplC.

Download Full-text

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

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01274-15 ◽

2015 ◽

Vol 59 (9) ◽

pp. 5841-5843 ◽

Cited By ~ 33

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.

Download Full-text

The Order Bacillales Hosts Functional Homologs of the WorrisomecfrAntibiotic Resistance Gene

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.00673-12 ◽

2012 ◽

Vol 56 (7) ◽

pp. 3563-3567 ◽

Cited By ~ 25

Author(s):

Lykke H. Hansen ◽

Mercè H. Planellas ◽

Katherine S. Long ◽

Birte Vester

Keyword(s):

Escherichia Coli ◽

Resistance Gene ◽

Gel Electrophoresis ◽

Inducible Promoter ◽

23S Rrna ◽

Peptidyl Transferase ◽

Whole Cell ◽

Content Type ◽

Cell Extracts ◽

Veterinary Importance

ABSTRACTThecfrgene encodes the Cfr methyltransferase that methylates a single adenine in the peptidyl transferase region of bacterial ribosomes. The methylation provides resistance to several classes of antibiotics that include drugs of clinical and veterinary importance. This paper describes a first step toward elucidating natural residences of the worrisomecfrgene and functionally similar genes. Threecfr-like genes from the orderBacillaleswere identified from BLAST searches and cloned into plasmids under the control of an inducible promoter. Expression of the genes was induced inEscherichia coli, and MICs for selected antibiotics indicate that thecfr-like genes confer resistance to PhLOPSa (phenicol, lincosamide, oxazolidinone, pleuromutilin, and streptogramin A) antibiotics in the same way as thecfrgene. In addition, modification at A2503 on 23S rRNA was confirmed by primer extension. Finally, expression of the Cfr-like proteins was verified by SDS gel electrophoresis of whole-cell extracts. The work shows thatcfr-like genes exist in the environment and thatBacillalesare natural residences ofcfr-like genes.

Download Full-text