Non-stressful death of 23S rRNA mutant G2061C defective in puromycin reaction.

Catalysis of peptide bond formation in the peptidyl transferase center is a major enzymatic activity of the ribosome. Mutations limiting peptidyl transferase activity are mostly lethal. However, cellular processes triggered by peptidyl transferase deficiency in the bacterial cell are largely unknown.

Methyltransferase that modifies guanine 966 of the 16 S rRNA: functional identification and tertiary structure.

N(2)-Methylguanine 966 is located in the loop of Escherichia coli 16 S rRNA helix 31, forming a part of the P-site tRNA-binding pocket. We found yhhF to be a gene encoding for m(2)G966 specific 16 S rRNA methyltransferase. Disruption of the yhhF gene by kanamycin resistance marker leads to a loss of modification at G966.

Identification of Escherichia coli m2G methyltransferases: II. The ygjO gene encodes a methyltransferase specific for G1835 of the 23 S rRNA.

Escherichia coli ribosomal RNA contains five guanosine residues methylated at N2. The ygjO gene was previously predicted to methylate 16 S rRNA residue G966 due to its high sequence homology with the protein RsmC, responsible for G1207 methylation. We have identified the target of YgjO as being m2G1835 of the 23 S rRNA and not m2G966 of the 16 S rRNA as expected.

Identification of Escherichia coli m2G methyltransferases: I. the ycbY gene encodes a methyltransferase specific for G2445 of the 23 S rRNA.

N2-methylguanosine 2445 of the 23 S rRNA is located in a cluster of modified nucleotides concentrated at the peptidyl transferase center of the ribosome. Here we describe the identification of a gene, ycbY, as encoding an enzyme responsible for methylation of G2445. Knock-out of the ycbY gene leads to loss of modification at G2445 as revealed by reverse transcription.

Function of the ribosomal E-site: a mutagenesis study.

Ribosomes synthesize proteins according to the information encoded in mRNA. During this process, both the incoming amino acid and the nascent peptide are bound to tRNA molecules. Three binding sites for tRNA in the ribosome are known: the A-site for aminoacyl-tRNA, the P-site for peptidyl-tRNA and the E-site for the deacylated tRNA leaving the ribosome.

The conserved A-site finger of the 23S rRNA: just one of the intersubunit bridges or a part of the allosteric communication pathway?

During the translocation of tRNAs and mRNA relative to the ribosome, the B1a, B1b and B1c bridges undergo the most extensive conformational changes among the bridges between the large and the small ribosomal subunits. The B1a bridge, also called the "A-site finger" (ASF), is formed by the 23S rRNA helix 38, which is located right above the ribosomal A-site. Here, we deleted part of the ASF so that the B1a intersubunit bridge could not be formed (DeltaB1a).

Alteration in location of a conserved GTPase-associated center of the ribosome induced by mutagenesis influences the structure of peptidyltransferase center and activity of elongation factor G.

Translocation catalyzed by elongation factor G occurs after the peptidyltransferase reaction on the large ribosomal subunit. Deacylated tRNA in the P-site stimulates multiple turnover GTPase activity of EF-G. We suggest that the allosteric signal from the peptidyltransferase center that activates EF-G may involve the alteration in the conformation of elongation factor binding center of the ribosome.