Methylation of bacterial release factors RF1 and RF2 is required for normal translation termination in vivo.
Bacterial release factors RF1 and RF2 are methylated on the Gln residue of a universally conserved tripeptide motif GGQ, which interacts with the peptidyl transferase center of the large ribosomal subunit, triggering hydrolysis of the ester bond in peptidyl-tRNA and releasing the newly synthesized polypeptide from the ribosome. In vitro experiments have shown that the activity of RF2 is stimulated by Gln methylation. The viability of Escherichia coli K12 strains depends on the integrity of the release factor methyltransferase PrmC, because K12 strains are partially deficient in RF2 activity due to the presence of a Thr residue at position 246 instead of Ala. Here, we study in vivo RF1 and RF2 activity at termination codons in competition with programmed frameshifting and the effect of the Ala-246 --> Thr mutation. PrmC inactivation reduces the specific termination activity of RF1 and RF2(Ala-246) by approximately 3- to 4-fold. The mutation Ala-246 --> Thr in RF2 reduces the termination activity in cells approximately 5-fold. After correction for the decrease in level of RF2 due to the autocontrol of RF2 synthesis, the mutation Ala-246 --> Thr reduced RF2 termination activity by approximately 10-fold at UGA codons and UAA codons. PrmC inactivation had no effect on cell growth in rich media but reduced growth considerably on poor carbon sources. This suggests that the expression of some genes needed for optimal growth under such conditions can become growth limiting as a result of inefficient translation termination.
Mora
Liliana
L
CNRS, UPR 9073, Institut de Biologie Physico-Chimique, 13 rue Pierre et Marie Curie, Paris 75005, France.
Heurgué-Hamard
Valérie
V
de Zamaroczy
Miklos
M
Kervestin
Stephanie
S
Buckingham
Richard H
RH
D020816:Amino Acid Motifs / Q000502:physiology; D019943:Amino Acid Substitution; D018388:Codon, Terminator / Q000235:genetics / Q000378:metabolism; D048168:Escherichia coli K12 / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D018965:Frameshifting, Ribosomal / Q000502:physiology; D008745:Methylation; D020125:Mutation, Missense; D010443:Peptide Chain Termination, Translational / Q000502:physiology*; D010454:Peptide Termination Factors / Q000235:genetics / Q000378:metabolism*; D010458:Peptidyl Transferases / Q000235:genetics / Q000378:metabolism; D011496:Protein Methyltransferases / Q000235:genetics / Q000378:metabolism; D011499:Protein Processing, Post-Translational / Q000502:physiology*; D012346:RNA, Transfer, Amino Acyl / Q000235:genetics / Q000378:metabolism*; D054681:Ribosome Subunits, Large, Bacterial / Q000235:genetics / Q000378:metabolism
D018388:Codon, Terminator; D029968:Escherichia coli Proteins; D010454:Peptide Termination Factors; D012346:RNA, Transfer, Amino Acyl; C489005:prfA protein, E coli; C489006:prfB protein, E coli; C019037:tRNA, peptidyl-; D011496:Protein Methyltransferases; C092404:prmC protein, E coli; D010458:Peptidyl Transferases