TY - JOUR
T1 - Protein synthesis factors (RF1, RF2, RF3, RRF, and tmRNA) and peptidyl-tRNA hydrolase rescue stalled ribosomes at sense codons
AU - Vivanco-Domínguez, Serafín
AU - Bueno-Martínez, José
AU - León-Avila, Gloria
AU - Iwakura, Nobuhiro
AU - Kaji, Akira
AU - Kaji, Hideko
AU - Guarneros, Gabriel
N1 - Funding Information:
This research was supported by Creative Biomedical Research Institute (to H.K. and A.K.) and by grant no. 54206 of Consejo Nacional de Ciencia y Tecnología, Mexico (to G.G.). S.V.-D. was supported by A.K. and H.K. We thank the following individuals who gave us strains or constructs used in this work: Drs. L. A. Isaksson and R. Buckingham for US486, Y. Nakamura and K. Ito for YN2971, Carol Gross for CAG12025, C. Yanofsky and R. Cruz-Vera for SVS1144, R. T. Sauer for W3110 ssrA∷Kan, and B. J. Del Tito, Jr. for pDC952.
PY - 2012/4/13
Y1 - 2012/4/13
N2 - During translation, ribosomes stall on mRNA when the aminoacyl-tRNA to be read is not readily available. The stalled ribosomes are deleterious to the cell and should be rescued to maintain its viability. To investigate the contribution of some of the cellular translation factors on ribosome rescuing, we provoked stalling at AGA codons in mutants that affected the factors and then analyzed the accumulation of oligopeptidyl (peptides of up to 6 amino acid residues, oligopep-)-tRNA or polypeptidyl (peptides of more than 300 amino acids in length, polypep-)-tRNA associated with ribosomes. Stalling was achieved by starvation for aminoacyl-tRNAArg4 upon induced expression of engineered lacZ (β-galactosidase) reporter gene harboring contiguous AGA codons close to the initiation codon or at internal codon positions together with minigene ATGAGATAA accompanied by reduced peptidyl-tRNA hydrolase (Pth). Our results showed accumulations of peptidyl-tRNA associated with ribosomes in mutants for release factors (RF1, RF2, and RF3), ribosome recycling factor (RRF), Pth, and transfer-messenger RNA (tmRNA), implying that each of these factors cooperate in rescuing stalled ribosomes. The role of these factors in ribosome releasing from the stalled complex may vary depending on the length of the peptide in the peptidyl-tRNA. RF3 and RRF rescue stalled ribosomes by "drop-off" of peptidyl-tRNA, while RF1, RF2 (in the absence of termination codon), or Pth may rescue by hydrolyzing the associated peptidyl-tRNA. This is followed by the disassembly of the ribosomal complex of tRNA and mRNA by RRF and elongation factor G.
AB - During translation, ribosomes stall on mRNA when the aminoacyl-tRNA to be read is not readily available. The stalled ribosomes are deleterious to the cell and should be rescued to maintain its viability. To investigate the contribution of some of the cellular translation factors on ribosome rescuing, we provoked stalling at AGA codons in mutants that affected the factors and then analyzed the accumulation of oligopeptidyl (peptides of up to 6 amino acid residues, oligopep-)-tRNA or polypeptidyl (peptides of more than 300 amino acids in length, polypep-)-tRNA associated with ribosomes. Stalling was achieved by starvation for aminoacyl-tRNAArg4 upon induced expression of engineered lacZ (β-galactosidase) reporter gene harboring contiguous AGA codons close to the initiation codon or at internal codon positions together with minigene ATGAGATAA accompanied by reduced peptidyl-tRNA hydrolase (Pth). Our results showed accumulations of peptidyl-tRNA associated with ribosomes in mutants for release factors (RF1, RF2, and RF3), ribosome recycling factor (RRF), Pth, and transfer-messenger RNA (tmRNA), implying that each of these factors cooperate in rescuing stalled ribosomes. The role of these factors in ribosome releasing from the stalled complex may vary depending on the length of the peptide in the peptidyl-tRNA. RF3 and RRF rescue stalled ribosomes by "drop-off" of peptidyl-tRNA, while RF1, RF2 (in the absence of termination codon), or Pth may rescue by hydrolyzing the associated peptidyl-tRNA. This is followed by the disassembly of the ribosomal complex of tRNA and mRNA by RRF and elongation factor G.
KW - peptidyl-tRNA hydrolase Pth
KW - release factors RF1, RF2, RF3
KW - rescue of stalled ribosomes
KW - ribosome recycling factor RRF
KW - transfer-messenger RNA tmRNA
UR - http://www.scopus.com/inward/record.url?scp=84858298050&partnerID=8YFLogxK
U2 - 10.1016/j.jmb.2012.02.008
DO - 10.1016/j.jmb.2012.02.008
M3 - Artículo
C2 - 22326347
SN - 0022-2836
VL - 417
SP - 425
EP - 439
JO - Journal of Molecular Biology
JF - Journal of Molecular Biology
IS - 5
ER -