Recycling of ribosomal complexes stalled at the step of elongation in Escherichia coli.
Translating ribosomes often stall during elongation. The stalled ribosomes are known to be recycled by tmRNA (SsrA)-mediated trans-translation. Another process that recycles the stalled ribosomes is characterized by peptidyl-tRNA release. However, the mechanism of peptidyl-tRNA release from the stalled ribosomes is not well understood. We used a defined system of an AGA-minigene containing a small open reading frame (ATG AGA AGA). Translation of the AGA-minigene mRNA is toxic to Escherichia coli because it stalls ribosomes during elongation and sequesters tRNA(Arg4) as a short-chain peptidyl-tRNA(Arg4) in the ribosomal P-site. We show that a ribosome recycling factor (RRF)-mediated process rescues the host from the AGA-minigene toxicity by releasing the peptidyl-tRNA(Arg4) from the ribosomes. The growth phenotypes of E. coli strains harboring mutant alleles of RRF and initiation factor 3 (IF3) genes and their consequences on lambdaimmP22 phage replication upon AGA-minigene expression reveal that IF3 facilitates the RRF-mediated processing of the stalled ribosomes. Additionally, we have designed a uracil DNA glycosylase gene construct, ung-stopless, whose expression is toxic to E. coli. We show that the RRF-mediated process also alleviates the ung-stopless construct-mediated toxicity to the host by releasing the ung mRNA from the ribosomes harboring long-chain peptidyl-tRNAs.
Singh
Nongmaithem Sadananda
NS
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore-560012, India.
Ahmad
Rais
R
Sangeetha
Ramachandran
R
Varshney
Umesh
U
D000483:Alleles; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*; D010957:Plasmids; D039665:Prokaryotic Initiation Factor-3 / Q000235:genetics / Q000502:physiology; D012329:RNA, Bacterial / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism; D012269:Ribosomal Proteins / Q000235:genetics / Q000378:metabolism; D012270:Ribosomes / Q000235:genetics* / Q000378:metabolism*
D039665:Prokaryotic Initiation Factor-3; D012329:RNA, Bacterial; D012346:RNA, Transfer, Amino Acyl; D012269:Ribosomal Proteins; C087267:ribosome releasing factor; C019037:tRNA, peptidyl-