Kinetics of paused ribosome recycling in Escherichia coli.
The bacterial tmRNA.SmpB system recycles stalled translation complexes in a process termed 'ribosome rescue.' tmRNA.SmpB specifically recognizes ribosomes that are paused at or near the 3' end of truncated mRNA; therefore, nucleolytic mRNA processing is required before paused ribosomes can be rescued from full-length transcripts. Here, we examine the recycling of ribosomes paused on both full-length and truncated mRNAs. Peptidyl-tRNAs corresponding to each paused translation complex were identified, and their turnover kinetics was used to estimate the half-lives of paused ribosomes in vivo. Ribosomes were paused at stop codons on full-length mRNA using a nascent peptide motif that interferes with translation termination and elicits tmRNA.SmpB activity. Peptidyl-tRNA turnover from these termination-paused ribosomes was slightly more rapid in tmRNA(+) cells (T(1/2)=22+/-2.2 s) than in DeltatmRNA cells (T(1/2)=32+/-1.6 s). Overexpression of release factor (RF) 1 greatly accelerated peptidyl-tRNA turnover from termination-paused ribosomes in both tmRNA(+) and DeltatmRNA cells, whereas other termination factors had little or no effect on recycling. In contrast to inefficient translation termination, ribosome recycling from truncated transcripts lacking in-frame stop codons was dramatically accelerated by tmRNA.SmpB. However, peptidyl-tRNA still turned over from nonstop-paused ribosomes at a significant rate (t(1/2)=61+/-7.3 s) in DeltatmRNA cells. Overexpression of RF-1, RF-3, and ribosome recycling factor in DeltatmRNA cells failed to accelerate ribosome recycling from nonstop mRNA. These results indicate that tmRNA.SmpB activity is rate limited by mRNA cleavage, and that RF-3 and ribosome recycling factor do not constitute a tmRNA-independent rescue pathway, as previously suggested. Peptidyl-tRNA turnover from nonstop-paused ribosomes in DeltatmRNA cells suggests the existence of another uncharacterized ribosome rescue pathway.
Janssen
Brian D
BD
Department of Molecular, Cellular, and Developmental Biology, University of California, Santa Barbara, CA 93106-9610, USA.
Hayes
Christopher S
CS
D004926:Escherichia coli / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D009690:Nucleic Acid Conformation; D010443:Peptide Chain Termination, Translational; D010454:Peptide Termination Factors / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012346:RNA, Transfer, Amino Acyl / Q000378:metabolism*; D012270:Ribosomes / Q000378:metabolism*
D029968:Escherichia coli Proteins; D010454:Peptide Termination Factors; D012333:RNA, Messenger; D012346:RNA, Transfer, Amino Acyl; C489005:prfA protein, E coli