Shutdown in protein synthesis due to the expression of mini-genes in bacteria.
Mutants of Escherichia coli partially deficient in peptidyl-tRNA hydrolase are killed by the expression of certain very short open reading frames (mini-genes), encoded by the wild-type bar regions of phage lambda. According to the current hypothesis, protein synthesis is shut off, and the host cells die, after essential tRNA species become sequestered due to abnormal translation termination (drop-off) of mini-gene-encoded peptides as peptidyl-tRNA. Here we study variants of bar mini-genes, both in vivo and in vitro, in order to identify the structural elements that influence this inhibition of protein synthesis. Three parameters were measured during the expression of these variants: the rates of normal translation termination, peptidyl-tRNA dissociation from the ribosome and hydrolysis of peptidyl-tRNA by peptidyl-tRNA hydrolase were measured. Previous observations that RRF, EF-G and RF3 stimulated drop-off were confirmed and extended; stimulation by these factors can reach 30-fold. Both factor-stimulated and spontaneous drop-off depended on the nature of the stop signal. The degree of inhibition of cell growth following induction of mini-gene expression could be accounted for in terms of a toxicity index comprising the three parameters above. Inhibition was greatly reduced in cells lacking RF3. Mini-genes with more efficient Shine/Dalgarno sequences killed cells even with normal peptidyl-tRNA hydrolase activity. It is proposed that the retranslation by ribosomes of mini-gene transcripts with efficient ribosome binding (Shine/Dalgarno) sequences strongly contributes to the inhibitory effects of mini-gene expression on protein synthesis.
Dinçbas
V
V
Department of Cell and Molecular Biology, BMC, Uppsala, S-75124, Sweden.
Heurgué-Hamard
V
V
Buckingham
R H
RH
Karimi
R
R
Ehrenberg
M
M
D001426:Bacterial Proteins / Q000096:biosynthesis*; D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D002265:Carboxylic Ester Hydrolases / Q000235:genetics / Q000378:metabolism; D003062:Codon / Q000235:genetics; D018388:Codon, Terminator / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics* / Q000378:metabolism*; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009154:Mutation; D016366:Open Reading Frames; D010443:Peptide Chain Termination, Translational / Q000235:genetics; D012329:RNA, Bacterial / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D012270:Ribosomes / Q000378:metabolism
D001426:Bacterial Proteins; D003062:Codon; D018388:Codon, Terminator; D017931:DNA Primers; D012329:RNA, Bacterial; D012333:RNA, Messenger; D002265:Carboxylic Ester Hydrolases; C019674:aminoacyl-tRNA hydrolase