Inhibition of translation and cell growth by minigene expression.
A random five-codon gene library was used to isolate minigenes whose expression causes cell growth arrest. Eight different deleterious minigenes were isolated, five of which had in-frame stop codons; the predicted expressed peptides ranged in size from two to five amino acids. Mutational analysis demonstrated that translation of the inhibitory minigenes is essential for growth arrest. Pulse-labeling experiments showed that expression of at least some of the selected minigenes results in inhibition of cellular protein synthesis. Expression of the deleterious minigenes in cells deficient in peptidyl-tRNA hydrolase causes accumulation of families of peptidyl-tRNAs corresponding to the last minigene codon; the inhibitory action of minigene expression could be suppressed by overexpression of the tRNA corresponding to the last sense codon in the minigene. Experimental data are compatible with the model that the deleterious effect of minigene expression is mediated by depletion of corresponding pools of free tRNAs.
Tenson
T
T
Center for Pharmaceutical Biotechnology, University of Illinois, Chicago, Illinois 60607, USA.
Herrera
J V
JV
Kloss
P
P
Guarneros
G
G
Mankin
A S
AS
D001483:Base Sequence; D002265:Carboxylic Ester Hydrolases / Q000235:genetics / Q000378:metabolism; D003001:Cloning, Molecular; D003062:Codon; D018388:Codon, Terminator; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics* / Q000254:growth & development*; D015870:Gene Expression; D005822:Genetic Vectors; D015698:Genomic Library; D016297:Mutagenesis, Site-Directed; D010455:Peptides / Q000737:chemistry; D010957:Plasmids; D016133:Polymerase Chain Reaction; D014176:Protein Biosynthesis; D012329:RNA, Bacterial / Q000235:genetics; D012346:RNA, Transfer, Amino Acyl / Q000235:genetics
D003062:Codon; D018388:Codon, Terminator; D017931:DNA Primers; D010455:Peptides; D012329:RNA, Bacterial; D012346:RNA, Transfer, Amino Acyl; C019037:tRNA, peptidyl-; D002265:Carboxylic Ester Hydrolases; C019674:aminoacyl-tRNA hydrolase