The frequency of transcriptional and translational errors at nonsense codons in the lacZ gene of Escherichia coli

Abstract

Nonsense alleles in the lacZ gene of E. coli do not completely eliminate enzyme activity as errors during protein synthesis allow some chains to be completed. The relative contributions of transcriptional and translational errors to this leakiness were investigated by two methods: the introduction of rho- alleles into extreme-polar mutants and the kinetics of beta-galactosidase induction. Virtually all the errors appeared to be transcriptional in the case of two extreme-polar and one non-polar mutation. These alleles should prove useful for further in vivo investigations of RNA polymerase accuracy. With two other non-polar alleles, transcriptional mistakes were low and translational ones high. The frequency of RNA polymerase errors was context-dependent and varied for different nonsense codons in the same position and for the same codon in different positions. The reasons why some alleles showed no activity due to translational errors could not be clearly established. However, increasing the rates of ribosomal errors from one such allele with streptomycin raised the contribution of ribosomal errors to activity markedly and non-linearly. Translational mistakes may give rise to active enzyme only if the monomers are formed at a rate sufficient for effective aggregation to the normal tetramer.

Visualizing high error levels during gene expression in living bacterial cells

“…Several studies have reported large fluctuations in the level of errors among different codons encoding the same protein (14,15,17,18), suggesting a different degree of susceptibility to errors along the coding sequence. In this view, evolutionary pressure that shapes codon usage could allow different genes to be prone to unequal error rates according to their cellular function (35).…”

“…In vivo measurements of errors in gene expression in bacteria have been estimated to occur at a rate of 10 −4 to 10 −5 per nucleotide during transcription and ≈10 −3 to 10 −4 per codon during translation. These error rates are significantly higher than that attributed to the highfidelity replication process, where the frequency of errors is as low as 10 −8 to 10 −9 per nucleotide (8,9,(12)(13)(14)(15)(16)(17)(18)(19)(20).…”

“…For the most part, in vivo error rates in bacterial gene expression have been estimated by measuring the activity of a mutated reporter gene (such as lacZ) (14,17,18) in a large cell population. However, the resultant measurements represent the average error rates, and thus do not detect the actual error level per cell and the variability, if any, among individuals.…”

Use of an in Vivo Reporter Assay to Test for Transcriptional and Translational Fidelity in Yeast

“…Parameters that influence translational fidelity include genetic background, antibiotics, and epigenetic states. Typically, the fidelity of translation has been considered less stringent than that of transcription (19,(23)(24)(25).In an effort to examine the contribution of transcriptional and translational errors and SII's potential role in proofreading, we have designed a reporter system to detect misincorporation events mediated by RNA polymerase II at an artificial stop codon engineered into a plasmid introduced into yeast. Using yeast strains with a deletion or a disruption of the SII-encoding gene (DST1), we were unable to find evidence that this cleavage-activating factor participates in proofreading.…”

“…Parameters that influence translational fidelity include genetic background, antibiotics, and epigenetic states. Typically, the fidelity of translation has been considered less stringent than that of transcription (19,(23)(24)(25).…”