Connections between translation, transcription and replication error-rates

To monitor inaccuracy in gene expression in living cells, we designed an experimental system in the bacterium Bacillus subtilis whereby spontaneous errors can be visualized and quantified at a single-cell level. Our strategy was to introduce mutations into a chromosomally encoded gfp allele, such that errors in protein production are reported in real time by the formation of fluorescent GFP molecules. The data reveal that the amount of errors can greatly exceed previous estimates, and that the error rate increases dramatically at lower temperatures and during stationary phase. Furthermore, we demonstrate that when facing an antibiotic threat, an increase in error level is sufficient to allow survival of bacteria carrying a mutated antibiotic-resistance gene. We propose that bacterial gene expression is error prone, frequently yielding protein molecules that differ slightly from the sequence specified by their DNA, thus generating a cellular reservoir of nonidentical protein molecules. This variation may be a key factor in increasing bacterial fitness, expanding the capability of an isogenic population to face environmental challenges.Bacillus subtilis | translational errors | variations in living cells | translation fidelity D NA is duplicated with remarkable fidelity to ensure that accurate genetic information is transmitted from one generation to the next. This information is passed from DNA to RNA and from RNA to protein during gene expression; however, the accuracy of these downstream events is relatively less understood. Although RNA and proteins are generally considered short-lived noninherited molecules, several diseases are now known to arise from errors occurring during transcription and translation (1-3), implying that faithful transfer of genetic information from DNA to proteins is crucial for maintaining proper cellular functions.

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confidence: 84%

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confidence: 99%

Visualizing high error levels during gene expression in living bacterial cells

Meyerovich

Mamou

Ben-Yehuda

2010

“…RNA polymerase (RNAP) incorporates nucleotides into nascent RNA chains at rates of 10-70 s −1 (1,2), but only inserts the wrong nucleotide approximately once per 100,000 bases, on average (3). Although the energetics of base-pairing is fundamental to transcription fidelity, the discrimination for correctly matched nucleotide substrates is kinetically controlled, and accomplished by active site conformational changes centered on the trigger loop (TL) (4-7).…”

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confidence: 99%

Trigger loop dynamics mediate the balance between the transcriptional fidelity and speed of RNA polymerase II

Larson

Zhou

Kaplan

et al. 2012

123

187

During transcription, RNA polymerase II (RNAPII) must select the correct nucleotide, catalyze its addition to the growing RNA transcript, and move stepwise along the DNA until a gene is fully transcribed. In all kingdoms of life, transcription must be finely tuned to ensure an appropriate balance between fidelity and speed. Here, we used an optical-trapping assay with high spatiotemporal resolution to probe directly the motion of individual RNAPII molecules as they pass through each of the enzymatic steps of transcript elongation. We report direct evidence that the RNAPII trigger loop, an evolutionarily conserved protein subdomain, serves as a master regulator of transcription, affecting each of the three main phases of elongation, namely: substrate selection, translocation, and catalysis. Global fits to the force-velocity relationships of RNAPII and its trigger loop mutants support a Brownian ratchet model for elongation, where the incoming NTP is able to bind in either the pre-or posttranslocated state, and movement between these two states is governed by the trigger loop. Comparison of the kinetics of pausing by WT and mutant RNAPII under conditions that promote base misincorporation indicate that the trigger loop governs fidelity in substrate selection and mismatch recognition, and thereby controls aspects of both transcriptional accuracy and rate.optical trap | optical tweezers | Pol II T he transcription of genetic information stably encoded in DNA into a transient RNA message occurs with remarkable fidelity. RNA polymerase (RNAP) incorporates nucleotides into nascent RNA chains at rates of 10-70 s −1 (1, 2), but only inserts the wrong nucleotide approximately once per 100,000 bases, on average (3). Although the energetics of base-pairing is fundamental to transcription fidelity, the discrimination for correctly matched nucleotide substrates is kinetically controlled, and accomplished by active site conformational changes centered on the trigger loop (TL) (4-7). The TL is an evolutionarily conserved mobile element that stabilizes substrate NTPs in the active site of a variety of multisubunit RNA polymerases, including eukaryotic RNAP I, II, and III, as well as bacterial and archaeal RNAP (8). In the case of RNAPII, alteration of the TL has important consequences for activity and fidelity. TL residue His1085, for example, interacts with the bound NTP substrate, and is fully conserved among multisubunit RNA polymerases. The importance of this residue is underscored by the lethality of the H1085A substitution in yeast (7) and catalytic defects resulting from substitutions at this position for both the yeast and bacterial enzymes (7, 9-11). Additionally, a substitution for residue Glu1103 of the TL has been found not only to promote nucleotide misincorporation (6, 7), but also to increase the elongation rate (6,7,12), properties that are jointly consistent with the notion of kinetic proofreading (13). To explore further the interplay between elongation rate and transcriptional fidelity, we determined the effec...

“…In E. coli cells under standard growth conditions, the error rate of transcription is 10 Ϫ5 . Theoretical considerations argue in favor of the importance of transcriptional fidelity for the cell (5). Several experimental approaches have confirmed this importance in vivo and especially under conditions favoring nucleotide triphosphate (NTP) misincorporation (6)(7)(8).…”

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confidence: 99%

Selectivity and proofreading both contribute significantly to the fidelity of RNA polymerase III transcription

Alic

Ayoub²,

Landrieux³

et al. 2007

We examine here the mechanisms ensuring the fidelity of RNA synthesis by RNA polymerase III (Pol III). Misincorporation could only be observed by using variants of Pol III deficient in the intrinsic RNA cleavage activity. Determination of relative rates of the reactions producing correct and erroneous transcripts at a specific position on a tRNA gene, combined with computational methods, demonstrated that Pol III has a highly efficient proofreading activity increasing its transcriptional fidelity by a factor of 10 3 over the error rate determined solely by selectivity (1.8 ؋ 10 ؊4 ). We show that Pol III slows down synthesis past a misincorporation to achieve efficient proofreading. We discuss our findings in the context of transcriptional fidelity studies performed on RNA Pols, proposing that the fidelity of transcription is more crucial for Pol III than Pol II.nucleotide selectivity ͉ transcriptional fidelity