Misincorporation by RNA polymerase is a major source of transcription pausingin vivo

Abstract

The transcription error rate estimated from mistakes in end product RNAs is 10−3–10−5. We analyzed the fidelity of nascent RNAs from all actively transcribing elongation complexes (ECs) in Escherichia coli and Saccharomyces cerevisiae and found that 1–3% of all ECs in wild-type cells, and 5–7% of all ECs in cells lacking proofreading factors are, in fact, misincorporated complexes. With the exception of a number of sequence-dependent hotspots, most misincorporations are distributed relatively randomly. Misincorporation at hotspots does not appear to be stimulated by pausing. Since misincorporation leads to a strong pause of transcription due to backtracking, our findings indicate that misincorporation could be a major source of transcriptional pausing and lead to conflicts with other RNA polymerases and replication in bacteria and eukaryotes. This observation implies that physical resolution of misincorporated complexes may be the main function of the proofreading factors Gre and TFIIS. Although misincorporation mechanisms between bacteria and eukaryotes appear to be conserved, the results suggest the existence of a bacteria-specific mechanism(s) for reducing misincorporation in protein-coding regions. The links between transcription fidelity, human disease, and phenotypic variability in genetically-identical cells can be explained by the accumulation of misincorporated complexes, rather than mistakes in mature RNA.

A link between transcription fidelity and pausing in vivo

“…This earlier study suggested that, in sequence-dependent pausing, a C −1 G +1 motifs (non-template DNA sequence) increase the rate of G>A misincorporation at +1 position 10 . Indeed, at G>A hotspots, we also observed a clear bias for C preceding the misincorporated 3′ end, however, we did not see strong pausing at these C −1 positions 41 . Therefore, misincorporations might occur at those positions without the involvement of a pause.…”

“…These values are considerably higher than expected from the overall error rate of RNAP, 10 −3 –10 −6 42 . A somewhat lower proportion (0.1% and 1% for wild-type and mutant E. coli strains) of misincorporated ECs was reported by a different study, 10 and we have discussed the possible cause of such differences elsewhere 41 . Given that misincorporation causes stable backtracking, it is likely that these higher proportions are mainly caused by the accumulation of misincorporated complexes due to their slow resolution, even in wild-type cells.…”

TFS and Spt4/5 accelerate transcription through archaeal histone‐based chromatin

“…The inability of TFS DE-AA to properly donate acidic residues to the active site of RNAP abrogates its function as a cleavage stimulatory factor. Backtracking can result from extended pausing (Nudler, 2012;Imashimizu et al, 2013;Weixlbaumer et al, 2013;Hein et al, 2014;Imashimizu et al, 2015;James et al, 2017;Lerner et al, 2016;Gabizon et al, 2018), and the configuration of mobile-domains of RNAP is known to modulate the propensity to pause and the duration of pausing Martinez-Rucobo et al, 2011;Chakraborty et al, 2012;Weixlbaumer et al, 2013;Hein et al, 2014;Jun et al, 2014;Schulz et al, 2016;Sheppard et al, 2016;Bernecky et al, 2017;Feklistov et al, 2017;Kang et al, 2017;Duchi et al, 2018). We thus examined whether addition of Spt4 and/or Spt5 would influence the efficiency of RNA cleavage, with transcript cleavage also serving as a proxy for the propensity to, and depth of backtracking.…”

How Acts of Infidelity Promote DNA Break Repair: Collision and Collusion Between DNA Repair and Transcription

“…Eukaryotic TFIIS is functionally similar to Gre in restarting backtracked RNAPII complexes and preserving of transcription fidelity suggesting that these functions are conserved across all kingdoms of life. In Gre and TFIIS mutants, misincorporation events increase by ≈2‐ and ≈7‐fold over that observed in wild‐type strains, respectively . Although mRNA is considered to be transient, the consequences of transcription errors produce heritable phenotypes and can contribute to stochastic variability between bacterial cells .…”