Fidelity of RNA polymerase II transcription controlled by elongation factor TFIIS

Abstract

Fidelity of DNA and protein synthesis is regulated by a proofreading mechanism but function of a similar mechanism during RNA synthesis has not been demonstrated. Analysis of transcriptional fidelity and its control has been hampered by the necessity to employ complex DNA templates requiring either a promoter and initiation factors or 3-extended templates. To circumvent this difficulty, we have created an RNA-DNA dumbbell template that can be recognized as a template-primer and extended by RNA polymerase II. By employing this system, we demonstrate that RNA polymerase II can misincorporate a nucleotide and carry out template-dependent elongation at the mispaired end. The transcripts containing misincorporated residues can be cleaved by the very slow 3 3 5 ribonuclease activity of the RNA polymerase II, but enhancement of this activity by the elongation factor TFIIS generates RNA with a high degree of fidelity. This enhanced preferential cleavage of misincorporated transcripts suggests an important role for TFIIS in maintaining transcriptional fidelity.Maintenance of fidelity in macromolecular information transfer is important for precise replication and differentiation of an organism. DNA is replicated with astounding precision (10 Ϫ9to 10 Ϫ10 errors per base replicated); the high fidelity is achieved by a DNA polymerase capable of both base-pair discrimination and proofreading (1). The analogous question of how transcriptional fidelity might be maintained remains largely unanswered. Maintenance of fidelity during elongation is likely to be an important target for gene regulation in eukaryotes. As has been shown for Escherichia coli, the elongation͞cleavage factor GreA controls the fidelity of RNA polymerase transcription (2-4). Several eukaryotic elongation factors, TFIIF (5), TFIIS (6-10), P-TEFb (11), and elongin SIII (12), have been shown to enhance the ability of RNA polymerase II (pol II) to produce full-length mRNA transcripts, but their role in transcriptional fidelity has not yet been explored. The recent discovery that pol II harbors an intrinsic 3Ј 3 5Ј ribonuclease activity that can be potentiated by TFIIS (9, 10, 13, 14) led us to investigate the role of this factor in transcriptional fidelity.Assessment of transcriptional fidelity has been impeded by the difficulties in generating functional elongation complexes halted at specific sites and in precise analysis of the RNA products. While promoter-dependent transcription by E. coli RNA polymerase of a DNA template containing PR promoter allowed study of the fidelity of transcription and its control by elongation factor GreA, these studies are complex and require sophisticated kinetic analysis to resolve the data (2). Other templates not requiring any accessory factors such as 3Ј-extended DNAs and the synthetic RNA-DNA bubble duplexes can be accurately initiated and elongated by pol II but they also pose difficulties in simple biochemical analysis of the RNA products to identify misincorporation (7,15).In the present studies, we have designed ...

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

“…This may be the extreme case of the preference to cut the nascent RNA just 5Ј of an unstable RNA-DNA base pair, demonstrated for Pol III (30). This extends observations made on RNA-DNA ''dumbbell'' templates, using Pol IIϩTFIIS (22). Importantly, in our study, the rates of both cleavage and extension are measured under physiologically more relevant conditions, within authentic ternary complexes halted on a class III gene.…”

“…Only the fidelity of the mRNA-synthesizing Pol II has been investigated to date. The selectivity of Pol II for the correct NTP was measured in vitro (21), and, paralleling the situation in E. coli, the role of TFIIS, the Pol II cleavage-stimulatory factor, in permitting efficient proofreading was determined (21,22). Interestingly, at least in the yeast Saccharomyces cerevisiae, the errors committed by Pol II during the synthesis of mRNAs may be masked by the higher error-rate of mRNA translation (23).…”

Single-Molecule Studies of RNA Polymerase: Motoring Along

“…This backtracking is followed by endonucleolytic cleavage of the 3′ RNA fragment carrying the error, which can be promoted by transcription factors GreA and GreB in prokaryotes, or by TFIIS in eukaryotes (96,125,126). Single-molecule experiments provide compelling support for this model, showing that the frequency of long pauses increases in the presence of the nucleotide analog ITP, which mimics misincorporation, and that long pauses lead to enzyme backtracking by an average of ~5 bp.…”

Splice-site pairing is an intrinsically high fidelity process

“…It has been demonstrated that RNA transcription and translation work with a high degree of fidelity (17)(18)(19). Defining errors during the process of RNA transcription is conceptually straightforward because the incorporation of new nucleotides is template-directed.…”