Dissecting Chemical Interactions Governing RNA Polymerase II Transcriptional Fidelity

Abstract

Maintaining high transcriptional fidelity is essential to life. For all eukaryotic organisms, RNA polymerase II (Pol II) is responsible for messenger RNA synthesis from the DNA template. Three key checkpoint steps are important in controlling Pol II transcriptional fidelity: nucleotide selection and incorporation, RNA transcript extension, and proofreading. Some types of DNA damage significantly reduce transcriptional fidelity. However, the chemical interactions governing each individual checkpoint step of Pol II transcriptional fidelity and the molecular basis of how subtle DNA base damage leads to significant losses of transcriptional fidelity are not fully understood. Here we use a series of “hydrogen bond deficient” nucleoside analogs to dissect chemical interactions governing Pol II transcriptional fidelity. We find that whereas hydrogen bonds between a Watson-Crick base pair of template DNA and incoming NTP are critical for efficient incorporation, they are not required for efficient transcript extension from this matched 3’-RNA end. In sharp contrast, the fidelity of extension is strongly dependent on the discrimination of an incorrect pattern of hydrogen bonds. We show that U:T wobble base interactions are critical to prevent extension of this mismatch by Pol II. Additionally, both hydrogen bonding and base stacking play important roles in controlling Pol II proofreading activity. Strong base stacking at the 3’-RNA terminus can compensate for loss of hydrogen bonds. Finally, we show that Pol II can distinguish very subtle size differences in template bases. The current work provides the first systematic evaluation of electrostatic and steric effects in controlling Pol II transcriptional fidelity.

Strand-specific (asymmetric) contribution of phosphodiester linkages on RNA polymerase II transcriptional efficiency and fidelity

“…The 2′-5′-linked oligos were gel purified and purchased from Gene Link Inc. The pol II elongation complexes for transcription assays were assembled using established methods (51,55).…”

“…The pol II elongation complexes for transcription assays were assembled using established methods (51,55). Briefly, an aliquot of 5′-32 P-labeled RNA was annealed with a 1.5-fold amount of template DNA and twofold amount of nontemplate DNA to form RNA/DNA scaffold in elongation buffer [20 mM Tris·HCl (pH = 7.5), 40 mM KCl, 5 mM MgCl 2 ].…”

“…The assay was carried out as previously described (51,55). Briefly, nucleotide incorporation assays were conducted by preincubating 50 nM scaffold with 200 nM pol II for 10 min in elongation buffer at 22°C.…”

“…Recently, we used synthetic nucleic acid analogs to reveal the importance of chemical interactions and the intrinsic structural features of nucleic acids in controlling pol II transcriptional fidelity. To this end, we revealed the distinct contributions of hydrogen bonds between nucleobases in controlling individual fidelity checkpoint steps using hydrogen bond-deficient nucleotide analogues (51). Recently, we revealed that the sugar backbone is a dominant factor in controlling all three fidelity checkpoint steps of pol II transcriptional fidelity using sugar-ring disrupted mutant (52).…”

“…A general theme from these studies is to use synthetic nucleic acid chemistry to chemically modify the specific functional groups or motifs of nucleic acids. This chemical mutation approach allows us to systematically dissect the individual roles of chemical interactions and intrinsic structural motifs of nucleic acids in controlling pol II transcriptional fidelity, which cannot be achieved by conventional biochemical and genetic approaches (50)(51)(52)(53).…”