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

Abstract

Nonenzymatic RNA polymerization in early life is likely to introduce backbone heterogeneity with a mixture of 2′-5′ and 3′-5′ linkages. On the other hand, modern nucleic acids are dominantly composed of 3′-5′ linkages. RNA polymerase II (pol II) is a key modern enzyme responsible for synthesizing 3′-5′-linked RNA with high fidelity. It is not clear how modern enzymes, such as pol II, selectively recognize 3′-5′ linkages over 2′-5′ linkages of nucleic acids. In this work, we systematically investigated how phosphodiester linkages of nucleic acids govern pol II transcriptional efficiency and fidelity. Through dissecting the impacts of 2′-5′ linkage mutants in the pol II catalytic site, we revealed that the presence of 2′-5′ linkage in RNA primer only modestly reduces pol II transcriptional efficiency without affecting pol II transcriptional fidelity. In sharp contrast, the presence of 2′-5′ linkage in DNA template leads to dramatic decreases in both transcriptional efficiency and fidelity. These distinct effects reveal that pol II has an asymmetric (strand-specific) recognition of phosphodiester linkage. Our results provided important insights into pol II transcriptional fidelity, suggesting essential contributions of phosphodiester linkage to pol II transcription. Finally, our results also provided important understanding on the molecular basis of nucleic acid recognition and genetic information transfer during molecular evolution. We suggest that the asymmetric recognition of phosphodiester linkage by modern nucleic acid enzymes likely stems from the distinct evolutionary pressures of template and primer strand in genetic information transfer during molecular evolution.2′-5′ phosphodiester linkage | trigger loop | synthetic nucleic acid analogues T he remarkable capacity of RNA as the functional catalyst as well as the genetic information carrier provides a strong support for the RNA world hypothesis, in which RNA is proposed to play a key role in the early evolution of primitive life before DNA and protein (enzyme) evolved (1, 2). One critical issue for RNA replication in early life is backbone heterogeneity because RNA contains both 2′-OH and 3′-OH, in which both can form phosphodiester bond during RNA polymerization. Indeed, previous studies revealed that nonenzymatic RNA replication would lead to a mixture of 2′-5′ and 3′-5′ linkages (Fig. 1A) (3-9). The 2′-5′-linked nucleic acids can form duplex structures by themselves or by pairing with natural nucleic acids (10-15). The 2′-5′ linkage substitutions in some functional RNAs retain molecular recognition and catalytic properties (16)(17)(18)(19). These discoveries suggested that the 2′-5′ linkage in nucleic acids could be an important alternative linkage during early evolution.This backbone heterogeneity problem is largely eliminated during evolution. Modern nucleic acids are dominated by 3′-5′ linkages. The 2′-5′ RNA linkages are only present in a few specific processes. One example is the formation of lariat RNA intron during splicing, where the 2′-OH of an...

Impact of template backbone heterogeneity on RNA polymerase II transcription

“…We observed three strong pol II pausing sites near the 2′-5′ dT site (pausing at 10–12 nt positions). However, pol II can eventually bypass the 2′-5′ dT linkage site to generate longer transcripts upon extended incubation as previously observed ( 30 ). Taken together, we revealed that pol II tolerates 3′-5′ rU substitution (sugar backbone heterogeneity), whereas a single substitution with 2′-5′ rU causes strong block for pol II elongation.…”

“…Previous studies have revealed the molecular basis of how RNA pol II recognizes functional groups and structural features of nucleic acids during transcription ( 25 – 29 ). Particularly, our very recent work used a synthetic 2′-5′ DNA template as a model to dissect the phosphodiester linkage impact on pol II transcription ( 30 ). However, it is unclear how pol II processes the template DNA containing ‘natural’ backbone heterogeneity (3′-5′DNA, 3′-5′ RNA and 2′-5′ RNA), which could exist during evolution and whether pol II recognizes these three types of backbone differently.…”

Dual coding potential of a 2′,5′-branched ribonucleotide in DNA

“…Our qPCR data showed that a single 2 ′ ,5 ′ -linked ribonucleotide in a DNA template decreases DNA synthesis by a factor of 5. Consistent with the findings of Xu et al (2014Xu et al ( , 2015, these data indicate that 2 ′ ,5 ′ -linked ribonucleotides in genomic DNA templates would slow down DNA replication and transcription in prokaryotic and eukaryotic cells. Moreover, because RNA 2 ′ ,5 ′ linkages are not a substrate for RNase H (Kandimalla et al 1997), it is likely that such rNMPs would not be removed by the cellular type 2 RNase H-initiated repair mechanism (Williams and Kunkel 2014;Williams et al 2016) and would persist in DNA.…”

“…A backbone heterogeneity with 2 ′ ,5 ′ -linked RNA is thought to exist in DNA during molecular evolution (Xu et al 2015). Whether contemporary template-dependent nucleic acid polymerases still tolerate 2 ′ ,5 ′ -linked rNMPs in DNA (Xu et al 2014(Xu et al , 2015 has become of interest. Our qPCR data showed that a single 2 ′ ,5 ′ -linked ribonucleotide in a DNA template decreases DNA synthesis by a factor of 5.…”

Functional interplay between NTP leaving group and base pair recognition during RNA polymerase II nucleotide incorporation revealed by methylene substitution

“…These nucleic acid analogs enabled us to investigate the individual contributions of the nucleobase and sugar moieties to pol II transcription. These studies have advanced our understanding of how the intrinsic structural features of nucleic acid moieties are recognized and how specific interactions are involved in substrate selection and incorporation during pol II transcription ( 21 – 24 ). However, the interactions involved at the triphosphate moiety of NTPs during nucleotide selection and incorporation have not yet been extensively explored.…”