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.

Keywords: 2′–5′ phosphodiester linkage; synthetic nucleic acid analogues; trigger loop.

Fig. 1.

Nucleic acid structures with 3′–5′ and 2′–5′ phosphodiester linkage. (A) Scheme of 3′–5′– and 2′–5′–linked DNA and RNA. (B) Wild-type, primer mutant, and template mutant scaffolds for RNA pol II transcription.

Fig. 2.

The 2′–5′ phosphodiester linkage in RNA primer end reduces RNA pol II transcriptional efficiency but does not affect fidelity. (A) Scaffold of RNA, template DNA, and nontemplate DNA for runoff elongation from the 2′–5′–linked primer end. The red star refers to the 2′–5′ linkage. (B) RNA pol II transcription products of 3′–5′–linked (left part of the gel) and 2′–5′–linked (right part of the gel) scaffolds in the presence of 25 μM NTP. Time points are 0, 30 s, 5 min, 20 min, 1 h, 2 h, and 4 h from left to right. The boxed area shows the major difference with linkage alteration. (C) Catalytic constants (k_pol) and specificity constants (k_pol/K_d,app) of pol II transcription from the 3′–5′ primer (blue bars) and the 2′–5′ primer (gray bars). (D) Specificity constants for both correct incorporation (ATP) and incorrect incorporation (UTP) (Left) and the discrimination power (Right).

Fig. 3.

Transcriptional efficiency of RNA pol II is greatly reduced by the linkage alteration in the DNA template. (A) Scaffold of RNA, template DNA, and nontemplate DNA for runoff elongation through the 2′–5′ linkage site in the template. The red star refers to the 2′–5′ linkage in the DNA. (B) RNA pol II transcription products of 3′–5′–linked (left part of the gel) and 2′–5′–linked (right part of the gel) scaffolds in the presence of 25 μM NTP. Time points are 30 s, 2 min, 5 min, 20 min, and 1 h from left to right. Numbers on the left refer to lengths of RNA elongation products; positions of these RNA products on the DNA template are partly shown on the right. The boxed area shows the major difference with linkage alteration. (C) Scaffolds used to study every transcriptionally slowed down step. Three primers refer to three stalled positions in the DNA template. The red star refers to the 2′–5′ linkage. (D) Catalytic constants (k_pol) and specificity constants (k_pol/K_d,app) of pol II transcription through 3′–5′ linkage (blue) and 2′–5′ linkage (red).

Fig. 4.

Transcriptional fidelity of RNA pol II is also reduced by the linkage alteration in the DNA template. (A) Fidelity decreases in the first checkpoint: nucleotide selection and incorporation. (Left) Specificity constants between correct incorporation (ATP) and incorrect incorporation (UTP) and (Right) the discrimination power. (B) Fidelity decreases in the second checkpoint: subsequent extension. (Left) Specificity constants between extension after the correct 3′-terminus (11A) and the incorrect 3′-terminus (11U) and (Right) the discrimination power. (C) Fidelity decreases in the third checkpoint: proofreading. (Left) Rate constants for cleavage of the correct 3′-terminus (11A) and the incorrect 3′-terminus (11U) and (Right) the discrimination power.

Fig. 5.

Distinct effects of α-amanitin on RNA pol II in the 2′–5′ linkage primer and template. (A) Nucleotide incorporation rates in the absence (–) and presence (+) of α-amanitin. (B) Effects of α-amanitin on nucleotide incorporation. The effects of α-amanitin refer to folds of rate change before and after α-amanitin treatment.

Fig. 6.

Superimposition of RNA pol II elongation complex (PDB ID: 2E2J) with 2′–5′ phosphodiester linkage in RNA primer (PDB ID: 4MSB) (A) and DNA template (B). RNA primer is shown in red, and DNA template is shown in blue. The superimposed RNA and DNA with 2′–5′ phosphodiester linkages are shown in yellow (A) and magenta (B), respectively. The misaligned template or primer is highlighted by arrows.

Fig. 7.

Phosphodiester linkages in RNA primer and DNA template play two distinct roles during pol II transcription. (A) RNA pol II transcription on the wild-type RNA/DNA scaffold. Nucleotide incorporation has a good alignment with the upstream duplex, and the trigger loop of pol II (colored in green) is in the closed state, ensuring high efficiency and fidelity. (B) RNA pol II transcription after a 2′–5′–linked primer end. The trigger loop of pol II can be closed but the misaligned 3′-terminus lowers the primer extension efficiency. Hence, fidelity is maintained but the overall enzymatic efficiency is reduced. (C) RNA pol II transcription through a 2′–5′–linked position in the DNA template. During incorporation opposite the 2′–5′ linkage, the trigger loop of pol II cannot be closed because of the linkage alteration induced base and sugar shift. Therefore, both transcriptional efficiency and fidelity decreased.