The chemical nature of the 5′ end of RNA is a key determinant of RNA stability, processing, localization, translation efficiency1,2, and has been proposed to provide a layer of “epitranscriptomic” gene regulation3. Recently it has been shown that some bacterial RNA species carry a 5′-end structure reminiscent of the 5′ 7-methylguanylate “cap” in eukaryotic RNA. In particular, RNA species containing a 5′-end nicotinamide adenine dinucleotide (NAD+) or 3′-desphospho-coenzyme A (dpCoA) have been identified in both Gram-negative and Gram-positive bacteria3–6. It has been proposed that NAD+, reduced NAD+ (NADH), and dpCoA caps are added to RNA after transcription initiation, in a manner analogous to the addition of 7-methylguanylate caps6–8. Here, we show instead that NAD+, NADH, and dpCoA are incorporated into RNA during transcription initiation, by serving as non-canonical initiating nucleotides (NCINs) for de novo transcription initiation by cellular RNA polymerase (RNAP). We further show that both bacterial RNAP and eukaryotic RNAP II incorporate NCIN caps, that promoter DNA sequences at and upstream of the transcription start site determine the efficiency of NCIN capping, that NCIN capping occurs in vivo, and that NCIN capping has functional consequences. We report crystal structures of transcription initiation complexes containing NCIN-capped RNA products. Our results define the mechanism and structural basis of NCIN capping, and suggest that NCIN-mediated “ab initio capping” may occur in all organisms

The gas-phase acidities of the N1 and N3 sites of uracil have been bracketed to provide an understanding of the intrinsic reactivity of this nucleic base. The experiments indicate that in the gas phase, the N3 site is far less acidic (∆H acid ) 347 ( 4 kcal mol -1 ) than the N1 site (∆H acid ) 333 ( 4 kcal mol -1 ), in direct contrast to in solution, where the two sites are so close in acidity as to be unresolvable. This intrinsic difference and the coalescence in solution is interpreted through gas-phase and dielectric-medium calculations. The results point to a possible chemical reason that N1 is the preferred glycosylation site in nature: nature may simply take advantage of the differential N1 and N3 acidities in a nonpolar environment to achieve selectivity.

5-methylcytosine (mC) is an epigenetic mark that impacts transcription, development, and genome stability, and aberrant DNA methylation contributes to aging and cancer. Active DNA demethylation involves stepwise oxidation of mC to 5-hydroxymethylcytosine, 5-formylcytosine (fC), and potentially 5-carboxylcytosine (caC), excision of fC or caC by thymine DNA glycosylase (TDG), and restoration of cytosine via follow-on base excision repair. Here, we investigate the mechanism for TDG excision of fC and caC. We find that 5-carboxyl-2′-deoxycytidine ionizes with pKa values of 4.28 (N3) and 2.45 (carboxyl), confirming that caC exists as a monoanion at physiological pH. Calculations do not support the proposal that G·fC and G·caC base pairs adopt a wobble structure that is recognized by TDG. Previous studies show that N-glycosidic bond hydrolysis follows a stepwise (SN1) mechanism, and that TDG activity increases with pyrimidine N1 acidity, i.e., leaving-group quality of the target base. Calculations here show that fC and the neutral tautomers of caC are acidic relative to other TDG substrates, but the caC monoanion exhibits poor acidity and likely resists TDG excision. While fC activity is independent of pH, caC excision is acid catalyzed, and the pH profile indicates that caC ionizes in the enzyme-substrate complex with an apparent pKa of 5.8, likely at N3. Mutational analysis reveals that Asn191 is essential for excision of caC but dispensable for fC activity, indicating that N191 may stabilize N3-protonated forms of caC to facilitate acid catalysis, and suggesting that N191A-TDG could potentially be useful for studying DNA demethylation in cells.

A mechanism is proposed to explain the activity of orotidine 5'-monophosphate decarboxylase (ODCase). This enzyme is the one of the most proficient known, with a catalytic proficiency (kcat/Km)/knon = 10(23) M-1. Quantum mechanical calculations predict a mechanism involving a stabilized carbene intermediate, which represents a previously unrecognized mode of enzymatic activity for ODCase. The proposed mechanism involves proton transfer from a weak acid (pKa = 7, where Ka is the acid constant) concerted with decarboxylation, in a nonpolar enzyme environment. Such a mechanism makes possible different approaches to the design of ODCase inhibitors. Furthermore, the prediction that general acid catalysis may only be effective in low dielectric media is of general significance for understanding the activity of many enzymes.

The gas phase acidities of a series of uracil derivatives (1-methyluracil, 3-methyluracil, 6-methyluracil, 5,6-dimethyluracil, and 1,3-dimethyluracil) have been bracketed to provide an understanding of the intrinsic reactivity of uracil. The experiments indicate that in the gas phase, uracil has four sites more acidic than water. Among the uracil analogs, the N1-H sites have ⌬H acid values of 331-333 kcal mol Ϫ1 ; the acidity of the N3 sites fall between 347-352 kcal mol Ϫ1 . The vinylic C6 in 1-methyluracil and 3-methyluracil brackets to 363 kcal mol Ϫ1 , and 369 kcal mol Ϫ1 in 1,3-dimethyluracil; the C5 of 1,3-dimethyluracil brackets to 384 kcal mol Ϫ1 . Calculations conducted at B3LYP/6-31ϩG* are in agreement with the experimental values. The bracketing of several of these sites involved utilization of an FTMS protocol to measure the less acidic site in a molecule that has more than one acidic site, establishing the generality of this method. In molecules with multiple acidic sites, only the two most acidic sites were bracketable, which is attributable to a kinetic effect. The measured acidities are in direct contrast to in solution, where the two most acidic sites of uracil (N1 and N3) are indifferentiable. The vinylic C6 site is also particularly acidic, compared to acrolein and pyridine. The biological implications of these results, particularly with respect to enzymes for which uracil is a substrate, are discussed. (J Am Soc Mass Spectrom 2002, 13, 985-995)

Short, strong hydrogen bonds are common in charged systems in the gas phase, but the importance of such bonding in enzymatic catalysis has been the subject of considerable controversy. Confusion has arisen about the relationship among bond strength, the "low-barrier" or "no-barrier" nature of the hydrogen bonding, the role of pK a matching, the covalent or electrostatic nature of the bonding, and the role of solvation on the strengths of these types of hydrogen bonds. We have attempted to strip away the "Alice in Wonderland" quality of the definitions in this field by defining, through high-level calculations, when short-strong hydrogen bonds do and do not occur. The strengths and geometries of several types of hydrogen bonds involving anions have been investigated by ab initio quantum mechanical calculations. For a series of enols hydrogen-bonded to enolates, the strengths of the short, strong gas-phase hydrogen bonds are linearly related to the differences between the proton affinities (PA) of the two anions which share the proton. The bond strength is also related to the O‚‚‚O distance between them. There is no discontinuity at ∆PA ) 0, and hydrogen-bonding becomes even stronger in a computational experiment when the PA of the H-bond acceptor exceeds that of the donor. "Low-barrier" hydrogen bonds with single-well minima after inclusion of zero-point energies occur when ∆PA is near 0, but no special stability accrues when the double-well minimum becomes single-well. The maleic/fumaric and mesaconic/citraconic systems studied by Drueckhammer have been investigated computationally. The influence of solvation on hydrogen-bond strength was studied using solvent cavity models. Small increases in dielectric constant from the gas-phase value ( ) 1) rapidly reduce the strengths of charged hydrogen bonds. Short, strong hydrogen bonds occur only with charged systems, and only then in nonpolar ( < 10) environments. Alternative mechanisms are often available to account for enzymatic catalysis; the example of orotidine monophosphate decarboxylase is discussed.

The gas-phase acidities of adenine, 9-ethyladenine, and 3-methyladenine have been investigated for the first time, using computational and experimental methods to provide an understanding of the intrinsic reactivity of adenine. Adenine is found to have two acidic sites, with the N9 site being 19 kcal mol(-1) more acidic than the N10 site; the bracketed acidities are 333 +/- 2 and 352 +/- 4 kcal mol(-1), respectively. Because measurement of the less acidic site can be problematic, we benchmarked the adenine N10 measurement by bracketing the acidity of 9-ethyladenine, which has the N9 site blocked and allows for exclusive measurement of the N10 site. The acidity of 9-ethyladenine brackets to 352 +/- 4 kcal mol(-1), comparable to that of the N10 site of the parent adenine. Calculations and experiments with 3-methyladenine, a harmful mutagenic nucleobase, uncovered the surprising result that the most commonly written tautomer of 3-methyladenine is not the most stable in the gas phase. We have found that the most stable tautomer is the "N10 tautomer" 10, as opposed to the imine tautomer 3. The bracketed acidity of 10 is 347 +/- 4 kcal mol(-1). Since 10 is not a viable species in DNA, 3 is a likely tautomer; calculations indicate that this form has an extremely high acidity (320-323 kcal mol(-1)). The biological implications of these results, particularly with respect to enzymes that cleave alkylated bases from DNA, are discussed.

Hypoxanthine is a mutagenic purine base that most commonly arises from the oxidative deamination of adenine. Damaged bases such as hypoxanthine are associated with carcinogenesis and cell death. This inevitable damage is counteracted by glycosylase enzymes, which cleave damaged bases from DNA. Alkyladenine DNA glycosylase (AAG) is the enzyme responsible for excising hypoxanthine from DNA in humans. In an effort to understand the intrinsic properties of hypoxanthine, we examined the gas-phase acidity and proton affinity using quantum mechanical calculations and gas-phase mass spectrometric experimental methods. In this work, we establish that the most acidic site of hypoxanthine has a gas-phase acidity of 332 +/- 2 kcal mol-1, which is more acidic than hydrochloric acid. We also bracket a less acidic site of hypoxanthine at 368 +/- 3 kcal mol-1. We measure the proton affinity of the most basic site of hypoxanthine to be 222 +/- 3 kcal mol-1. DFT calculations of these values are consistent with the experimental data. We also use calculations to compare the acidic and basic properties of hypoxanthine with those of the normal bases adenine and guanine. We find that the N9-H of hypoxanthine is more acidic than that of adenine and guanine, pointing to a way that AAG could discriminate damaged bases from normal bases. We hypothesize that AAG may cleave certain damaged nucleobases as anions and that the active site may take advantage of a nonpolar environment to favor deprotonated hypoxanthine as a leaving group versus deprotonated adenine or guanine. We also show that an alternate mechanism involving preprotonation of hypoxanthine is energetically less attractive, because the proton affinity of hypoxanthine is less than that of adenine and guanine. Last, we compare the acidity in the gas phase versus that in solution and find that a nonpolar environment enhances the differences in acidity among hypoxanthine, adenine, and guanine.