Base stacking and unstacking as determined from a DNA decamer containing a fluorescent base

Wu, Pengguang; Nordlund, Thomas M.; Gildea, Brian; McLaughlin, Larry W.

doi:10.1021/bi00479a024

Cited by 60 publications

(58 citation statements)

References 31 publications

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“…2-AP can substitute for adenine and pair with thymine without changing much of the physicochemical properties of double-stranded polynucleotide (22)(23)(24)(25)(26). 2-AP acts as an adenine and pairs with thymine when present in the template strand during polynucleotide synthesis by DNA polymerase (see the experiments mentioned in the Fig.…”

Section: Resultsmentioning

confidence: 99%

A “master” in base unpairing during isomerization of a promoter upon RNA polymerase binding

Lim

Lee

Roy

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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Isomerization of a closed to open complex of a promoter upon RNA polymerase binding involves base unpairing at the ؊10 region. After potassium permanganate sensitivity of unpaired thymine residues, we studied base unpairing at the ؊10 region during isomerization upon RNA polymerase binding at the P1 and P3 promoters of the gal operon. Substitution of adenine by 2-amino purine (2-AP) at the invariable A⅐T base pair at the ؊11 position of P1 and P3 prevented unpairing not only at that position but also at the other downstream positions, suggesting a ''master'' role of the adenine base at ؊11 of the template strand in overall base unpairing. 2-AP at ؊11 did not inhibit the formation of RNA polymerase⅐promoter complex and subsequent isomerization of the polymerase. Substitution of adenine by 2-AP at several other positions did not affect thymine unpairing. Changing the position of the amino group from C6 in adenine to C2 in 2-AP is mutational only at the master switch position, ؊11. U nder ordinary physiological conditions, transcription inEscherichia coli is executed by the RNA polymerase holoenzyme comprising ␣ 2 ␤␤Ј 70 subunits. Transcription initiation follows specific binding of RNA polymerase to the promotor. The initial binary complex (closed complex) undergoes structural changes (isomerization) in both proteins and DNA to make an open complex that is competent for subsequent templatedependent polymerization of ribonucleotides (1, 2). Whether isomerization of RNA polymerase and DNA occurs simultaneously or one after the other is unknown. The 70 subunit plays a key role in recognizing and making specific contacts with base pairs at the Ϫ35 and Ϫ10 regions of the promoter (3). In the open complex, DNA at the Ϫ10 region presumably becomes singlestranded for RNA polymerase to start reading the exposed template strand and polymerizing ribonucleotides (4-10). The bases in the nontemplate strand make specific interactions with amino acid residues of 70 (11). The consequence of these interactions is believed to stabilize the unstable nature of the unpaired DNA region (11-13). The consensus DNA sequence of the Ϫ10 region is 5Ј-TATAAT-3Ј in the nontemplate strand, with the first T being at the Ϫ12 position (1). To elucidate the molecular procedures of base unpairing during open complex formation, altered forms of DNA (depurinated, nicked, forked, or with single-stranded gap) in the Ϫ10 region were used as templates (10,(14)(15)(16)(17)(18)(19). These studies suggested that base unpairing involves an initial distortion or localized melting event around Ϫ11 (16,20). In this paper, we provide results by using intact duplex DNA to demonstrate that the adenine residue itself at the position Ϫ11 of the nontemplate strand plays a master role in the initiation of base unpairing. Materials and MethodsMaterials. E. coli RNA polymerase holoenzyme was purchased from Epicentre Technologies (Madison, WI). cAMP receptor protein (CRP), purified to 98% homogeneity by FPLC (Amersham Pharmacia) from an E. coli strain carrying the crp gen...

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Section: Resultsmentioning

confidence: 99%

A “master” in base unpairing during isomerization of a promoter upon RNA polymerase binding

Lim

Lee

Roy

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

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“…[9] As part of an effort to study the Dewar formation, herein we report the photophysical properties of the pyrimidinone chromophore. For a nucleoside bearing 5-methyl-2(1H)-pyrimidinone, Wu et al [19] determined a fluorescence lifetime of 4 ns in aqueous solution, that is, a much larger value than reported for native nucleosides. Photochemical transformations were not mentioned for this pyrimidinone.…”

Section: Introductionmentioning

confidence: 90%

The Excited‐State Decay of 1‐Methyl‐2(1H)‐pyrimidinone is an Activated Process

et al. 2011

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The photophysics of 1-methyl-2(1H)-pyrimidinone (1MP) dissolved in water is investigated by steady-state and time-resolved fluorescence, UV/Vis absorption, and IR spectroscopy. In the experiments, excitation light is tuned to the lowest-energy absorption band of 1MP peaking at 302 nm. At room temperature (291 K) its fluorescence lifetime amounts to 450 ps. With increasing temperature this lifetime decreases and equals 160 ps at 338 K. Internal conversion (IC) repopulating the ground state and intersystem crossing (ISC) to a triplet state are the dominant decay channels of the excited singlet state. At room temperature both channels contribute equally to the decay, that is, the quantum yields of IC and ISC are both approximately 0.5. The temperature dependence of UV/Vis transient absorption signals shows that the activation energy of the IC process (2140 cm(-1)) is higher than that of the ISC process (640 cm(-1)).

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“…It specifically Watson-Crick base-pairs with thymine or uracil and has proven its utility as a probe to monitor local conformational changes in nucleic acid structure, for example, in catalytic RNA and upon formation of nucleic acid-protein complexes (Millar 1996;O'Neill and Barton 2002;Walter et al 2002;Patel and Bandwar 2003;Roy 2003;Bradrick and Marino 2004;Clerte and Hall 2004). Several fluorescent pyrimidine analogs also have been developed, although they have not yet reached a similar level of popularity (Inoue et al 1985;Wu et al 1990;Godde et al 1998;Wilhelmsson et al 2001).…”

Section: Introductionmentioning

confidence: 99%

Pyrrolo-C as a fluorescent probe for monitoring RNA secondary structure formation

Tinsley¹,

Walter²

2006

RNA

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Pyrrolo-C (PC), or 3-[b-D-2-ribofuranosyl]-6-methylpyrrolo [2,3-d]pyrimidin-2(3H)-one, is a fluorescent analog of the nucleoside cytidine that retains its Watson-Crick base-pairing capacity with G. Due to its red-shifted absorbance, it can be selectively excited in the presence of natural nucleosides, making it a potential site-specific probe for RNA structure and dynamics. Similar to 2-aminopurine nucleoside, which base-pairs with uridine (or thymidine), PC's fluorescence becomes reversibly quenched upon base-pairing, most likely due to stacking interactions with neighboring bases. To test its utility as an RNA probe, we examined PC's fluorescent properties over a wide range of ionic strengths, pH, organic cosolvents, and temperatures. Incorporation of PC into a single-stranded RNA results in an $60% reduction of fluorescence intensity, while duplex formation reduces the fluorescence by $75% relative to the free ribonucleoside. We find that the fluorescence intensity of PC is only moderately affected by ionic strength, pH, and temperature, while it is slightly enhanced by organic cosolvents, making it a versatile probe for a broad range of buffer conditions. We demonstrate two applications for PC: fluorescent measurements of the kinetics of formation and dissociation of an RNA/DNA complex, and fluorescent monitoring of the thermal denaturation of the central segment of an RNA duplex. Taken together, our data showcase the potential of pyrrolo-C as an effective fluorescent probe to study RNA structure, dynamics, and function, complementary to the popular 2-aminopurine ribonucleoside.

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Base stacking and unstacking as determined from a DNA decamer containing a fluorescent base

Cited by 60 publications

References 31 publications

A “master” in base unpairing during isomerization of a promoter upon RNA polymerase binding

A “master” in base unpairing during isomerization of a promoter upon RNA polymerase binding

The Excited‐State Decay of 1‐Methyl‐2(1H)‐pyrimidinone is an Activated Process

Pyrrolo-C as a fluorescent probe for monitoring RNA secondary structure formation

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