Thiation of Nucleosides. I. Synthesis of 2-Amino-6-mercapto-9-β-D-ribofuranosylpurine (“Thioguanosine”) and Related Purine Nucleosides<sup>1</sup>

Fox, Jack J.; Wempen, Iris; Hampton, Alexander; Doerr, Iris L.

doi:10.1021/ja01540a041

Cited by 212 publications

(102 citation statements)

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“…2-AP is a fluorescent base analog of dA that base pairs with dT. The fluorescence of 2-AP is sensitive to local changes that result from melting of dsDNA (9,14,15). This is evident from the fluorescence spectra in Fig.…”

Section: Resultsmentioning

confidence: 99%

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Equilibrium and Stopped-flow Kinetic Studies of Interaction between T7 RNA Polymerase and Its Promoters Measured by Protein and 2-Aminopurine Fluorescence Changes

Jia

Kumar

Patel

1996

Journal of Biological Chemistry

112

120

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The mechanism of bacteriophage T7 RNA polymerase binding to its promoter DNA was investigated using stopped-flow and equilibrium methods. To measure the kinetics of protein-DNA interactions in real time, changes in tryptophan fluorescence in the polymerase and 2-aminopurine (2-AP) fluorescence in the promoter DNA upon binary complex formation were used as probes. The protein fluorescence changes measured conformational changes in the polymerase whereas the fluorescence changes of 2-AP base, substituted in place of dA in the initiation region (؊4 to ؉4), measured structural changes in the promoter DNA, such as DNA melting. The kinetic studies, carried out in the absence of the initiating nucleotide, are consistent with a two-step DNA binding mechanism,where the RNA polymerase forms an initial weak ED a complex rapidly with an equilibrium association constant K 1 . The ED a complex then undergoes a conformational change to ED b , wherein RNA polymerase is specifically and tightly bound to the promoter DNA. Both the polymerase and the promoter DNA may undergo structural changes during this isomerization step. The isomerization of ED a to ED b is a fast step relative to the rate of transcription initiation and its rate does not limit transcription initiation. To understand how T7 RNA polymerase modulates its transcriptional efficiency at various promoters at the level of DNA binding, comparative studies with two natural T7 promoters, ⌽10 and ⌽3.8, were conducted. The results indicate that kinetics, the bimolecular rate constant of DNA binding, k on (K 1 k 2 ), and the dissociation rate constant, k off (k ؊2 ), and thermodynamics, the equilibrium constants of the two steps (K 1 and k 2 /k ؊2 ) both play a role in modulating the transcriptional efficiency at the level of DNA binding. Thus, the 2-fold lower k on , the 4-fold higher k off , and the 2-5-fold weaker equilibrium interactions together make ⌽3.8 a weaker promoter relative to ⌽10.Bacteriophage T7 RNA polymerase is a 98-kDa single subunit polymerase that catalyzes synthesis of RNA complementary in sequence to the template DNA (1). The phage enzymes are among the simplest RNA polymerases known, as no accessory proteins are necessary for specific initiation, elongation, or termination of transcription (2, 3). The 17 promoters of bacteriophage T7 direct specific initiation of RNA synthesis that occurs in a rapid and processive manner (4, 5). Due to their simplicity these enzymes serve as model systems to understand, in depth, the mechanisms of transcription initiation, elongation, or termination.Initiation of transcription occurs by recognition and binding of the RNA polymerase to a promoter DNA sequence. This event is recognized as one of the important steps at which transcription and gene expression is regulated. The 17 bacteriophage T7 promoters share consensus sequence from Ϫ17 to ϩ6 position relative to the transcription start site at ϩ1 (6). The class III gene promoters of T7 are absolutely conserved in DNA sequence, whereas the class II gene promoters diff...

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Section: Resultsmentioning

confidence: 99%

“…The extinction coefficient of 2-AP, at 260 nm, equal to 1000 M Ϫ1 cm Ϫ1 was used in the calculation of 2-AP DNA concentration (9). The double-stranded (ds) DNAs were prepared by annealing the individual single-stranded DNA strands.…”

mentioning

confidence: 99%

Equilibrium and Stopped-flow Kinetic Studies of Interaction between T7 RNA Polymerase and Its Promoters Measured by Protein and 2-Aminopurine Fluorescence Changes

Jia

Kumar

Patel

1996

Journal of Biological Chemistry

112

120

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“…Deprotection of the carbohydrate residue and subsequent preparation of the dimethoxytrityl phosphoramidite derivative 6 (Scheme 1), produced a compound suitable for incorporation 5637 I H2NNH2 into oligodeoxynucleotides using phosphite triester synthesis procedures (32). We have found that silver oxide oxidation of the hydrazino derivative i (Scheme 1) is a simpler and more convenient route to the 2-aminopurine chromophore than previously described hydrogenation (27) or elimination reactions (28,29). Hydrazine reacts rapidly with the sulfonated nucleoside by attack at the 6-position.…”

Section: Resuitsmentioning

confidence: 88%

A new approach to the synthesis of a protected 2-aminopurine derivative and its incorporation into oligodeoxynucleotides containing the Eco RI and Bam HI recognition sites

et al. 1988

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A protected 2-aminopurine nucleoside suitable for incorporation into oligodeoxynucleotides using phosphite triester chemical synthesis procedures has been prepared via oxidation of a purine hydrazino derivative with silver (I) oxide. Five oligodeoxynucleotides containing Eco RI and Bam HI recognition sites have been prepared such that, in the double stranded form, the 2-aminopurine base has either a complementary thymine or cytosine nucleobase. The helix character and thermodynamic parameters for helix formation have been examined.

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“…In addition to elucidation of the elementary processes, characterization of the chemical states of the intermediates is possible, which will reflect the protein structure. It is likely that 2-amino-6-mercapto-9-flribofuranosylpurine analogues will be particularly useful in this respect, since the absorption band is well removed from that of protein (Fox et al, 1958) and they should quench protein fluorescence. The fluorescent properties of various adenosine analogues that are suitable for such studies have been described (Ward et al, 1969a).…”

Section: Discussionmentioning

confidence: 99%

Elementary processes of the magnesium ion-dependent adenosine triphosphatase activity of heavy meromysin. A transient kinetic approach to the study of kinases and adenosine triphosphatases and a colorimetric inorganic phosphate assay in situ

Trentham

Bardsley

Eccleston

et al. 1972

Biochemical Journal

161

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Transient kinetic studies of Mg2+-dependent heavy-meromyosin ATPase (adenosine triphosphatase) were done by monitoring the release of both ADP and Pi into the reaction medium by using linked assay systems. The release of Pi was monitored by its quantitative transfer to ADP, with concomitant reduction of NAD+ in the presence of D-glyceraldehyde 3-phosphate, D-glyceraldehyde 3-phosphate dehydrogenase and phosphoglycerate kinase. The dissociation rates of the products, ADP and Pi, from heavy meromyosin were shown to be faster than the rate-controlling process, which occurs after the initial bond cleavage of ATP. The chromophoric ATP analogue, 6-mercapto-9-f-D-ribofuranosylpurine 5'-triphosphate (thioATP) was used as a substrate and spectral changes associated with a single turnover of heavy meromyosin could be assigned to elementary processes ofthe mechanism. It was shown that the dissociation rate ofthioADP was not the rate-controlling process of the thioATPase, whose catalytic-centre activity was 7.6 times that of the ATPase at pH8. The dissociation rate of ADP from heavy meromyosin was measured by using thioATP as displacing agent and was found to be 2.3 s-1, which is about 50 times the catalytic-centre activity of the ATPase at pH8. Transient kinetic studies with chromophoric adenosine phosphate analogues have general application for kinases and ATPases both in characterizing the chemical states of the intermediates and in delineating the elementary processes of the enzyme mechanism.

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Thiation of Nucleosides. I. Synthesis of 2-Amino-6-mercapto-9-β-D-ribofuranosylpurine (“Thioguanosine”) and Related Purine Nucleosides¹

Cited by 212 publications

References 12 publications

Equilibrium and Stopped-flow Kinetic Studies of Interaction between T7 RNA Polymerase and Its Promoters Measured by Protein and 2-Aminopurine Fluorescence Changes

Equilibrium and Stopped-flow Kinetic Studies of Interaction between T7 RNA Polymerase and Its Promoters Measured by Protein and 2-Aminopurine Fluorescence Changes

A new approach to the synthesis of a protected 2-aminopurine derivative and its incorporation into oligodeoxynucleotides containing the Eco RI and Bam HI recognition sites

Elementary processes of the magnesium ion-dependent adenosine triphosphatase activity of heavy meromysin. A transient kinetic approach to the study of kinases and adenosine triphosphatases and a colorimetric inorganic phosphate assay in situ

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Thiation of Nucleosides. I. Synthesis of 2-Amino-6-mercapto-9-β-D-ribofuranosylpurine (“Thioguanosine”) and Related Purine Nucleosides1

Cited by 212 publications

References 12 publications

Equilibrium and Stopped-flow Kinetic Studies of Interaction between T7 RNA Polymerase and Its Promoters Measured by Protein and 2-Aminopurine Fluorescence Changes

Equilibrium and Stopped-flow Kinetic Studies of Interaction between T7 RNA Polymerase and Its Promoters Measured by Protein and 2-Aminopurine Fluorescence Changes

A new approach to the synthesis of a protected 2-aminopurine derivative and its incorporation into oligodeoxynucleotides containing the Eco RI and Bam HI recognition sites

Elementary processes of the magnesium ion-dependent adenosine triphosphatase activity of heavy meromysin. A transient kinetic approach to the study of kinases and adenosine triphosphatases and a colorimetric inorganic phosphate assay in situ

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Thiation of Nucleosides. I. Synthesis of 2-Amino-6-mercapto-9-β-D-ribofuranosylpurine (“Thioguanosine”) and Related Purine Nucleosides¹