Fluorescence and absorption spectroscopic properties of RNA 5′-cap analogues derived from 7-methyl-, N2,7-dimethyl- and N2,N2,7-trimethyl-guanosines

Wieczorek, Z; Stępiński, Janusz; Jankowska, Marzena; Lönnberg, Harri

doi:10.1016/1011-1344(94)07098-9

Cited by 32 publications

(34 citation statements)

References 21 publications

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“…Charge on the monophosphate group is essential: a 5´-monophosphate methyl ester influences the acidity less than a dianionic monophosphate group. It may, hence, be suggested that the increase of pK a values upon 5´-phosphorylation results, at least partially, from an electrostatic interaction between the phosphate and the cationic base [20]. Consistent with such an interaction, 7-methyl group slightly decreases the pK a of the phosphate group of 7-methylguanosine 5´-monophosphate (2a), whereas a similar effect is not observed with guanosine 5´-monophosphate (2b) [21].…”

Section: Structure and Interactions Of 5´-cap And 7-methylguanine Nucmentioning

confidence: 78%

Preparation and Properties of mRNA 5-cap Structure

Mikkola¹,

Salomaki²,

Zhang³

et al. 2005

COC

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The 5´-terminus of eukaryotic messenger RNA (mRNA) molecules contains an unique structure, viz. a dinucleoside 5´,5´-triphosphate moiety where the terminal nucleoside is 7-methylguanosine. This 5´-cap structure serves as a site of recognition for numerous enzymes involved in splicing, transport and translation of mRNA, and protects mRNA against intracellular exonucleases. In addition, viral RNA polymerases use capped mRNA sequences of the host cell as primers for viral RNA synthesis. Understanding of molecular mechanism of all these processes requires detailed information on the chemical properties of the cap structure, including capability to prepare conjugates and structural analogs of the cap for research tools. This review tends to summarize the present knowledge on various aspects of the chemistry of the cap structure, including chemical stability and mechanisms of breakdown, protolytic and complexing equilibria, stacking interactions and synthetic methodology.

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Section: Structure and Interactions Of 5´-cap And 7-methylguanine Nucmentioning

confidence: 78%

Preparation and Properties of mRNA 5-cap Structure

Mikkola¹,

Salomaki²,

Zhang³

et al. 2005

COC

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“…The emission maximum was found to red-shift relative to that of free L-tryptophan, which might be caused by the change of microenvironments around the fluorophores (38). The absorption spectra of different nucleotides are well known to overlap considerably with the absorption spectra of the three fluorescent amino acids (33,39,40) leading to significant inner filter effect. Therefore the absorption spectra of different potential nucleotides to be used in the binding experiments were measured at relevant concentrations, in search of an alternative excitation wavelength for the recombinant proteins.…”

Section: Resultsmentioning

confidence: 99%

Kinetic and Physical Characterization of the Inducible UDP-N-acetylglucosamine Pyrophosphorylase from Giardia intestinalis

Mok

Edwards

2005

Journal of Biological Chemistry

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The UDP-N-acetylglucosamine pyrophosphorylase in Giardia intestinalis (GiUAP) is one of the five inducible enzymes to synthesize UDP-GalNAc, which is an important precursor for cyst wall synthesis. The recombinant UDP-N-acetylglucosamine pyrophosphorylase (rGiUAP) and its mutants G108A and G210A were expressed and identified by SDS-PAGE, size-exclusion chromatography, Western hybridization, and MALDI mass spectrometry. Sequence comparison with other eukaryotic UAPs has identified three specific motifs. Within these motifs alanine substitution for Gly 108 or Gly 210 dramatically reduced the pyrophosphate synthesis, suggesting these amino acids are catalytic residues. Besides, the rGi-UAP was found to have relaxed binding to other uridine-based nucleotides, suggesting the substrate binding pocket is specific to uridine rather than phosphate group(s). Moreover, thermal denaturation analysis showed a significant increase in T m for the rGiUAP and G108A upon binding of the substrate Mg-UTP. In contrast, G210A showed a decreased T m upon binding of Mg-UTP. These results showed that binding of Mg-UTP increases protein stability of the rGiUAP, and the catalytic residue Gly 210 plays a significant role in stabilizing the protein structure. Such stabilization effect induced by substrate binding might be physiologically important as it favors the production of UDP-GlcNAc and hence the downstream GalNAc, which is crucial to survival of Giardia. These results help to define the essential amino acids for catalysis in the GiUAP and reveal the role of Mg-UTP binding in regulation of protein stability.Giardia intestinalis has long been recognized as one of the most early branching eukaryotes (1). It is also one of the most common causes of gastrointestinal infection in human and other mammals (2). When this parasite travels down the intestine in the host, the transformation from a vegetative trophozoite to an infectious cyst requires the synthesis of a rigid cyst wall to survive the adverse environmental conditions outside the host. As this transformation represents a basic adaptive response of a eukaryote to the environment for propagation, G. intestinalis provides a simple eukaryotic model for differentiation and, the biochemical mechanisms of encystation and the cyst wall synthesis have been the subject of intensive investigations in the past decade.Previous studies have shown that cyst wall contains both carbohydrate and protein components (3-9). One of the major components of the outer cyst wall has been identified as a [D-GalNAc(␤133)-D-GalNAc] n homopolymer by chemical methods, mass spectrometry, and hydrogen nuclear magnetic resonance spectroscopy (10). This GalNAc in G. intestinalis is synthesized from endogenous Glc rather than salvaged from external source (5). The precursor of GalNAc, UDP-GalNAc, is synthesized by five inducible enzymes: glucosamine-6-phosphate isomerase (EC 3.5.99.6); glucosamine-6-phosphate N-acetylase (EC 2.3.1.4); phosphoacetylglucosamine mutase (EC 5.4.2.3, AGM) 2 ; UDP-N-acetylglucosamine py...

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“…Decreasing affinity for phosphorylated eIF4E K as (phos)/ K as (wt) [%] versus negative charge of the cap analogs. The resultant charges at given pH were estimated from the experimental pK a1 values for dissociation of the N1 proton at of 7-methyloguanosine (Rhoads et al 1983;Wieczorek et al 1995), and from the experimental pK a2 values for dissociation of the second proton of the terminal phosphate group in the mononucleotide cap analogs (Dawson et al 1969). …”

Section: Discussionmentioning

confidence: 99%

Phosphorylation of eIF4E attenuates its interaction with mRNA 5′ cap analogs by electrostatic repulsion: Intein-mediated protein ligation strategy to obtain phosphorylated protein

Zuberek¹,

Wysłouch‐Cieszyńska²,

Niedzwiecka³

et al. 2003

RNA

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120

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Phosphorylation of the eukaryotic initiation factor eIF4E in response to mitogenic stimuli and cytokines is implicated in the regulation of the initiation step of translation. It still remains unclear how the phosphorylation of eIF4E regulates the translation. To address this problem, we applied a unique technique in protein engineering, intein-mediated protein ligation, to synthesize eIF4E, which is selectively phosphorylated at Ser 209. Using selectively chosen synthetic cap analogs, we compared quantitatively the cap affinity for phosphorylated and unphosphorylated eIF4E by a fluorometric time-synchronized titration method. A 1.5-to 4.5-fold reduction of the cap affinity for phosphorylated eIF4E was observed, depending on the negative charge of the 5-to-5 phosphate chains as well as the presence of a longer tetraribonucleotide strand. Possible implications for understanding the regulation of eIF4E functioning, cap complex formation, and stability, are discussed.

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Fluorescence and absorption spectroscopic properties of RNA 5′-cap analogues derived from 7-methyl-, N2,7-dimethyl- and N2,N2,7-trimethyl-guanosines

Cited by 32 publications

References 21 publications

Preparation and Properties of mRNA 5-cap Structure

Preparation and Properties of mRNA 5-cap Structure

Kinetic and Physical Characterization of the Inducible UDP-N-acetylglucosamine Pyrophosphorylase from Giardia intestinalis

Phosphorylation of eIF4E attenuates its interaction with mRNA 5′ cap analogs by electrostatic repulsion: Intein-mediated protein ligation strategy to obtain phosphorylated protein

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