Event Title

New Assays to Analyze the Thermodynamics of RNA Binding to Enzymes that Remove RNA Caps

Presenter Information

Gaelle Sehi
Basil Hable

Mentor 1

David Frick

Location

Union Wisconsin Room

Start Date

27-4-2018 1:00 PM

Description

Recently, the cofactor nicotinamide adenine dinucleotide (NAD) has been reported as a covalent modification of bacterial RNA. Given the central role of NAD in redox biochemistry, posttranslational protein modification and signaling, its attachment to RNA indicates that there are unknown functions of RNA in these processes and undiscovered pathways in RNA metabolism and regulation. Analogous to a eukaryotic cap, 5′-NAD modification is shown in vitro to stabilize RNA against 5′-processing by the RNA-pyrophosphohydrolase RppH and against endonucleolytic cleavage by ribonuclease (RNase) E. This is where the nudix phosphohydrolase NudC plays an important role to decap NAD-RNA and thereby triggering RNase-E-mediated RNA decay. Nudix enzymes hydrolyze phosphoanhydride bonds linked to nucleotides. One subclass of Nudix enzymes cleaves dinucleotides to yield two nucleoside monophosphates. The prototype enzyme of this Nudix subclass is the product of the E. coli nudC gene (aka NudC or Orf257), which has been previously shown to cleave NAD(P)H, NAD(P)+, ADP-ribose, AppA, and NAD-capped RNA. Nudix hydrolases have been known to co-purify with nucleic acids. In this study, we have developed novel assays to analyze the thermodynamics of RNA binding to NudC, by proving that NudC binds to both single stranded and double stranded oligomers using techniques like fluorescence spectroscopy and native gel electrophoresis.

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Apr 27th, 1:00 PM

New Assays to Analyze the Thermodynamics of RNA Binding to Enzymes that Remove RNA Caps

Union Wisconsin Room

Recently, the cofactor nicotinamide adenine dinucleotide (NAD) has been reported as a covalent modification of bacterial RNA. Given the central role of NAD in redox biochemistry, posttranslational protein modification and signaling, its attachment to RNA indicates that there are unknown functions of RNA in these processes and undiscovered pathways in RNA metabolism and regulation. Analogous to a eukaryotic cap, 5′-NAD modification is shown in vitro to stabilize RNA against 5′-processing by the RNA-pyrophosphohydrolase RppH and against endonucleolytic cleavage by ribonuclease (RNase) E. This is where the nudix phosphohydrolase NudC plays an important role to decap NAD-RNA and thereby triggering RNase-E-mediated RNA decay. Nudix enzymes hydrolyze phosphoanhydride bonds linked to nucleotides. One subclass of Nudix enzymes cleaves dinucleotides to yield two nucleoside monophosphates. The prototype enzyme of this Nudix subclass is the product of the E. coli nudC gene (aka NudC or Orf257), which has been previously shown to cleave NAD(P)H, NAD(P)+, ADP-ribose, AppA, and NAD-capped RNA. Nudix hydrolases have been known to co-purify with nucleic acids. In this study, we have developed novel assays to analyze the thermodynamics of RNA binding to NudC, by proving that NudC binds to both single stranded and double stranded oligomers using techniques like fluorescence spectroscopy and native gel electrophoresis.