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Related Concept Videos

Transfer RNA Synthesis02:36

Transfer RNA Synthesis

One of the unique features of tRNA is the presence of modified bases. In some tRNAs, modified bases account for nearly 20% of the total bases in the molecule. Altogether, these unusual bases protect the tRNA from enzymatic degradation by RNases.
Each of these chemical modifications is carried by a specific enzyme, post-transcription. All of these enzymes have unique base and site-specificity. Methylation, the most common chemical modification, is carried by at least nine different enzymes, with...
Transcription Attenuation in Prokaryotes02:42

Transcription Attenuation in Prokaryotes

Transcriptional attenuation occurs when RNA transcription is prematurely terminated due to the formation of a terminator mRNA hairpin structure.  Bacteria use these hairpins to regulate the transcription process and control the synthesis of several amino acids including histidine, lysine, threonine, and phenylalanine. Transcription attenuation takes place in the non-coding regions of mRNA.
There are several different mechanisms used to attenuate transcription. In ribosome mediated...
tRNA Activation02:26

tRNA Activation

Aminoacyl-tRNA synthetases are present in both eukaryotes and bacteria. Though eukaryotes have 20 different aminoacyl-tRNA synthetases to couple to 20 amino acids, many bacteria do not have genes for all of these aminoacyl-tRNA synthetases. Despite this, they still use all 20 amino acids to synthesize their proteins. For instance, some bacteria do not have the gene encoding the enzyme that couples glutamine with its partner tRNA. In these organisms, one enzyme adds glutamic acid to all of the...
tRNA Activation02:26

tRNA Activation

Aminoacyl-tRNA synthetases are present in both eukaryotes and bacteria. Though eukaryotes have 20 different aminoacyl-tRNA synthetases to couple to 20 amino acids, many bacteria do not have genes for all of these aminoacyl-tRNA synthetases. Despite this, they still use all 20 amino acids to synthesize their proteins. For instance, some bacteria do not have the gene encoding the enzyme that couples glutamine with its partner tRNA. In these organisms, one enzyme adds glutamic acid to all of the...
Repressible Operon: trp Operon01:21

Repressible Operon: trp Operon

The trp operon in Escherichia coli exemplifies a repressible operon. It regulates the synthesis of tryptophan through repressor-mediated transcriptional control and attenuation. This dual regulatory mechanism ensures tryptophan biosynthesis occurs only when needed, conserving cellular resources.Structure of the trp OperonThe trp operon consists of five structural genes (trpE, trpD, trpC, trpB, and trpA) that encode enzymes for tryptophan biosynthesis. These genes are transcribed as a single...
Allosteric Proteins-ATCase01:19

Allosteric Proteins-ATCase

Binding sites linkages can regulate a protein's function.  For example, enzyme activity is often regulated through a feedback mechanism where the end product of the biochemical process serves as an inhibitor.
Aspartate transcarbamoylase (ATCase) is a cytosolic enzyme that catalyzes the condensation of L-aspartate and carbamoyl phosphate to  N-carbamoyl-L-aspartate. This reaction is the first step in pyrimidine biosynthesis. UTP and CTP, the end products of the pyrimidine synthesis pathway,...

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Exploring Sequence Space to Identify Binding Sites for Regulatory RNA-Binding Proteins
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tRNA binding, positioning, and modification by the pseudouridine synthase Pus10.

Rajashekhar Kamalampeta1, Laura C Keffer-Wilkes, Ute Kothe

  • 1Department of Chemistry and Biochemistry, University of Lethbridge, 4401 University Drive, Lethbridge, AB, T1K 3M4, Canada.

Journal of Molecular Biology
|June 8, 2013
PubMed
Summary

Pyrococcus furiosus Pus10, a pseudouridine synthase, binds tRNA with high affinity and efficiently forms pseudouridine. Its catalytic mechanism involves a thumb loop and a novel arginine residue, suggesting an induced-fit binding model.

Keywords:
Michaelis–Menten kineticsRNA bindingRNA modificationtRNAthumb loop

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Pus10 is a recently discovered pseudouridine synthase in archaea and eukaryotes.
  • It catalyzes the modification of uridine 55 in the TΨC arm of transfer RNAs (tRNAs).

Purpose of the Study:

  • To perform the first quantitative biochemical analysis of tRNA binding and pseudouridine formation by Pyrococcus furiosus Pus10.
  • To elucidate the catalytic mechanism and structural determinants of Pus10 activity.

Main Methods:

  • Quantitative biochemical assays to determine binding affinities (Kd) and kinetic parameters (Km, kcat).
  • Site-directed mutagenesis to investigate the role of specific residues and domains.
  • Analysis of tRNA binding and pseudouridine formation.

Main Results:

  • Pus10 exhibits high affinity for both substrate and product tRNA (Kd = 30 nM).
  • Efficient catalysis with Km = 400 nM and kcat = 0.9 s(-1).
  • Mutagenesis identified the thumb loop as crucial for catalysis and a novel arginine (R208) for uridine flipping; the THUMP domain aids tRNA binding.

Conclusions:

  • The thumb loop and arginine 208 are key for Pus10 catalysis and substrate positioning.
  • The THUMP domain facilitates tRNA binding.
  • TRNA binding by Pus10 likely follows an induced-fit mechanism, essential for pseudouridine formation.