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Related Concept Videos

Ribozymes02:47

Ribozymes

The term ribozyme is used for RNA that can act as an enzyme. Ribozymes are mainly found in selected viruses, bacteria, plant organelles, and lower eukaryotes. Ribozymes were first discovered in 1982 when Tom Cech’s laboratory observed Group I introns acting as enzymes. This was shortly followed by the discovery of another ribozyme, Ribonulcease P, by Sid Altman’s laboratory. Both Cech and Altman received the Nobel Prize in chemistry in 1989 for their work on ribozymes.
Ribozymes can be...
Ribozymes02:47

Ribozymes

The term ribozyme is used for RNA that can act as an enzyme. Ribozymes are mainly found in selected viruses, bacteria, plant organelles, and lower eukaryotes. Ribozymes were first discovered in 1982 when Tom Cech’s laboratory observed Group I introns acting as enzymes. This was shortly followed by the discovery of another ribozyme, Ribonulcease P, by Sid Altman’s laboratory. Both Cech and Altman received the Nobel Prize in chemistry in 1989 for their work on ribozymes.
Ribozymes can be...
Introduction to Mechanisms of Enzyme Catalysis01:13

Introduction to Mechanisms of Enzyme Catalysis

For many years, scientists thought that enzyme-substrate binding took place in a simple "lock-and-key" fashion. This model stated that the enzyme and substrate fit together perfectly in one instantaneous step. However, current research supports a more refined view scientists call induced fit. The induced-fit model expands upon the lock-and-key model by describing a more dynamic interaction between enzyme and substrate. As the enzyme and substrate come together, their interaction causes a mild...
Introduction to Mechanisms of Enzyme Catalysis01:13

Introduction to Mechanisms of Enzyme Catalysis

For many years, scientists thought that enzyme-substrate binding took place in a simple "lock-and-key" fashion. This model stated that the enzyme and substrate fit together perfectly in one instantaneous step. However, current research supports a more refined view scientists call induced fit. The induced-fit model expands upon the lock-and-key model by describing a more dynamic interaction between enzyme and substrate. As the enzyme and substrate come together, their interaction causes a mild...
ATP Synthase: Mechanism01:48

ATP Synthase: Mechanism

In animals, the mitochondrial F1F0 ATP synthase is the key protein that synthesizes ATP molecules through a complex catalytic mechanism. While the nuclear genome encodes the majority of ATP synthase subunits, the mitochondrial genome encodes some of the enzyme's most critical components. The formation of this multi-subunit enzyme is a complex multi-step process regulated at the level of transcription, translation, and assembly. Defects in one or more of these steps can result in decreased ATP...
Improving Translational Accuracy02:07

Improving Translational Accuracy

Base complementarity between the three base pairs of mRNA codon and the tRNA anticodon is not a failsafe mechanism. Inaccuracies can range from a single mismatch to no correct base pairing at all. The free energy difference between the correct and nearly correct base pairs can be as small as 3 kcal/ mol. With complementarity being the only proofreading step, the estimated error frequency would be one wrong amino acid in every 100 amino acids incorporated. However, error frequencies observed in...

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Probing general base catalysis in the hammerhead ribozyme.

Jason M Thomas1, David M Perrin

  • 1Department of Chemistry, University of British Columbia, 2036 Main Mall, Vancouver, British Columbia, Canada, V6T 1Z1.

Journal of the American Chemical Society
|October 28, 2008
PubMed
Summary

Researchers identified a key functional group in the hammerhead ribozyme using an affinity label. This study reveals that guanine-12 (G12) acts as a general base catalyst in RNA cleavage.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Enzymology

Background:

  • Hammerhead ribozymes are crucial RNA enzymes involved in RNA cleavage.
  • Previous studies suggested specific functional groups participate in general/acid-base catalysis.
  • The precise role of these groups in the catalytic mechanism remained unclear.

Purpose of the Study:

  • To identify the functional general base in the *S. mansoni* hammerhead ribozyme.
  • To test the hypothesis that specific ribozyme functional groups are directly involved in catalysis.
  • To investigate the role of guanine-12 (G12) in the catalytic mechanism.

Main Methods:

  • Development of an affinity label (2'-bromoacetamide) to target the general base.
  • Reaction of the hammerhead ribozyme with the substrate analogue.
  • Identification of the alkylated residue using footprinting analysis.
  • pH and magnesium ion ([Mg2+]) dependency studies.

Main Results:

  • Demonstrated alkylation of N1 of guanine-12 (G12) in a pH and [Mg2+] dependent manner.
  • Observed alkylation patterns consistent with the native RNA cleavage reaction.
  • Provided evidence that deprotonated N1 of G12 functions as a general base catalyst.
  • Indicated a downward perturbation of the pKa of G12 within the active site.
  • Identified pH-independent alkylations providing insights into ribozyme structure.

Conclusions:

  • Guanine-12 (G12) directly functions as a general base in hammerhead ribozyme catalysis.
  • The active site structure lowers the pKa of G12, facilitating its role.
  • The study provides significant evidence for the catalytic mechanism of hammerhead ribozymes.
  • Further insights into ribozyme conformation and structure were gained.