Ribozyme Cleavage

Internship Report , 2014 , 11 Pages , Grade: 1,0

Chemistry - Bio-chemistry

The hammerhead ribozyme is a catalytic active RNA molecule that catalyzes the cleavage of RNA. This reaction is part of the in vivo self-cleavage mechanism that is observed in the rolling cycle replication mechanism of viroids.

The chemically synthesized hammerhead ribozyme that is used in this experiment forms of two RNA strands that harbor the catalytic activity and a substrate RNA strand as shown in figure 1. In the presence of MgCl2, the ribozyme adopts its active conformation and cleaves substrate strand.
In order to demonstrate the cleavage, the substrate strand is radioactively labeled with 32P at the 5’ end by T4 polynucleotide kinase.

Inhaltsverzeichnis

• Introduction
• Material and methods
  • 5' tagging of the substrate strand with T4 polynucleotide kinase
  • Cleavage of the substrate strand by the enyzme strand
• Results
  • 5' tagging of the substrate strand with T4 polynucleotide kinase
  • Cleavage of the substrate strand by the enyzme strand
• Discussion
  • 5' tagging of the substrate strand with T4 polynucleotide kinase
  • Cleavage of the substrate strand by the enyzme strand

Zielsetzung und Themenschwerpunkte

This experiment aims to demonstrate the catalytic activity of the hammerhead ribozyme, a self-cleaving RNA molecule. The experiment focuses on the cleavage of a substrate RNA strand by the ribozyme, which is composed of two RNA strands. The experiment utilizes radioactive labeling of the substrate strand to track the cleavage reaction.

• Catalytic activity of the hammerhead ribozyme
• RNA cleavage mechanism
• Radioactive labeling of RNA
• Enzymatic activity of T4 polynucleotide kinase
• Analysis of cleavage products

Zusammenfassung der Kapitel

The experiment begins with the 5' end labeling of the substrate strand with radioactive 32P using T4 polynucleotide kinase. This step ensures that the substrate strand can be easily tracked during the cleavage reaction. The labeled substrate strand is then incubated with the ribozyme in the presence of MgCl2, which promotes the ribozyme's active conformation and facilitates the cleavage reaction. The cleavage products are then analyzed using denaturing polyacrylamide gel electrophoresis, allowing for the visualization and quantification of the cleaved substrate strand.

Schlüsselwörter

The keywords and focus themes of the text include hammerhead ribozyme, RNA cleavage, catalytic activity, radioactive labeling, T4 polynucleotide kinase, substrate strand, enzyme strand, denaturing polyacrylamide gel electrophoresis, and analysis of cleavage products.

• College: Free University of Berlin (Institut für Chemie und Biochemie)
• Course: Methodenmodul Nukleinsäuren
• Grade: 1,0
• Publication Year: 2014
• Pages: 11
• Catalog Number: V282480
• ISBN (eBook): 9783656823452
• ISBN (Book): 9783656823445
• Language: English
• Tags: ribozyme, cleavage