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Related Concept Videos

RNA Splicing01:32

RNA Splicing

Splicing is the process by which eukaryotic RNA is edited before its translation into protein. The RNA strand transcribed from eukaryotic DNA is called the primary transcript. The primary transcripts that become mRNAs are called precursor messenger RNAs (pre-mRNAs). Eukaryotic pre-mRNA contains alternating sequences of exons and introns. Exons are nucleotide sequences that code for proteins, whereas introns are the non-coding regions. In RNA splicing, introns are removed and exons are bonded...
RNA Splicing01:32

RNA Splicing

Splicing is the process by which eukaryotic RNA is edited before its translation into protein. The RNA strand transcribed from eukaryotic DNA is called the primary transcript. The primary transcripts that become mRNAs are called precursor messenger RNAs (pre-mRNAs). Eukaryotic pre-mRNA contains alternating sequences of exons and introns. Exons are nucleotide sequences that code for proteins, whereas introns are the non-coding regions. In RNA splicing, introns are removed and exons are bonded...
Mitochondrial Precursor Proteins01:39

Mitochondrial Precursor Proteins

Mitochondrial precursors are partially unfolded or loosely folded polypeptide chains. Newly synthesized precursors are inhibited from spontaneously folding into their native conformation by the cytosolic chaperones, heat shock proteins 70 (Hsp70), and mitochondrial import stimulation factors (MSFs). Precursors bound to MSFs are guided to the TOM70-TOM37 receptors, while precursors bound to Hsp70  chaperones are targetted to TOM20-TOM22 receptor complexes.
Most of the mitochondrial precursors...
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...
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...
Translocation of Proteins into the Mitochondria01:19

Translocation of Proteins into the Mitochondria

Mitochondrial precursors are translocated to the internal subcompartments via independent mechanisms involving distinct protein machineries called translocases.
Sorting of outer membrane proteins:
Mitochondrial outer membrane proteins are of two types: the transmembrane, beta-barrel porins, and the membrane-anchored, alpha-helical proteins. Beta-barrel porin precursors are translocated by the TOM complex and inserted into the outer mitochondrial membrane by the SAM complex. In contrast,...

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Related Experiment Video

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ACT1-CUP1 Assays Determine the Substrate-Specific Sensitivities of Spliceosomal Mutants in Budding Yeast
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A conserved threonine spring-loads precursor for intein splicing.

Albert K Dearden1, Brian Callahan, Patrick Van Roey

  • 1Department of Physics, Applied Physics, and Astronomy, Rensselaer Polytechnic Institute, Troy, New York 12180, USA.

Protein Science : a Publication of the Protein Society
|February 21, 2013
PubMed
Summary

Protein splicing, a key biological process, involves inteins. This study reveals how a specific threonine residue induces strain, accelerating the crucial first step of protein splicing for biotechnology.

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Structural Biology

Background:

  • Protein splicing is a self-catalytic process mediated by inteins, essential in all domains of life.
  • Inteins are widely used in biotechnology for protein ligation and manipulation.
  • The initial N-S acyl shift mechanism in protein splicing remains incompletely understood.

Purpose of the Study:

  • To elucidate the structural origins and mechanistic role of conformational strain in protein splicing.
  • To identify the specific residues responsible for inducing strain at the N-terminal splice junction.
  • To understand how this strain influences the rate of the N-S acyl shift.

Main Methods:

  • Quantum mechanical simulations to model reaction pathways.
  • Site-directed mutagenesis to alter specific amino acid residues.
  • X-ray crystallography to determine high-resolution structures of intermediates.

Main Results:

  • Identified a conserved threonine residue (Thr69) in the Ssp DnaE intein as a key mediator of conformational strain.
  • Demonstrated that Thr69 induces strain via hydrogen bonding, positioning the splice junction optimally.
  • Showed that this strain significantly enhances the rate of the N-S acyl shift, the first step of splicing.

Conclusions:

  • The study provides fundamental insights into the mechanism controlling the initial step of protein splicing.
  • The findings highlight the role of conformational strain, mediated by specific residues, in enzyme catalysis.
  • Understanding this mechanism has significant implications for protein engineering and biotechnological applications involving inteins.