Intein Mediated Ligation

A technique known as intein-mediated ligation (Iwai and Pluckthun 1999) allows for the in vitro cyclization of proteins. In this method the protein to be cyclized is fused at its C-terminus to a disabled Saccharomyces cerevisiae VMA intein (Hirata et al. 1990 Kane et al. 1990), which is itself fused at its C-terminus to a chitin-binding domain (CBD Chong et al. 1997). The intein is disabled by mutation of the C-terminal asparagine residue to alanine, thus disabling the splicing function at the...

Scale Down of Intein Processes

As mentioned above, inteins are expected to have wide use in proteomics research for recovery, isolation and analysis using miniaturized channels on a microfluidics platform and for orienting and tagging proteins in microarrays and on chips. Scaling down intein technology will have application in meeting the requirements of fast, low-volume, and parallel processing for proteomics analysis. Miniaturization is a key component with any high-throughput technology and requires substantial...

DNA Binding to the Splicing and Endonuclease Domains of PISceI

The X-ray structure of the intein PI-SceI in complex with a 36-bp duplex DNA that contains its 31-bp recognition sequence (Fig. 2) is the only structure determined thus far for an intein-associated homing endonuclease in complex with its DNA target (Moure et al. 2002). The interactions of other inteins with their substrates could show similar features, especially those that have a substantial degree of sequence homology, such as the HO endonuclease and other VMA1 inteins which recognize similar...

General Characteristics of Protein Splicing

Protein biosynthesis was initially thought to be a simple process the genetic information in DNA is directly copied into messenger RNAs, which in turn direct the biosynthesis of proteins. However, an unexpected discovery was made by two groups independently in 1990 (Hirata et al. 1990 Kane et al. 1990). In Saccharomyces cerevisiae, the nascent 120-kDa translation product of the VMA1 gene autocatalytically excises a 50-kDa site-specific endonucle-ase (VMAl-derived endonuclease VDE, also called...

Binding of the IEP to the Intron RNA

The properties of group II intron homing endonucleases are dictated by the interaction between the intron RNA and the IEP. This interaction starts when the IEP binds the intron in unspliced precursor RNA and promotes its splicing by stabilizing the catalytically active RNA structure. The Ll.LtrB IEP binds to the intron RNA as a dimer, and the binding of the cognate intron is very tight, with the apparent Kd measured as koff kon in the pM range (Saldanha et al. 1999 Rambo and Doudna 2004)....

The HNH Family A Tree of Three Branches 21 The HNH Consensus Sequence

The -34 amino acid HNH motif gained its name from the invariant histidine and asparagine residues that are characteristic of the motif. Since first described, many new sequences have been added to the group allowing a more Fig. 1. Consensus sequence and secondary structure of the HNH motif. 1 Consensus sequence for the HNH motif. Residues in bold are those that denote the motif itself, underlined residues indicate positions where hydrophobic residues stabilize the fold in the context of the...

The His Cys Box Family of Homing Endonucleases

Similar to members of other homing endonuclease families, the His-Cys box homing endonucleases are encoded by group I introns that exhibit mobility into intron-less alleles of their host genes. For all His-Cys box family members identified thus far, the host genes are nuclear rDNA loci from several species of protists including slime molds, fungi, algae, and amoebae. This endonuclease family is characterized by a series of conserved histidine and cysteine residues over -100 amino acid stretch...

References

Bayley H, Chang CY, Miller WT, Niblack B, Pan P (1998) Caged peptides and proteins by targeted chemical modification. Methods Enzymol 291 117-135 Bolduc JM, Spiegel PC, Chatterjee P, et al (2003) Structural and biochemical analyses of DNA and RNA binding by a bifunctional homing endonuclease and group I intron splicing factor. Genes Dev 17 2875-2888 Bremer MCD, Gimble FS, Thorner J, Smith CL (1992) VDE endonuclease cleaves Saccha-romyces cerevisiae genomic DNA at a single site physical mapping...

Reporter Protein Reconstitution

Minibeasts Facts For Kids Pictures

Fluorescent or bioluminescent reporter proteins have proven to be of great use for detecting a variety of cellular activities Grimm 2004 . A typical example is the reporter gene assay with a firefly or Renilla luciferase reporter gene. Activation of transcription factors leads to gene expression of the luciferase, which leads to a detectable light signal in the presence of the substrate, d-luciferin or coelenterazine. Green fluorescent protein GFP and its variants are also used as a marker of...