Catalytic Properties of the Intron RNA

Group II intron RNAs are ribozymes capable of self-splicing in vitro via two sequential transesterification reactions that result in the formation of an intron lariat (Fig. lb). Chemically, the splicing reactions are the same as those of spliceosomal introns, which are believed to be evolutionary descendants Fig. 1. Group II intron RNA secondary structure and splicing mechanism, a Conserved RNA secondary structure. The structure consists of six double-helical domains (DI-DVI) radiating from a...

The Canonical Protein Splicing Mechanism

In the protein-splicing reaction, the intein is excised and the N- and C-exteins are linked directly by a peptide bond. Early in vitro studies of protein splicing using inteins from extreme thermophiles expressed as fusion proteins in E. coli led to two general conclusions about the nature of protein splicing (1) protein splicing is mediated entirely by amino acid residues within the intein and by the adjacent C-extein residue and requires no additional protein catalysts, and (2) protein...

ScaleUp of Intein Processes

Although the advantages of intein-based bioseparations are well established at the laboratory scale, only a few very recent reports support their use at large scale (Sharma et al. 2003 Ma and Cooney 2004). Furthermore, the economic viability of inteins in the commercial production of any real therapeutic or commodity protein has yet to be proven directly. However, the practical issues associated with this type of process can be partially assessed through a theoretical analysis with a few basic...

How Intertwined Are the DNA and RNA Activities in Maturases

The fact that bifunctional LAGLIDADG DNA endonucleases RNA maturases exist raises the question of whether or not such proteins use a single binding site to interact with their nucleic acid ligands. Indeed, the similarity between respective DNA-binding sites and the RNA structure around the 5' and 3' SS (P1 P10 pseudoknot Fig. 2b) has fueled speculation that such proteins may use analogous interactions to perform both functions (reviewed in Lam-bowitz et al. 1999). However, in vitro experiments...

Engineering Homing Enzymes with Novel Functions

4.1 Changing the Recognition Site Specificity of Homing Endonucleases Homing endonucleases have become invaluable tools to study complex genomes. By virtue of its ability to introduce unique breaks into complex genomes that mimic lesions made by DNA-damaging agents, I-Scel is commonly used to study DNA repair pathways in organisms ranging from yeast to humans (see, for example, Johnson and Jasin 2001). Moreover, this enzyme stimulates gene targeting over 1000-fold in mammalian systems through...

Applications Turning Junk into Gold

The title of this subsection has been borrowed from that of an essay on the practical application of introns and inteins (Wickelgren 2003). Basically, the topic breaks down into the utility of homing endonucleases, group II intron RNPs, and inteins. 5.1 Site-Specific Group I Intron and Intein Endonucleases The engineering of DNA often demands cleavage at rare sites and, to some extent, the highly site-specific endonucleases of group I introns and inteins fulfill the requirement. One need simply...

The HO Endonuclease Site in MATal

The origin of HO is intimately associated with the structure of the MAT locus. In S. cerevisiae, the a and a idiomorphs (versions) of the MAT locus differ only by their Ya or Ya regions. Ya contains the complete coding region of the a-specific MATal gene, but Ya contains only the 5' portions of the a-specific genes MATa 1 and MATal which are transcribed divergently (Fig. 1). This arrangement ensures that each of the three MAT genes is only expressed in the correct cell type, but it also means...

The Mechanism of Maturase Assisted Splicing

Maturases are relatively small proteins (-30 kDa) that interact with much larger RNAs e.g., -380 kDa for the average size of maturase encoding introns, 1200nucleotides (nt) . How might such a small protein recognize a large RNA and facilitate splicing Since maturases are intron-specific cofactors (see above), they must recognize idiosyncratic features of their cognate introns but what governs maturase specificity remains a mystery. The yeast maturases are expressed as fusion proteins with the...

Mating Type Genes in S cerevisiae

Many fungi use some form of homothallic mating-type switching to produce diploids from haploid meiotic segregants. In budding yeast MATalMATa diploids are created by conjugation of haploid MATa and MATa cells. The MATa Fig. 1. Arrangement of mating-type genes in S. cerevisiae. The MAT locus is approximately 2400 bp. Mating-type-specific Ya and Ya sequences are about 650 and 750 bp, respectively, and are surrounded by 700-bp W and X regions, a 230-bp Z1 region and a 90-bp Z2 region. The...

Enzymology of HNHpaMe Motif Endonucleases

3.1 The HNH pp -Me Motif Is Functionally Adaptable Enzymes that contain the HNH ppa-Me motif and no other DNA recognition domains, as is the case for the bacterial colicins Fig. 4.1 , CAD and Serratia nuclease, show little cleavage specificity. This is consistent with the biological roles for these proteins whose function it is to degrade intracellular DNA colicins, CAD , or, as in the case of Serratia nuclease, catabolize extracellular nucleic acids for nutrient uptake Friedhoff et al. 1999...

HNH and His Cys Box Homing Endonucleases

The HNH family of endonucleases is characterized by a defining motif that spans 25 residues of a well-conserved sequence. The homing endonucleases I-Cmoel, I-TevIII, I-Hmul, and I-HmuII Eddy and Gold 1991 Goodrich-Blair and Shub 1996 are members of the HNH family, and I-Cmoel has been well characterized biochemically Drouin et al. 2000 for a full description of these enzymes, see Keeble et al., this Volume . The non-specific DNase colicin E9 was the first HNH enzyme to have its structure solved...

Selection and Reporter Systems for Intein Function

Direct observation of intein products through SDS-PAGE and by Western blot are still used in many cases, particularly for the verification of splicing with newly discovered inteins in their native hosts and for the observation of splicing from artificial precursors in Escherichia coli Liu and Yang 2004 Mills et al. 2004 Southworth et al. 2004 . They have even been used to screen small libraries of up to several hundred mutants for loss of splicing function Kawasaki et al. 1997 . However, these...

Protein transSplicing and Cleavage Systems

Protein Cleavage

The N- and C-terminal segments of an artificially or naturally split intein, each segment being fused to a foreign protein sequence, are able to assemble and mediate protein trans-splicing, yielding a native peptide bond between the two foreign protein sequences Lew et al. 1998 Mills et al. 1998 South- worth et al. 1998 Wu et al. 1998a,b Yamazaki et al. 1998 Evans et al. 2000 . In particular, studies of a naturally occurring split intein from the dnaE gene of Synechocystis sp. PCC6803 Ssp DnaE...

Biochemical Activities of Group II Intron Encoded Proteins

Group II IEPs, examples of which are shown in Fig. 2, contain several domains associated with different biochemical activities. The RT domain at the N-ter-minus contains conserved sequence blocks RT-1 to -7, characteristic of the fingers and palm of retroviral RTs, along with an additional upstream motif, RT-0 Xiong and Eickbush 1990 Malik et al. 1999 Zimmerly et al. 2001 . The latter is found only in non-LTR-retroelement RTs and may be part of an extended fingers subdomain used for specific...

References

Aylon Y, Kupiec M 2004 DSB repair the yeast paradigm. DNA Repair Amst 3 797-815 Bakhrat A, Jurica MS, Stoddard BL, Raveh D 2004 Homology modeling and mutational analysis of Ho endonuclease of yeast. Genetics 166 721-728 Bressan DA, Vazquez J, Haber JE 2004 Mating type-dependent constraints on the mobility of the left arm of yeast chromosome III. J Cell Biol 164 361-371 Butler G, Kenny C, Fagan A, Kurischko C, Gaillardin C, Wolfe KH 2004 Evolution of the MATlocus and its Ho endonuclease in yeast...

Intein Mediated Protein Ligation IPL

Intein Protein Ligation

Unlike other affinity fusion systems that mainly serve to simplify protein purification, the intein fusion systems can be modulated to perform numerous other tasks, one of which is intein-mediated protein ligation IPL Evans et al. 1998 Evans and Xu 1999 or expressed protein ligation Muir et al. 1998 Severinov and Muir 1998 . The IPL technique has been used for incorporation of non-coded amino acids into a protein sequence, segmental labeling of proteins for NMR analysis, addition of fluorescent...

Intein Fusion Systems for Protein Purification

Intein Fusion Protein

Protein purification can be simplified by fusion of the protein of interest to an affinity tag, such as poly-His tag, MBP tag, etc. LaVallie and McCoy 1995 . However, removal of the affinity tag by protease treatment and subsequent purification steps are often costly and time-consuming. The discovery of inteins and the elucidation of protein-splicing mechanisms provided a novel approach to affinity protein purification Xu et al. 2000 . The ability of an intein to catalyze peptide bond cleavage...

Mechanisms of DNA Target Site Recognition and Specificity

As catalysts of the genetic mobility of introns and inteins, LAGLIDADG homing endonucleases as well as other enzymes with the same biological func tion must balance two somewhat contradictory requirements they need to be highly sequence-specific, in order to promote precise intron transfer in their host genomes which are most often a chloroplast or mitochondrial genome, and yet must retain sufficient site recognition flexibility to allow successful lateral transfer in the face of sequence...

The Group I Intron Encoded Homing Endonuclease IScel

I knew that I-Scel could not be similar to the bacterial restriction enzymes for two reasons. First, E. coli survived my plasmid expression experiments. Second, more importantly, during mitochondrial crosses, the novel intron copy was always inserted at the appropriate location, not elsewhere in the mitochondrial genome. Therefore, my enzyme had to be specific enough to cleave only one site in the mitochondrial genome. The parallel with HO was also suggestive Kostriken and Heffron 1984 . My...

Homing Endonucleases and the Yeast Mitochondrial Locus A Historical Perspective

Outside the laboratory, near Paris, nature was exulting in its colorful mid-spring glory. The last technical details and experimental pitfalls had been fixed in the preceding weeks. Now, the site-specific endonucleolytic activity of the intron-encoded protein that I had carefully engineered to express in Escherichia coli was detectable. According to my autoradiogram, it was cleaving the intron-less DNA exactly where I expected. This experiment opened the way to a series of yet...

TevI as the Model Giyyig Enzyme Structure and Function

I-TevI is the most extensively studied GIY-YIG endonuclease. It is encoded by a group I intron found in the thymidylate synthase td gene of bacteriophage T4. As described in other chapters in this volume, homing endonucleases are site-specific enzymes that recognize fairly lengthy DNA targets with some degree of sequence tolerance. Even given this description, I-TevI is extremely unusual in that the 28-kDa protein recognizes a 37-bp target site as a monomer Mueller et al. 1995 and that it can...

Maturases or DNA Endonucleases

All group I intron maturases contain two copies of a conserved amino acid motif designated LAGLIDADG. Over 200 other proteins also contain one or two copies of this motif and most of these proteins are site-specific DNA endonucleases that function in genetic mobility a process called homing Dujon 1989 Chevalier and Stoddard 2001 . Genes coding for these proteins are found in self-splicing polypeptide inteins, and group I and II introns as well as free standing ORFs Chevalier and Stoddard 2001...

Use of the IScel Endonuclease in Heterologous Systems

At the end of 1987,1 moved my laboratory from the CNRS at Gif-sur-Yvette to the Institut Pasteur in Paris. Given the exceptionally long recognition sequence of I-Scel, I was foreseeing a tool to cleave chromosomes, and possibly entire genomes, at a single predetermined site. Given the then emerging genome programs, especially the yeast sequencing project, YAC cloning and the first gene replacements in mammalian cells, this sounded to me like an attractive idea. One difficulty was the partial...

Structures of Laglidadg Homing Endonucleases

The structures of six LAGLIDADG enzymes bound to their DNA targets have been determined. These include two isoschizomeric homodimers I-Crel Heath et al. 1997 Jurica et al. 1998 Chevalier et al. 2001,2003 and I-Msol Chevalier et al. 2003 , which are both encoded within group I introns in the 23S rDNA of the green algae Chlamydomonas reinhardtii and Monomastix two pseudo-symmetric monomers I-Anil Bolduc et al. 2003 and I-Scel Moure et al. 2003 , which are encoded in mitochondrial introns of the...