Cleavage site recognition

Signal peptide identification, like DNA intron/exon sequence discrimination, involves the two related problems of signal peptide discrimination (search for content) and cleavage site recognition (search for signal). It is well suited to neural network methods for several reasons. The functional units are encoded by local, linear sequences of amino acids rather than global 3-dimensional structures (Claros et al., 1997). The ambiguity of... 

The maturation of the precursor protein involves their proteolytic cleavage. There are two proteins important in this cleavage, so-called processing protease and protease enhancing peptide. These are now believed to be nonidentical subunits of the same enzyme. The structural requirements for recognition of the cleavage site are not fully understood and except for a positively charged residue at position (-2) there is no consensus sequence around this site. 

Although they often share little sequence similarity and have quite different specificiities, many restriction enzymes have similar three-dimensional structures as well as mechanisms of action. This is true for the EcoRI, BamHl (Fig. 26-5),83/90 EcoRV,91/91a and C/r 101 enzymes,84 and presumably many others. The specifically shaped and tightly packed active sites in the enzyme-substrate complexes ensure specificity. For example, the EcoRV endonuclease cleaves DNA at its recognition site at least a million times faster than at any other DNA sequence.91 As mentioned in Chapter 12, restriction endonucleases require a metal ion, preferably Mg2+, and probably act via a hydroxyl ion generated from Mg2+-OH2 at the active site. Three conserved active site residues, Asp 91, Glu 111, and Lys 113, in the EcoRI endonuclease interact with the DNA near the cleavage site. Lys 113 is replaced by Glu 113 in the BamHl enzyme.83 90... 

Finishing the transcripts. Additional modifications must be made to some mRNAs, and there will doubtless be many surprises as the details are worked out. One detail, which was discovered in the 1980s, is the specific function of snRNA U7 in recognition of the 3 end of pre-mRNAs for histones. The U7 RNA apparently base-pairs with a sequence near the 3 end cleavage site, acting as a cutting guide.47 672 673... 

A similar strategy was used to examine the potential role of a reverse turn as a recognition element adjacent to the cleavage site of substrates of HIV protease.197 A series of inhibitors was prepared, the synthesis of which involved the solution coupling of the statine-like transition state mimic to the (3-turn mimetic to provide 46 (Scheme 22). Subsequent sodium in ammonia reduction provided analogue 47. One of the compounds was a reasonably potent inhibitor of protease activity (IC50=2.6 x 10-8 M) (Table 1). 

The recognition sequences of type II restriction endonucleases are shown with the arrow (J.) indicating the cleavage site. 

To search for an appropriate restriction enzyme and its restriction profile, subject the query DNA to Webcutter at http //www.firstmarket.com/cutter/ cut2.html. Upload the sequence file (enter drive directoryseqfilename) or paste the sequence into the query box. Indicate your preferences with respect to the type of analysis, site display, and restriction enzymes to include in the analysis. After clicking the Analyze Sequence button, the restriction map (duplex sequence with restriction enzymes at the cleavage sites), as shown in Figure 9.3, is returned if Map of restriction sites is selected for display. You may also select Table of sites, sorted alphabetically by enzyme name for display which lists number of cuts, positions of sites, and recognition sequences. 

Cleavage sites for restriction enzymes occur randomly in the DNA. The frequency of a particular restriction site is dependent on the size of the DNA and the size of the recognition sequence. The probability of finding a particular four-base run in DNA is 1/256 (l/4>base-specific restriction sites in the 5243 base pair, circular genome of the simian virus SV40 is shown in Table 2.7. 

Figure 7.1 Amino acid sequences of ViP (upper box) and substance P and NkA (iower box). Note that the carboxy terminus of these peptides is amidated and amidatlon Is required for receptor recognition. Cleavage sites for NEP, ACE and tryptase are shown.

More recently, the substrate specificity of /9-secretase has been explored and compared with that of other aspartic proteases using a range of dodecameric substrates based mainly on the j3 -cleavage site of APP (67). The substrate recognition site of /3-secretase extended over several amino acids, and /9-secretase accepted a wide range of peptidic substrates. In common with other aspartic proteases, /9-secretase prefers a leucine residue at position PI. However, unlike these enzymes, /3-sccrctasc accepts polar or even acidic residues at positions PI and P2. and prefers bulky hydrophobic residues, preferably valine, at position P3. 

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