Ribonuclease modified

Further computational studies on adenines and adenosines concern the reaction mechanism of ribonuclease A with cytidyl-3,5 -adenosine [99BP697] and the molecular recognition of modified adenine nucleotides [99JMC5338]. 

Cotham WE, Metz TO, Ferguson PL, et al. Proteomic analysis of arginine adducts on glyoxal-modified ribonuclease. Mol. Cell. Proteomics 2004 3 1145-1153. 

If polyribonucleotides are treated simultaneously with methoxylamine and bisulphite, cytidine residues are converted into 5,6-dihydro-7V4-methoxycytidine-6-sulphonate,154 and uridine into 5,6-dihydrouridine-6-sulphonate.155 Treatment with dilute ammonia regenerates the uridine residues, leaving the dihydrocytidine derivatives unaffected. When only the cytidine residues have been derivatized, pancreatic ribonuclease becomes uridyl ribonuclease, since it is unable to cleave the chain on the 3 -side of the modified cytidine.154 This allows the isolation of blocks of modified cytidine residues. T2 ribonuclease may also be used. Alternatively, a ribonuclease from Physarum polycephalum has been found to hydrolyse CpX links very slowly, allowing the isolation of cytidine blocks.156 If both uridine and cytidine residues are modified, T2 ribonuclease acts as puryl ribonuclease, allowing the isolation of cumulative blocks of pyrimidines.155 This ability to alter the specificity of nuclease cleavage is a useful tool in sequence analysis. 

The S-ribonuclease is the complex formed between an eicosapeptide and the S-RNAse. While replacement of various amino acids by fluorinated analogues does not modify the activity of the native complex, replacement of His-12 by 4-F-His has a strong influence. Indeed, the S-ribonuclease, formed between the bovine pancreatic S-RNAse and the fluoro peptide that contains 4-F-His, has no more catalytic activity, but it is stable. This loss of enzymatic activity is probably due to the significant lowering of the pAia of the catalytic His (2.5 units), which results from the presence of the fluorine atom. It is known that histidine plays an important role in nucleophilic and acid-base processes, which are connected to the catalytic activity of numerous enzymes. 

Scheme 16 Enzyme-Catalyzed Fragment Synthesis of Ribonuclease A in Aqueous Solution Using a Modified Subtiligase[771...

A primary transcript is a linear copy of a transcriptional unit—the segment of DNA between specific initiation and termination sequences. The primary transcripts of both prokaryotic and eukaryotic tRNAs and rRNAs are post-transcriptionally modified by cleavage of the original transcripts by ribonucleases. tRNAs are then further modified to help give each species its unique identity. In contrast, prokaryotic mRNA is generally identical to its primary transcript, whereas eukaryotic nrRNA is extensively modified posttranscriptionally. 

Ribosomal RNAs of both prokaryotic and eukaryotic cells are synthesized from long precursor molecules called preribosomal RNAs. The 23S, 16S, and 5S ribosomal RNAs of prokaryotes are produced from a single RNA precursor molecule, as are the 28S, 18S, and 5.8S rRNAs of eukaryotes (Figure 30.15). [Note Eukaryotic 5S rRNA is synthesized by RNA polymerase III and modified sepa-j rately.] The preribosomal RNAs are cleaved by ribonucleases to yield intermediate-sized pieces of rRNA, which are further "trimmed"... 

Prokaryotic and eukaryotic tRNAs are also made from longer precursor molecules. These must have an intervening sequence (intron) removed, and the 5 - and 3 -ends of the molecule are trimmed by ribonuclease. A 3 -CCA sequence is added and bases at specific positions are modified, producing "unusual" bases. 

Ribonuclease A was the first enzyme to be synthesized in the laboratory. Fully active ribonuclease has been synthesized,752 as have new modified enzymes. For example a 63-residue peptide made up of five segments of the native RNase sequence retained measurable catalytic activity.753 Using total synthesis, unnatural amino acids, such as 4-fluorohistidine, have been incorporated at specific positions in RNAse.752... 

The enzyme consists of a single polypeptide chain of Mr 13 680 and 124 amino acid residues.187,188 The bond between Ala-20 and Ser-21 may be cleaved by subtilisin. Interestingly, the peptide remains attached to the rest of the protein by noncovalent bonds. The modified protein, called ribonuclease S, and the native protein, now termed ribonuclease A, have identical catalytic activities. Because of its small size, its availability, and its ruggedness, ribonuclease is very amenable to physical and chemical study. It was the first enzyme to be sequenced.187 The crystal structures of both forms of the enzyme were solved at 2.0-A resolution several years ago.189,190 Subsequently, crystal structures of many complexes of the enzyme with substrate and transition analogues and products have been solved at very high resolution.191 Further, because the catalytic activity depends on the ionizations of two histidine residues, the enzyme has been extensively studied by NMR (the imidazole rings of histidines are easily studied by this method—see Chapter 5). 

The first attempts to determine the structure of a productively bound enzyme-substrate complex were based on extrapolation from the structures of stable enzyme-inhibitor complexes. (The classic example of this, lysozyme, is discussed in section F3.) Such extrapolation may be done in several ways. For example, a portion of the substrate may be bound to the enzyme and the structure of the remainder determined by model building. An alternative method is to use a substrate analogue that is unreactive because its reactive bond is modified. Typical examples are the binding of phosphoglycolohydroxamate, a substrate analogue, to triosephosphate isomerase,44 or a piece of DNA, which lacks the reactive 2 -OH groups, to a ribonuclease.45... 

In view of the high stability of the enzyme most samples have been prepared by the procedure described by Kunitz (16) and modified by McDonald (17) to remove all traces of proteolytic activity. During this procedure the minced bovine pancreas is exposed to 0.25 N sulfuric acid, ammonium sulfate precipitation, 10 min at 95°-100° and pH 3, and, finally, reprecipitation. The product can be crystallized it was also shown later to contain a number of components all with ribonuclease activity. A practical summary of all details is given by Kunitz and McDonald (18). 

The most common method employs the substitution of all pyrimidine nucleotides by their 2, -NH2- or 2 -F-analogs [31]. These confer increased stability to the RNA, because most ribonucleases need the 2 -OH for cleaving RNA. Substitution of only the pyrimidine nucleotides increases the half-life of RNA from seconds to days [28]. Such -modified RNAs are suitable for the detection of analytes in biological samples like blood, serum, or urine. 

Pieken, W.A., Olsen, D.B., Benseler, F., Aurup, H. and Eckstein, F. (1991) Kinetic characterization of ribonuclease-resistant 2 -modified hammerhead ribozymes. Science, 253, 314-317. 

E. coli has seven rRNA transcription units, each containing one copy each of the 23S, 16S and 5S rRNA genes as well as one to four tRNA genes. Transcription produces a 30S pre-rRNA transcript. This folds up to form stem-loop structures, ribosomal proteins bind, and a number of nucleotides become methylated. The modified pre-rRNA transcript is then cleaved at specific sites by RNase III and the ends are trimmed by ribonucleases M5, M6 and M23 to release the mature rRNAs. 

Fig. 6.18. Electrochromatogram of four basic proteins obtained by isocratic separation using a modified polychloromethylstyrene-based PLOT column (Reprinted with permission from [50]. Copyright 1999 Elsevier). Column 47 cm (active length 40 cm) x 20 pm, inner polymer layer 2 pm mobile phase 20% acetonitrile in 20 mmol/1 phosphate buffer pH 2.5 voltage -30 kV EOF velocity measured with dimethylsulfoxide (DMSO) -3.46 x 10"8 m2V ls 1, migration time for DMSO 3.10 min. Peaks a-chymotrypsinogen (1), ribonuclease (2), lysozyme (3), cytochrome C (4).

Fig. 7.10. Electrochromatogram of protein separation on an etched diol-modified capillary. L (total capillary length) = 45 cm, 1 (effective capillary length) = 25 cm, i.d. = 50 pm, V = 22 kV, i = 7 pm, pH = 4.41. Solutes 1 = cytochrome c 2 = lysozyme (turkey) 3 = myoglobin and 4 = ribonuclease A.

Several types of etched capillaries have been tested with respect to within column reproducibility. For example, the reproducibility of the migration times for 151 consecutive injections of the proteins lysozyme and ribonuclease A was tested on an etched Ci8 modified column at pH = 3.0 [20]. It was found that both solutes gave no discernible increase or decrease in migration time (tM) and its overall reproducibility... 

Cytochrome c, lysozyme, myoglobin, ribonuclease A C18, etched and modified column diol open tubular column 30 mMphosphate, pH 2.14, 30 mM citric acid, pH 3-25 mM p-alanine, 30 mM acetic acid, pH 4.14 450 mm x 50 pm i.d. 250 mm packed length 103... 

Mixture of peptides and proteins (lysozyme, angiotensin I and III, bradykinin, ribonuclease A) Cl 8-modified etched capillary 30 mM citric acid-24.5 mM P-alanine, pH 3.0 450 mm x 50 pm i.d. 250 mm effective length 105 ... 

Chemical synthesis has provided an additional route to peptides containing halogenat-ed amino acids. Early 19F-NMR studies of proteins were performed on semi-synthetic polypeptides prepared by attachment of fluorinated probes to the polypeptide. For example, Heustis and Raftery modified ribonuclease by trifluoroacetylation of Lys residues 1 and 7. They then used 19F-NMR to study conformational changes brought about by the presence of inhibitors200. In his review, Gerig provides several other examples of this strategy187. 

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