Recognition of cleavage site A(2) in the yeast pre-rRNA.

Processing of the yeast pre-rRNA at site A(2) internal transcribed spacer 1(ITS1) has been shown to require several small nucleolar ribonucleoprotein particles (snoRNPs) as trans-acting factors. Here we report a detailed mutational analysis of the cid-acting signals required to specify the site of A(2) lie in the 3'-flanking sequence; deletion or substitution of nucleotides in this region strongly inhibits processing, and residual cleavage is inaccurate at the nucleotide level. In contrast, the deletion of the 5'- flanking nucleotides has no detectable effect on processing. An evolutionarily conserved sequence, ACAC, is located at the site of cleavage. Substitution of the 3' AC leads to heterogeneous cleavage, with activation of cleavage at an upstream ACAC sequence, In all mutants that retain an ACAC element, a site of cleavage is detected immediately 5' to this sequence, showing that this element is recognized. An ACAC sequence is, however, not essential for accurate cleavage of site A(2). An additional signal is also present 3' to A(2), in a region that has the potential to form a stem-loop structure that is evolutionarily conserved, but of low stability. As has been found for site A(1) (the 5' end of the yeast 18S rRNA), the identification of the site of processing at A(2) relies on multiple recognition elements.     

Deoxyribonuclease II as a probe for chromatin structure. I. Location of cleavage sites

DNAase II has been shown to cleave condensed mouse liver chromatin at 100-bp² intervals while chromatin in the extended form is cleaved at 200-bp intervals (Altenburger et al., 1976). Evidence is presented here that DNA digestion patterns of a half-nucleosomal periodicity are also obtained upon DNAase II digestion of chicken erythrocyte nuclei and yeast nuclei, both of which differ in their repeat lengths (210 and 165 bp) from mouse liver chromatin. In the digestion of mouse liver nuclei a shift from the 100-bp to the 200-bp cleavage mode takes place when the concentration of monovalent cations present during digestion is decreased below 1 mM. When soluble chromatin prepared by micrococcal nuclease is digested with DNAase II the same type of shift occurs, albeit at higher ionic strength.In order to map the positions of the DNAase II cleavage sites on the DNA relative to the positions of the nucleosome cores, the susceptibility of DNAase II-derived DNA termini to exonuclease III was investigated. In addition, oligonucleosome fractions from HaeIII and micrococcal nuclease digests were end-labelled with polynucleotide kinase and digested with DNAase II under conditions leading to 100 and 200-bp digestion patterns. Analysis of the chain lengths of the resulting radioactively labelled fragments together with the results of the exonuclease assay allow the following conclusions. In the 200-bp digestion mode, DNAase II cleaves exclusively in the internucleosomal linker region. Also in the 100-bp mode cleavage occurs initially in the linker region. Subsequently, DNAase II cleaves at intranucleosomal locations, which are not, however, in the centre of the nucleosome but instead around positions 20 and 125 of the DNA associated with the nucleosome core. At late stages of digestion intranucleosomal cuts predominate and linkers that are still intact are largely resistant to DNAase II due to interactions between adjacent nucleosomes. These findings offer an explanation for the sensitivity of DNAase II to the higher-order structure of chromatin.     

Location of the primary sites of micrococcal nuclease cleavage on the nucleosome core

The positions and relative frequencies of the primary cleavages made by micrococcal nuclease on the DNA of nucleosome core particles have been found by fractionating the double-stranded products of digestion and examining their single-stranded compositions. This approach overcomes the problems caused by secondary events such as the exonucleolytic and pseudo-double-stranded actions of the nuclease and, combined with the use of high resolution gel electrophoresis, enables the cutting site positions to be determined with a higher precision than has been achieved hitherto. The micrococcal nuclease primary cleavage sites lie close (on average, within 0.5 nucleotide) to those previously determined by Lutter (1981) for the nucleases DNase I and DNase II. These similarities show that the accessible regions are the same for all three nucleases, the cleavage sites being dictated by the structure of the nucleosome core. The differences in the final products of the digestion are explained in terms of secondary cleavage events of micrococcal nuclease. While the strongly protected regions of the nucleosome core DNA are common to all three nucleases, there are differences in the relative degrees of cutting at the more exposed sites characteristic of the particular enzyme. In particular, micrococcal nuclease shows a marked polarity in the 3'-5' direction in the cutting rates as plotted along a single strand of the nucleosomal DNA. This is explained in terms of the three-dimensional structure of the nucleosome where, in any accessible region of the double helix, the innermost strand is shielded by the outermost strand on the one side and the histone core on the other. The final part of the paper is concerned with the preference of micrococcal nuclease to cleave at (A,T) sequences in chromatin.     

Location in bacteriophage lamdba DNA of cleavage sites of the site-specific endonuclease from Bacillus amyloliquefaciens H.

The sites in Escherichia coli bacteriophage lambda DNA cleaved by the site-specific endonuclease isolated from Bacillus amyloliquefaciens H (BamI) are found to be at 0.114, 0.466, 0.580, 0.713, and 0.861 lambda units. The sites were located by analysis of the products of digestion of lambda DNA and lambda-ara transducing phage DNA, and verified by double digestion with BamI and EcoRI.     

Production of collagenase by mouse peritoneal macrophages in vitro. Characterization of sites of cleavage of tropocollagen.

Macrophages from mineral oil-stimulated mice produce collagenase at a constant rate over several days in culture. Phagocytosis of latex does not increase production of enzyme. Gel electrophoretic and electron microscopic analyses indicate that the specificity of the macrophage enzyme is similar to that of other previously characterized mammalian collagenases.     

Characterization of a ribonuclease III-like protein required for cleavage of the pre-rRNA in the 3'ETS in Arabidopsis

Ribonuclease III (RNaseIII) is responsible for processing and maturation of RNA precursors into functional rRNA, mRNA and other small RNA. In contrast to bacterial and yeast cells, higher eukaryotes contain at least three classes of RNaseIII, including class IV or dicer-like proteins. Here, we describe the functional characterization of AtRTL2, an Arabidopsis thaliana RNaseIII-like protein that belongs to a small family of genes distinct from the dicer family. We demonstrate that AtRTL2 is required for 3'external transcribed spacer (ETS) cleavage of the pre-rRNA in vivo. AtRTL2 localizes in the nucleus and cytoplasm, a nuclear export signal (NES) in the N-terminal sequence probably controlling AtRTL2 cellular localization. The modeled 3D structure of the RNaseIII domain of AtRTL2 is similar to the bacterial RNaseIII domain, suggesting a comparable catalytic mechanism. However, unlike bacterial RNaseIII, the AtRTL2 protein forms a highly salt-resistant homodimer that is only disrupted on treatment with DTT. These data indicate that AtRTL2 may use a dimeric mechanism to cleave double-stranded RNA, but unlike bacterial or yeast RNase III proteins, AtRTL2 forms homodimers through formation of disulfide bonds, suggesting that redox conditions may operate to regulate the activity of RNaseIII.     

Lead cleavage sites in the core structure of group I intron-RNA.

Self-splicing of group I introns requires divalent metal ions to promote catalysis as well as for the correct folding of the RNA. Lead cleavage has been used to probe the intron RNA for divalent metal ion binding sites. In the conserved core of the intron, only two sites of Pb2+ cleavage have been detected, which are located close to the substrate binding sites in the junction J8/7 and at the bulged nucleotide in the P7 stem. Both lead cleavages can be inhibited by high concentrations of Mg2+ and Mn2+ ions, suggesting that they displace Pb2+ ions from the binding sites. The RNA is protected from lead cleavage by 2'-deoxyGTP, a competitive inhibitor of splicing. The two major lead induced cleavages are both located in the conserved core of the intron and at phosphates, which had independently been demonstrated to interact with magnesium ions and to be essential for splicing. Thus, we suggest that the conditions required for lead cleavage occur mainly at those sites, where divalent ions bind that are functionally involved in catalysis. We propose lead cleavage analysis of functional RNA to be a useful tool for mapping functional magnesium ion binding sites.     

Comparative analysis of neuropeptide cleavage sites in human, mouse, rat, and cattle

Neuropeptides are an important class of signaling molecules that result from complex and variable posttranslational processing of precursor proteins and thus are difficult to identify based solely on genomic information. Bioinformatics prediction of precursor cleavage sites can support effective biochemical characterization of neuropeptides. Neuropeptide cleavage models were developed using comprehensive human, mouse, rat, and cattle precursor data sets and used to compare predicted neuropeptide processing across these species. Logistic regression and artificial neural network models were used to predict cleavages based on amino acid and physiochemical properties of amino acids at precursor sequence locations proximal to cleavage. Correct cleavage classification rates across species and models ranged from 85% to 100%, suggesting that amino acid and amino acid properties have major impact on the probability of cleavage and that these factors have comparable effects in human, mouse, rat, and cattle. The variable accuracy of each species-specific model to predict cleavage sites indicated that there are species- and precursor-specific processing patterns. Prediction of mouse cleavages using rat models was highly accurate, yet the reverse was not observed. Sensitivity and specificity revealed that logistic models are well suited to maximize the rate of true noncleavage predictions with moderate rates of true cleavage predictions; meanwhile, artificial neural networks maximize the rate of true cleavage predictions with moderate to low true noncleavage predictions. Logistic models also provided insights into the strength of the amino acid associations with cleavage. Prediction of neuropeptide cleavage sites using human, mouse, rat, and cattle models are available at . Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Allison Tegge and Bruce Southey contributed equally to this work.     

EcoRI cleavage sites in the argECBH region of the Escherichia coli chromosome.

The EcoRI cleavage of deoxyribonucleic acids (DNAs) from lambdadarg phages, carrying argECBH, has been examined. The phages are derived from the heat-inducible, lysis-defective strain lambda y199, and their bacterial DNA, including argECBH, is derived from Escherichia coli K-12. Such cleavage of the phage DNAs, in each case, produces the D, E, and F segments of lambda. Additionally, these DNAs yield segments, ordered from left to right, of length (in kilobases [kb]) determined by electron microscopy and 0.7% agarose slab gel electrophoresis as follows: lambdadarg13 (ppc argECBH bfe), 13.9, 2.8, 1.5, and 5.6; lambdadarg14 (ppc argECBH), 3.0, 2.0, 17.3, and 6.2; and lambdadarg23 (argECBH), 18.4 and 6.2. For lambdadarg13 sup102 DNA, the segment analogous to the 13.9-kb segment measures 12.2 kb. The direction from left to right corresponds to the clockwise orientation of the E. coli genetic map. The EcoRI segments define five cleavage sites near the arg region of the E. coli chromosome. For each of the DNAs, the arg genes occur on the largest segment produced. The 17.3-kb segment, being entirely bacterial, represents the argECBH-bearing EcoRI segment of the E. coli chromosome. The location of the arg genes was demonstrated electron microscopically in heteroduplex experiments.     

Sphingosine kinase-1 is cleaved by cathepsin B in vitro: identification of the initial cleavage sites for the protease

Previous work has identified sphingosine kinase-1 (SK1) as a substrate for the cysteine protease cathepsin B in vitro. In this study, the mechanism of SK1 cleavage by cathepsin B was investigated. We identified two initial cleavage sites for the protease, the first at histidine 122 and the second at arginine 199. Mutation analysis showed that replacement of histidine 122 with a tyrosine maintained the activity of SK1 while significantly reducing cleavage by cathepsin B at the initial cleavage site. The efficacy of cleavage of SK1 at arginine 199, however, was not affected. These studies demonstrate that SK1 is cleaved by cathepsin B in a sequential manner after basic amino acids, and that the initial cleavages at the two identified sites occur independently of each other.     

Pancreatic DNAase cleavage sites in nuclei

The DNA of nuclei is cleaved by a variety of nucleases in such a way that the cuts on a given strand are always separated by an integral multiple of 10 nucleotides. However, the spacing between cutting sites on opposite strands is not known for any nuclease. In this paper, we describe the determination of the spacing, or stagger, between cuts on opposite strands produced by the action of pancreatic DNAase (DNAase I) on nuclei. When nuclei are digested with DNAase I and the resultant DNA is analyzed by gel electrophoresis without prior denaturation, a complex pattern of bands is observed. A method which gives better than 90% recovery of DNA from polyacrylamide gels was used to isolate the individual fractions corresponding to these bands. The structure of the fractions was then determined using single-strand-specific nuclease to digest single-stranded "tails" and using DNA polymerases to extend recessed 3'-OH termini of partially duplex regions. Our results show that each component consists of a double-stranded region terminating in single-stranded tails at both ends. Although both chains of every duplex are 10-n nucleotides long (n integer), the chains are never completely paired. The experiments with DNA polymerase show an abundance of structures in which the 3'-OH termini of these duplexes are recessed by 8 nucleotides, and by inference, there must be structures with 5'-P termini recessed by 2 or 12 nucleotides. Thus DNAase I acts on nuclei to produce DNA with staggered cuts on opposite strands, separated by (10-n + 8) and (10-n + 2) base pairs (with 5'-P and 3'-OH termini extending, respectively). Two classes of models of DNA folding in the nucleosome have been proposed by other investigators to account for the presence of DNAase I cleavage sites at 10-n intervals along each DNA chain. One class of models leads to the prediction that cuts should either be unstaggered or separated by 10 nucleotides, while the other class is consistent with staggers of 6 and 4 nucleotides. Neither prediction is verified by our data; however, all these models may be made consistent with the results by assuming that the enzyme's site of recognition on nucleosomal DNA is not the same as its site of cleavage.     

Location of ion-binding sites in the gramicidin channel by X-ray diffraction.

We report the first X-ray diffraction on gramicidin in its membrane-active form by using uniformly aligned multilayer samples of membranes containing gramicidin and ions (T1+, K+, Ba2+, Mg2+ or without ions). From the difference electron density profiles, we found a pair of symmetrically located ion-binding sites for T1- at 9.6 (+/- 0.3) A and for Ba2+ at 13.0 (+/- 0.2) A from the midpoint of the gramicidin channel. The location of Ba(2+)-binding sites is near the ends of the channel, consistent with the experimental observation that divalent cations do not permeate but block the channel. The location of T1(+)-binding sites is somewhat of a surprise. It was generally thought that monovalent cations bind to the first turn of the helix from the mouth of the channel. (It is now generally accepted that the gramicidin channel is a cylindrical pore formed by two monomers, each a single-stranded beta 6.3 helix and hydrogen-bonded head-to-head at their N termini.) But our experiment shows that the T1(+)-binding site is either near the bottom of or below the first helix turn.     

PIN domain of Nob1p is required for D-site cleavage in 20S pre-rRNA

Nob1p (Yor056c) is essential for processing of the 20S pre-rRNA to the mature 18S rRNA. It is part of a pre-40S ribosomal particle that is transported to the cytoplasm and subsequently cleaved at the 3' end of mature 18S rRNA (D-site). Nob1p is also reported to participate in proteasome biogenesis, and it was therefore unclear whether its primary activity is in ribosome synthesis. In this work, we describe a homology model of the PIN domain of Nob1p, which structurally mimics Mg(2+)-dependent exonucleases despite negligible similarity in primary sequence. Insights gained from this model were used to design a point mutation that was predicted to abolish the postulated enzymatic activity. Cells expressing Nob1p with this mutation failed to cleave the 20S pre-rRNA. This supports both the significance of the structural model and the idea that Nob1p is the long-sought D-site endonuclease.     

Prediction of neuropeptide cleavage sites in insects

MOTIVATION: The production of neuropeptides from their precursor proteins is the result of a complex series of enzymatic processing steps. Often, the annotation of new neuropeptide genes from sequence information outstrips biochemical assays and so bioinformatics tools can provide rapid information on the most likely peptides produced by a gene. Predicting the final bioactive neuropeptides from precursor proteins requires accurate algorithms to determine which locations in the protein are cleaved. RESULTS: Predictive models were trained on Apis mellifera and Drosophila melanogaster precursors using binary logistic regression, multi-layer perceptron and k-nearest neighbor models. The final predictive models included specific amino acids at locations relative to the cleavage sites. Correct classification rates ranged from 78 to 100% indicating that the models adequately predicted cleaved and non-cleaved positions across a wide range of neuropeptide families and insect species. The model trained on D.melanogaster data had better generalization properties than the model trained on A. mellifera for the data sets considered. The reliable and consistent performance of the models in the test data sets suggests that the bioinformatics strategies proposed here can accurately predict neuropeptides in insects with sequence information based on neuropeptides with biochemical and sequence information in well-studied species.     

Probing stability and dynamics of proteins by protease digestion. II: Identification of the initial chymotryptic cleavage sites of homologous cytochromes c

Three homologous cytochromes c from horse, rabbit and tuna were subjected to chymotryptic digestion and their initial cleavage sites were identified. The sites in oxidized cytochromes c are the COOH-terminal sides of Tyr-48, Phe-46 and Tyr-46 for horse, rabbit and tuna cytochromes c, respectively. The results show that the chymotrypsin attacks a single site in each protein; the sites are located at the almost identical position on the polypeptide chain. Through the time-course studies of digestion, it was found that the three cytochromes c have different chymotrypsin-susceptibility at the initial cleavage site in the order of horse less than rabbit less than tuna. Studies on chymotryptic digestion of tuna cytochrome c in the reduced form revealed that the haem-reduction does not alter the initial cleavage site but increases the resistance to the proteolysis at the site. The uniqueness of the initial cleavage site in each cytochrome c species suggests that the protease susceptibility reflects some overall properties of the protein. At the same time, it was clarified that the initial cleavage site is also affected by a neighboring region by the fact that another potential cleavage site is located near the site in question. In order to elucidate the initial cleavage site, several physical properties of tuna cytochrome c molecule deduced from the X-ray 3D structure, accessible surface area, temperature factor, effective hydrophobicity and electrostatic potential, were compared with the experimental results and it was concluded that these properties given by a residue have no direct relationship with the chymotrypsin susceptibility.     

Serine protease of pestiviruses: determination of cleavage sites.

The single-stranded genomic RNA of pestiviruses is of positive polarity and encompasses one large open reading frame of about 4,000 codons. The resulting polyprotein is processed co- and posttranslationally by virus-encoded and host cell proteases to give rise to the mature viral proteins. A serine protease residing in the nonstructural (NS) protein NS3 (p80) has been shown to be essential for the release of the NS proteins located downstream of NS3. In this report the NS3 serine protease-dependent cleavage sites for bovine viral diarrhea virus (BVDV) strain CP7 are described. Proteins used for analysis were generated in Escherichia coli or in eukaryotic cells by the use of the T7 vaccinia virus system. The N termini of NS4A, NS4B, NS5A, and NS5B were determined by protein sequencing. Analysis of the data obtained showed that leucine at P1 is the only position conserved for all cleavage sites. At P1' alanine is found at the NS4A-NS4B site, whereas serine resides at this position at the NS3-NS4A, NS4B-NS5A, and NS5A-NS5B cleavage sites. For all cleavage sites the amino acids found at P1 and P1' are conserved for different genotypes of pestiviruses, despite the high degree of sequence variation found between these viruses. It is therefore assumed that the cleavage sites determined for BVDV CP7 are representative of those for all pestiviruses.