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Transcriptional Repressor CopR: Use of SELEX To Study the copR Operator Indicates that Evolution Was Directed at Maximal Binding Affinity. Peggy Freede, 2004.CopR is one of the two copy number control elements of the streptococcal plasmid pIP501 . It represses transcription of the repR mRNA encoding the essential replication initiator protein about 10- to 20-fold by binding to its operator region upstream of therepR promoter pII . CopR binds at two consecutive sites in themajor groove of the DNA that share the consensus motif 5'-CGTG.Previously, the minimal operator was narrowed down to 17 bp,and equilibrium dissociation constants for DNA binding and dimerizationwere determined to be 0.4 nM and 1.4 µM, respectively.In this work, we used a SELEX procedure to study copR operatorsequences of different lengths in combination with electrophoreticmobility shift assays of mutated copR operators as well as copynumber determinations to assess the sequence requirements forCopR binding . The results suggest that in vivo evolution wasdirected at maximal binding affinity . Three simultaneous nucleotideexchanges outside the bases directly contacted by CopR onlyslightly affected CopR binding in vitro or copy numbers in vivo.Furthermore, the optimal spacer sequence was found to comprise7 bp, to be AT rich, and to need an A/T and a T at the 3' positions,whereas broad variations in the sequences flanking the minimal17-bp operator were well tolerated. Collagenolytic Serine-Carboxyl Proteinase from Alicyclobacillus sendaiensis Strain NTAP-1: Purification, Characterization, Gene Cloning, and Heterologous Expression. Naoki Tsuruoka, 2003.Enzymatic degradation of collagen produces peptides, the collagen peptides, which show a variety of bioactivities of industrial interest . Alicyclobacillus sendaiensis strain NTAP-1, a slightly thermophilic, acidophilic bacterium, extracellularly produces a novel thermostable collagenolytic activity, which exhibits its optimum at the acidic region (pH 3.9) and is potentially applicable to the efficient production of such peptides . Here, we describe the purification to homogeneity, characterization, gene cloning, and heterologous expression of this enzyme, which we call ScpA . Purified ScpA is a monomeric, pepstatin-insensitive carboxyl proteinase with a molecular mass of 37 kDa which exhibited the highest reactivity toward collagen (type I, from a bovine Achilles tendon) among the macromolecular substrates examined . On the basis of the sequences of the peptides obtained by digestion of collagen with ScpA, the following synthetic peptides were designed as substrates for ScpA and kinetically analyzed: Phe-Gly-Pro-Ala*Gly-Pro-Ile-Gly (kcat, 5.41 s-1; Km, 32 µM) and Met-Gly-Pro-Arg*Gly-Phe-Pro-Gly-Ser (kcat, 351 s-1; Km, 214 µM), where the asterisks denote the scissile bonds . The cloned scpA gene encoded a protein of 553 amino acids with a calculated molecular mass of 57,167 Da . Heterologous expression of the scpA gene in the Escherichia coli cells yielded a mature 37-kDa species after a two-step proteolytic cleavage of the precursor protein . Sequencing of the scpA gene revealed that ScpA was a collagenolytic member of the serine-carboxyl proteinase family (the S53 family according to the MEROPS database), which is a recently identified proteinase family on the basis of crystallography results . Unexpectedly, ScpA was highly similar to a member of this family, kumamolysin, whose specificity toward macromolecular substrates has not been defined . Sequence versus Structure for the Direct Detection of 16S rRNA on Planar Oligonucleotide Microarrays. Darrell P. Chandler, 2003.A two-probe proximal chaperone detection system consisting of a species-specific capture probe for the microarray and a labeled, proximal chaperone probe for detection was recently described for direct detection of intact rRNAs from environmental samples on oligonucleotide arrays . In this study, we investigated the physical spacing and nucleotide mismatch tolerance between capture and proximal chaperone detector probes that are required to achieve species-specific 16S rRNA detection for the dissimilatory metal and sulfate reducer 16S rRNAs . Microarray specificity was deduced by analyzing signal intensities across replicate microarrays with a statistical analysis-of-variance model that accommodates well-to-well and slide-to-slide variations in microarray signal intensity . Chaperone detector probes located in immediate proximity to the capture probe resulted in detectable, nonspecific binding of nontarget rRNA, presumably due to base-stacking effects . Species-specific rRNA detection was achieved by using a 22-nt capture probe and a 15-nt detector probe separated by 10 to 14 nt along the primary sequence . Chaperone detector probes with up to three mismatched nucleotides still resulted in species-specific capture of 16S rRNAs . There was no obvious relationship between position or number of mismatches and within- or between-genus hybridization specificity . From these results, we conclude that relieving secondary structure is of principal concern for the successful capture and detection of 16S rRNAs on planar surfaces but that the sequence of the capture probe is more important than relieving secondary structure for achieving specific hybridization .
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