The trp repressor of Escherichia coli specifically binds to operator DNAs in three operons involved in tryptophan metabolism. The NMR spectra of repressor and a chymotryptic fragment lacking the six amino-terminal residues are compared. Two-dimensional J-correlated spectra of the two forms of the protein are superimposable except for cross-peaks that are associated with the N-terminal region. The chemical shifts and relaxation behavior of the N-terminal resonances suggest mobile "arms". Spin-echo experiments on a ternary complex of repressor with L-tryptophan and operator DNA indicate that the termini are also disordered in the complex, although removal of the arms reduces the DNA binding energy. Relaxation measurements on the armless protein show increased mobility for several residues, probably due to helix fraying in the newly exposed N-terminal region. DNA binding by the armless protein does not reduce the mobility of these residues. Thus, it appears that the arms serve to stabilize the N-terminal helix but that this structural role does not explain their contribution to the DNA binding energy. These results suggest that the promiscuous DNA binding by the arms seen in the X-ray crystal structure is found in solution as well.
The resonances in the aromatic region of the 'H-NMR spectrum of the Escherichiu coli trp aporepressor have been assigned to amino acid type by two-dimensional correlated spectroscopy (COSY), homonuclear HartmannHahn (HOHAHA) spectroscopy and nuclear Overhauser enhancement spectroscopy (NOESY) techniques and studies of the pH dependence of the chemical shifts. in combination with selective deuteration of the protein.Complete sequence-specific assignments of the aromatic resonances have been made by comparing the observed inter-residue NOES with those expected on the basis of the crystal structure of the protein [Zhang, R.-G., Joachimiak, A., Lawson, C. L., Shevitz, R. W., Otwinowski, Z. & Sigler, P. B. (1987) Nature 327, 591 -5971. The latter experiments have also permitted the sequence-specific assignment of some of the high-field methyl resonances. The complete assignment of the aromatic region of the spectrum, in particular of resonances from residues at the dimer interface, opcns the way to detailed studies of the conformational effects of corepressor and operator binding.The E. coli trp repressor regulates transcription by binding to operator DNA in the presence of tryptophan, thereby controlling tryptophan biosynthesis [l -31. With two identical subunits of 107 amino acids [4], it is one of the smallest proteins which both binds to a specific DNA sequence and whose affinity for that sequence is allosterically controlled. It has been extensively studied both by genetic and, more recently, by physical methods. The crystal structures of the aporepressor, of two forms of the active protein containing bound tryptophan, and of the repressor complexed to an oligonucleotide containing the operator sequence, have been determined by Sigler and his colleagues [S -81. Each subunit of the protein has six a-helices; helices A, B, C and F form an interlocking core with the corresponding helices of the second subunit, while helices D and E form a helix-turn-helix domain similar to that in other prokaryotic DNA-binding proteins (reviewed in [9]). The conformations of the aporepressor and the active repressor in the crystal are similar, except for a difference in the position of the DNA-binding domain relative to the 'core'; however, the precise conformation of the DNAbinding domain differs in two different crystal forms of the repressor, suggesting that it is flexible [7]. In the crystal structure of the repressor-operator complex, there are no direct hydrogen bonds or non-polar contacts between the protein and bases known to be important for recognition in vivo [XI.This implies that specific recognition of the operator must arise from the sequence dependence of the energy required to Correspondence to G. C. K. Roberts, Biological N M R Centre, University of Leicester, P. 0. Box 138, Medical Sciences Building, University Rd, Leicester LEI 9HN, UK Ahhrrviations. COSY, two-dimensional J-correlated spectroscopy; HOHAHA spectroscopy, two-dimensional homonuclear Hartmann-Hahn spectroscopy; NOESY, two-dimensional nuclear Ovcrhaus...
T he HID EVOlutiondqPCR/STR Setup System enables automation of DNA quantitative real-time polymerase chain reaction (PCR) setup, normalization of DNA sample, and PCR setup for short tandem repeat (STR) analysis. The HID EVOlution System tracks sample and reagent information and facilitates data transfer of DNA quantification, normalization, and PCR setup for STR analysis steps, eliminating the need for manual processing and repetitive data entry. Instruments for the automated system include a Tecan Freedom EVO 150 robot for liquid handling, the 7500 Real-Time PCR System for DNA quantification, the GeneAmp PCR System 9700 for STR amplification, and the 3130xl Genetic Analyzer for the detection of amplified STR fragments. Validation studies including reproducibility, accuracy, correlation, and contamination studies were performed. Results demonstrated clean liquid-handling capabilities and maintenance of sample integrity. Variation in average allele peak height obtained using automated protocol was similar to that obtained using the manual protocol. ( JALA 2010;15:65-73)
The HID EVOlution—Extraction System (Tecan Group Ltd., Mannedorf, Switzerland) was developed to automate DNA extraction from biological samples using the PrepFiler Automated Forensic DNA Extraction Kit (Applied Biosystems, Foster City, CA). The system consists of a Tecan Freedom EVO 150 robot (Tecan Group Ltd., Mannedorf, Switzerland), a graphical user interface designed for use with Freedom EVOware software v 2.1 SPI (Tecan Group Ltd., Mannedorf, Switerland) as well as instrument hardware and plastic to support the PrepFiler reagents and protocol. The DNA quality and quantity obtained were comparable to that observed with the corresponding manual extraction protocol. Purified DNA was free of inhibitors and ready for downstream applications, such as real-time quantitative PCR and PCR for short tandem repeat (STR) analysis. The DNA quantity and quality obtained were consistent as demonstrated by the quantification and STR results. Our studies indicate that the HID EVOlution—Extraction System can easily be adopted in forensic laboratories to alleviate some of the bottlenecks of sample preparation in forensic laboratories.
The possibility of extending NMR methods for structure determination to larger proteins (MW > 10 kD) depends on the development of isotopic labeling protocols for the simplification of their NMR spectra (isotopic spectral editing). We describe here the successful use of selective deuteration to obtain sequence specific assignments for (thus far) more than 50% of the residues of the trp repressor protein (25 kD). This is the largest protein for which detailed sequence specific assignments have been attempted to‐date.
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