Polypyrimidine tract binding protein (PTB) is a 57 kD hnRNP protein (hnRNP I) that binds to the pyrimidine tract typically found near the 3' end of introns. Primary sequence analysis suggests that PTB contains four RNA recognition motifs (RRMs). Data from comparative structural and deletional analysis of PTB are consistent with the presence of a four reiterated domain structure. Since PTB exists in solution as a homodimer, it contains an oligomeric array of eight RRMs. Though the function of RRMs in a monomeric context has been addressed, the significance of their presence in an oligomeric context has not been investigated. To correlate structural motifs with function, we have analyzed the RNA binding properties of wild-type and deletion constructs of PTB that contain RRMs in both an oligomeric and monomeric context. These studies indicate that there is not a strong correlation between the RNA binding affinity and specificity upon oligomerization. However, the mode of RNA interaction and dimerization is linked. We have also found that the primary contributor to the free energy of PTB binding and the primary determinant for RNA binding specificity resides in RRM 3, while the primary contributor to dimer stabilization coincides with residues in RRM 2.
The genes for two Sac7 DNA-binding proteins, Sac7d and Sac7e, from the extremely thermophilic archaeon Sulfolobus acidocaldarius have been cloned into Escherichia coli and sequenced. The sac7d and sac7e open reading frames encode 66 amino acid (7608 Da) and 65 amino acid (7469 Da) proteins, respectively. Southern blots indicate that these are the only two Sac7 protein genes in S. acidocaldarius, each present as a single copy. Sac7a, b, and c proteins appear to be carboxy-terminal modified Sac7d species. The transcription initiation and termination regions of the sac7d and sac7e genes have been identified along with the promoter elements. Potential ribosome binding sites have been identified downstream of the initiator codons. The sac7d gene has been expressed in E. coli, and various physical properties of the recombinant protein have been compared with those of native Sac7. The UV absorbance spectra and extinction coefficients, the fluorescence excitation and emission spectra, the circular dichroism, and the two-dimensional double-quantum filtered 1H NMR spectra of the native and recombinant species are essentially identical, indicating essentially identical local and global folds. The recombinant and native proteins bind and stabilize double-stranded DNA with a site size of 3.5 base pairs and an intrinsic binding constant of 2 x 10(7) M-1 for poly[dGdC].poly[dGdC] in 0.01 M KH2PO4 at pH 7.0. The availability of the recombinant protein permits a direct comparison of the thermal stabilities of the methylated and unmethylated forms of the protein. Differential scanning calorimetry demonstrates that the native protein is extremely thermostable and unfolds reversibly at pH 6.0 with a Tm of approximately 100 degrees C, while the recombinant protein unfolds at 92.7 degrees C.
The thermodynamics of the binding of the Sac7d protein of Sulfolobus acidocaldarius to doublestranded DNA has been characterized using spectroscopic signals arising from both the protein and the DNA. Ligand binding density function analysis has been used to demonstrate that the fractional change in protein intrinsic tryptophan fluorescence quenching that occurs upon DNA binding is equal to the fraction of protein bound. Reverse titration data have been fit directly to the McGhee-von Hippel model [
Enterohemorrhagic Escherichia coli O157:H7 presents a serious threat to human health and sanitation and is a leading cause in many food- and waterborne ailments. While conventional bacterial detection methods such as PCR, fluorescent immunoassays and ELISA exhibit high sensitivity and specificity, they are relatively laborious and require sophisticated instruments. In addition, these methods often demand extensive sample preparation and have lengthy readout times. We propose a simpler and more sensitive diagnostic technique featuring multiparametric magneto-fluorescent nanosensors (MFnS). Through a combination of magnetic relaxation and fluorescence measurements, our nanosensors are able to detect bacterial contamination with concentrations as little as 1 colony-forming unit (CFU). The magnetic relaxation property of our MFnS allow for sensitive screening at low target CFU, which is complemented by fluorescence measurements of higher CFU samples. Together, these qualities allow for the detection and quantification of broad-spectrum contaminations in samples ranging from aquatic reservoirs to commercially produced food.
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