We employed salt-dependent differential scanning calorimetric measurements to characterize the stability of six oligomeric DNA duplexes (5'-GCCGGAXTGCCGG-3'/5'-CCGGCAYTCCGGC-3') that contain in the central XY position the GC, AT, GG, CC, AA, or TT base pair. The heat-induced helix-to-coil transitions of all the duplexes are associated with positive changes in heat capacity, DeltaC(p), ranging from 0.43 to 0.53 kcal/mol. Positive values of DeltaC(p) result in strong temperature dependences of changes in enthalpy, DeltaH degrees, and entropy, DeltaS degrees , accompanying duplex melting and cause melting free energies, DeltaG degrees, to exhibit characteristically curved shapes. These observations suggest that DeltaC(p) needs to be carefully taken into account when the parameters of duplex stability are extrapolated to temperatures distant from the transition temperature, T(M). Comparison of the calorimetric and van't Hoff enthalpies revealed that none of the duplexes studied in this work exhibits two-state melting. Within the context of the central AXT/TYA triplet, the thermal and thermodynamic stabilities of the duplexes in question change in the following order: GC> AT > GG > AA approximately TT > CC. Our estimates revealed that the thermodynamic impact of the GG, AA, and TT mismatches is confined within the central triplet. In contrast, the thermodynamic impact of the CC mismatch propagates into the adjacent helix domains and may involve 7-9 bp. We discuss implications of our results for understanding the origins of initial recognition of mismatched DNA sites by enzymes of the DNA repair machinery.
We have previously found that genes of the CFR:BI restriction-modification (R-M) system from Citrobacter freundii are oriented divergently and that their promoter regions overlap. The overlapping promoters suggest regulation of gene expression at the transcriptional level. In this study the transcription regulation of CFR:BI R-M genes was analyzed in vivo and in vitro in Escherichia coli. It was shown that in the presence of CFR:BI methyltransferase (M.CFR:BI), cell galactokinase activity decreases 10-fold when the galactokinase gene (galK) is under the control of the cfrBIM promoter and increases 20-fold when galK is under the control of the cfrBIR promoter. The CFR:BI site, proven to be unique for the entire CFR:BI R-M gene sequence, is located in the -35 cfrBIM promoter region and is in close vicinity of the -10 cfrBIR promoter region. A comparison of the cfrBIM and the cfrBIR promoter activities in the in vitro transcription system using methylated and unmethylated DNA fragments as templates demonstrated that the efficiency of CFR:BI R-M gene transcription is regulated by enzymatic modification at the N-4-position of cytosine bases of the CFR:BI site by M.CFR:BI. From the results of the in vivo and in vitro experiments we suggest a new model of gene expression regulation in type II R-M systems.
The primary structures of the plasmids pECL18 (5571 bp) and pKPN2 (4196 bp) from Escherichia coli and Klebsiella pneumoniae, respectively, which carry genes for a Type II restriction-modification system (RMS2) with the specificity 5'-CCNGG-3', were determined in order to elucidate the structural relationship between them. The data suggest a possible role for recombination events at bom (basis of mobility) regions and the sites of resolution of multimer plasmid forms (so-called cer sequences) in the structural evolution of multicopy plasmids. Analysis of the sequences of pECL18 and pKPN2 showed that the genes for RM* Ecl18kI and RM* Kpn2kI, and the sequences of the rep (replication) regions in the two plasmids, are almost identical. In both plasmids, these regions are localized between the bom regions and the cer sites. The rest of the pECL18 sequence is almost identical to that of the mob (mobilization) region of ColE1, and the corresponding segment of pKPN2 is almost identical to part of pHS-2 from Shigella flexneri. The difference in primary structures results in different mobilization properties of pECL18 and pKPN2. The complete sequences of pECL18, pKPN2 and the pairwise comparison of the sequences of pECL18, pKPN2, ColE1 and pHS-2 suggest that plasmids may exchange DNA units via site-specific recombination events at bom and cer sites. In the course of BLASTN database searches using the cer sites of pECL18 and pKPN2 as queries, we found twenty cer sites of natural plasmids. Alignment of these sequences reveals that they fall into two classes. The plasmids in each group possess related segments between their cer and bom sites.
We used high-precision density and ultrasonic velocity measurements to characterize the native (N), molten globule (MG), and unfolded (U) conformations of apomyoglobin. The molten globule states that were studied in this work include the MG(pH4)(NaCl) state observed at pH 4 and 20 mM NaCl, the MG(pH4)(NaTCA) state observed at pH 4 and 20 mM sodium trichloracetate (NaTCA), the MG(pH2)(NaCl) state observed at pH 2 and 200 mM NaCl, and the MG(pH2)(NaTCA) state observed at pH 2 and 20 mM NaTCA. We used our densimetric and acoustic data to evaluate changes in adiabatic compressibility associated with the acid- or salt-induced N-to-MG, MG-to-U, MG-to-MG, and U-to-MG transitions of the protein. The N-to-MG(pH4)(NaCl) and N-to-MG(pH4)(NaTCA) transitions are accompanied by decreases in compressibility of -(3.0 +/- 0.6) x 10(-6) and -(2.0 +/- 0.6) x 10(-6) cm3 g(-1)bar(-1), respectively. The N-to-MG(pH2)(NaCl) and N-to-MG(pH2)(NaTCA) transitions are associated with compressibility changes of -(4.9 +/- 1.1) x 10(-6) and (0.7 +/- 0.9) x 10(-6) cm3 g(-1) bar(-1), respectively. We interpret these data in terms of the degree of unfolding of the various molten globule forms of apomyoglobin. In general, our compressibility data reveal significant disparities between the various equilibrium molten globule states of apomyoglobin while also quantitatively characterizing each of these states. Volumetric insights provided by our data facilitate gaining a better understanding of the folding pathways, intermediates, and kinetics of apomyoglobin folding.
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