Interactions of the cold shock protein CspB from Bacillus subtilis with single-stranded DNA. Importance of the T base content and position within the template.
The cold shock protein CspB from Bacillus subtilis binds T-based single-stranded DNA (ssDNA) with high affinity (Lopez, M. M., Yutani, K., and Makhatadze, G. I. (1999) J. Biol. Chem. 274, 33601-33608). In this paper we report the results of CspB interactions with non-homogeneous ssDNA templates containing continuous and non-continuous stretches of T bases. The analysis of CspB-ssDNA interactions was performed using fluorescence spectroscopy, analytical centrifugation and isothermal titration calorimetry. We show that (i) there is a strong correlation between the CspB affinity and stoichiometry and the T content in the oligonucleotide that is independent of which other bases are incorporated into the sequence of ssDNA; (ii) the binding properties of CspB to ssDNA templates with continuous or non-continuous stretches of T bases with similar T content is very similar, and (iii) the mechanism of interaction between CspB and the T-based non-homogeneous ssDNA is mainly through the bases (a stretch of three T bases located in the middle of the ssDNA templates makes the binding independent of the ionic strength). The biological relevance of these results to the role of CspB as an RNA chaperone is discussed.
Department of Biochemistry and Molecular Biology, Penn State University, College of Medicine, Hershey, Pennsylvania 17033, USA.
M M
MM
Lopez
K
K
Yutani
G I
GI
Makhatadze
D001412:Bacillus subtilis / Q000378:metabolism*; D001426:Bacterial Proteins; D001483:Base Sequence; D002352:Carrier Proteins / Q000378:metabolism*; D017931:DNA Primers; D004277:DNA, Single-Stranded / Q000378:metabolism*; D006360:Heat-Shock Proteins; D009994:Osmolar Concentration; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D013698:Templates, Genetic; D014364:Tryptophan / Q000378:metabolism
D001426:Bacterial Proteins; D002352:Carrier Proteins; D017931:DNA Primers; D004277:DNA, Single-Stranded; D006360:Heat-Shock Proteins; C077188:cold-shock protein CspB, Bacteria; D014364:Tryptophan