Interactions of the Cold Shock Protein CspB fromBacillus subtilis with Single-stranded DNA: IMPORTANCE OF THE T BASE CONTENT AND POSITION WITHIN THE TEMPLATE

Maria M Lopez,Katsuhide Yutani,George I Makhatadze

doi:10.1074/jbc.m010474200

Maria M Lopez, Katsuhide Yutani + Show 1 more

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https://doi.org/10.1074/jbc.m010474200

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Abstract

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.

Highlights

The detailed mechanism by which living cells survive a cold shock remains controversial [1]
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 single-stranded DNA (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
1) How is CspB binding to continuous stretches of T bases affected by the presence of other bases in the oligonucleotide? 2) How is such a binding affected when the T bases form non-continuous stretches? To address these questions, we used fluorescence spectroscopy, analytical centrifugation, and isothermal titration calorimetry and measured the CspB binding properties to series of ssDNA (23-mers) in which the T bases were placed in the middle of the oligonucleotide (23CpTi, 23ApTi, and 23GpTi, where i ϭ 3, 5, 7, 11, 15, and 17), forming a continuous stretch but varying its length

Summary

Introduction

The detailed mechanism by which living cells survive a cold shock remains controversial [1]. We used fluorescence spectroscopy, analytical centrifugation, and isothermal titration calorimetry and measured the CspB binding properties to series of ssDNA (23-mers) in which the T bases were placed in the middle of the oligonucleotide (23CpTi, 23ApTi, and 23GpTi, where i ϭ 3, 5, 7, 11, 15, and 17), forming a continuous stretch but varying its length. The results of these experiments were compared with the effect of non-continuous stretches of T bases, the effect of location of the T bases within short ssDNA template and the effect of the total length of the oligonucleotide on the CspB binding. These observations combined with the sequence analysis of 5Ј-UTR of cold shock-inducible proteins suggests possible function of CspB as RNA chaperone

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