The major cold shock protein of Bacillus subtilis
 CspB binds with high affinity to the ATTGG- and CCAAT sequences in single stranded oligonucleotides

ABSTRACT Using immunofluorescence microscopy and a fusion of a cold shock protein (CSP), CspB, to green fluorescent protein (GFP), we showed that in growing cells Bacillus subtilis CSPs specifically localize to cytosolic regions surrounding the nucleoid. The subcellular localization of CSPs is influenced by the structure of the nucleoid. Decondensed chromosomes in smc mutant cells reduced the sizes of the regions in which CSPs localized, while cold shock-induced chromosome compaction was accompanied by an expansion of the space in which CSPs were present. As a control, histone-like protein HBsu localized to the nucleoids, while β-galactosidase and GFP were detectable throughout the cell. After inhibition of translation, CspB-GFP was still present around the nucleoids in a manner similar to that in cold-shocked cells. However, in stationary-phase cells and after inhibition of transcription, CspB was distributed throughout the cell, indicating that specific localization of CspB depends on active transcription and is not due to simple exclusion from the nucleoid. Furthermore, we observed that nucleoids are more condensed and frequently abnormal incspB cspC and cspB cspDdouble-mutant cells. This suggests that the function of CSPs affects chromosome structure, probably through coupling of transcription to translation, which is thought to decondense nucleoids. In addition, we found that cspB cspD and cspB cspC double mutants are defective in sporulation, with a block at or before stage 0. Interestingly, CspB and CspC are depleted from the forespore compartment but not from the mother cell. In toto, our findings suggest that CSPs localize to zones of newly synthesized RNA, coupling transcription with initiation of translation.

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In silico identification and evaluation of Bacillus subtilis cold shock protein B (cspB)-like plant RNA chaperones

Journal of Biomolecular Structure and Dynamics ◽

10.1080/07391102.2020.1719198 ◽

2020 ◽

pp. 1-10

Author(s):

Suman ◽

Meenakshi Chaudhary ◽

Vikrant Nain

Keyword(s):

Bacillus Subtilis ◽

In Silico ◽

Cold Shock ◽

Cold Shock Protein ◽

In Silico Identification ◽

Rna Chaperones ◽

Protein B

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RNA and DNA Binding Epitopes of the Cold Shock Protein TmCsp from the Hyperthermophile Thermotoga maritima

The Protein Journal ◽

10.1007/s10930-020-09929-6 ◽

2020 ◽

Vol 39 (5) ◽

pp. 487-500

Author(s):

Konstanze von König ◽

Norman Kachel ◽

Hans Robert Kalbitzer ◽

Werner Kremer

Keyword(s):

Nucleic Acids ◽

Cold Shock ◽

Rna Binding ◽

Dissociation Constants ◽

Thermotoga Maritima ◽

Cold Shock Protein ◽

Binding Motif ◽

Purine Base ◽

High Affinity ◽

Binding Epitope

AbstractProkaryotic cold shock proteins (CSPs) are considered to play an important role in the transcriptional and translational regulation of gene expression, possibly by acting as transcription anti-terminators and “RNA chaperones”. They bind with high affinity to single-stranded nucleic acids. Here we report the binding epitope of TmCsp from Thermotoga maritima for both single-stranded DNA and RNA, using heteronuclear 2D NMR spectroscopy. At “physiological” growth temperatures of TmCsp (≥ 343 K), all oligonucleotides studied have dissociation constants between 1.6 ((dT)7) and 25.2 ((dA)7) μM as determined by tryptophan fluorescence quenching. Reduction of the temperature to 303 K leads to a pronounced increase of affinity for thymidylate (dT)7 and uridylate (rU)7 heptamers with dissociation constants of 4.0 and 10.8 nM, respectively, whereas the weak binding of TmCsp to cytidylate, adenylate, and guanylate heptamers (dC)7, (dA)7, and (dT)7 is almost unaffected by temperature. The change of affinities of TmCsp for (dT)7 and (rU)7 by approximately 3 orders of magnitude shows that it represents a cold chock sensor that switches on the cold shock reaction of the cell. A temperature dependent conformational switch of the protein is required for this action. The binding epitope on TmCsp for the ssDNA and RNA heptamers is very similar and comprises β-strands 1 and 2, the loop β1–β2 as well as the loops connecting β3 with β4 and β4 with β5. Besides the loop regions, surprisingly, mainly the RNA-binding motif RNP1 is involved in ssDNA and RNA binding, while only two amino acids, H28 and W29, of the postulated RNA-binding motif RNP2 interact with the uridylate and thymidylate homonucleotides, although a high affinity in the nanomolar range is achieved. This is in contrast to the binding properties of other CSPs or cold shock domains, where RNP1 as well as RNP2 are involved in binding. TmCsp takes up a unique position since it is the only one which possesses a tryptophan residue instead of a usually highly conserved phenylalanine or tyrosine residue at the end of RNP2. NMR titrations suggest that neither (dT)7 nor (rU)7 represent the full binding motif and that non-optimal intercalation of W29 into these oligonucleotides blocks the access of the RNP2 site to the DNA or RNA. NMR-experiments with (dA)7 suggest an interaction of W29 with the adenine ring. Full binding seems to require at least one single purine base well-positioned within a thymine- or uracil-rich stretch of nucleic acids.

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Structure in solution of the major cold-shock protein from Bacillus subtilis

Nature ◽

10.1038/364169a0 ◽

1993 ◽

Vol 364 (6433) ◽

pp. 169-171 ◽

Cited By ~ 147

Author(s):

A. Schnuchel ◽

R. Wiltscheck ◽

M. Czisch ◽

M. Herrler ◽

G. Wllllmsky ◽

...

Keyword(s):

Bacillus Subtilis ◽

Cold Shock ◽

Cold Shock Protein ◽

Structure In Solution

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Single-stranded DNA binding of the cold-shock protein CspB from Bacillus subtilis: NMR mapping and mutational characterization

Protein Science ◽

10.1110/ps.0219703 ◽

2003 ◽

Vol 12 (1) ◽

pp. 112-123 ◽

Cited By ~ 26

Author(s):

M. Zeeb

Keyword(s):

Bacillus Subtilis ◽

Dna Binding ◽

Cold Shock ◽

Cold Shock Protein ◽

Single Stranded Dna

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Cold-Induced Putative DEAD Box RNA Helicases CshA and CshB Are Essential for Cold Adaptation and Interact with Cold Shock Protein B in Bacillus subtilis

Journal of Bacteriology ◽

10.1128/jb.188.1.240-248.2006 ◽

2006 ◽

Vol 188 (1) ◽

pp. 240-248 ◽

Cited By ~ 79

Author(s):

Karen Hunger ◽

Carsten L. Beckering ◽

Frank Wiegeshoff ◽

Peter L. Graumann ◽

Mohamed A. Marahiel

Keyword(s):

Bacillus Subtilis ◽

Cold Shock ◽

Resonance Energy Transfer ◽

Resonance Energy ◽

Cold Shock Protein ◽

Nucleic Acid Binding ◽

Rna Helicases ◽

Dead Box ◽

Initiation Of Translation

ABSTRACT The nucleic acid binding cold shock proteins (CSPs) and the cold-induced DEAD box RNA helicases have been proposed separately to act as RNA chaperones, but no experimental evidence has been reported on a direct cooperation. To investigate the possible interaction of the putative RNA helicases CshA and CshB and the CSPs from Bacillus subtilis during cold shock, we performed genetic as well as fluorescence resonance energy transfer (FRET) experiments. Both cshA and cshB genes could be deleted only in the presence of a cshB copy in trans, showing that the presence of one csh gene is essential for viability. The combined gene deletion of cshB and cspD resulted in a cold-sensitive phenotype that was not observed for either helicase or csp single mutants. In addition to the colocalization of the putative helicases CshA and CshB with CspB and the ribosomes in areas surrounding the nucleoid, we detected a strong FRET interaction in vivo between CshB and CspB that depended on active transcription. In contrast, a FRET interaction was not observed for CshB and the ribosomal protein L1. Therefore, we propose a model in which the putative cold-induced helicases and the CSPs work in conjunction to rescue misfolded mRNA molecules and maintain proper initiation of translation at low temperatures in B. subtilis.

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