Functional conservation of cold shock domains in bacteria and higher plants.
In Escherichia coli, a family of cold shock proteins (CSPs) function as transcription antiterminators or translational enhancers at low temperature by destabilizing RNA secondary structure. A wheat nucleic acid-binding protein (WCSP1) was found to contain a cold shock domain (CSD) bearing high similarity to E. coli cold shock proteins. In the present study, a series of mutations were introduced into WCSP1, and its functionality was investigated by using in vivo and in vitro assays in the context of functional conservation with E. coli CSPs. Constitutive expression of WT WCSP1 in an E. coli cspA, cspB, cspE, cspG quadruple deletion mutant complemented its cold-sensitive phenotype, suggesting that WCSP1 shares a function with E. coli CSPs for cold adaptation. In addition, transcription antitermination activity was demonstrated for WCSP1 by using an E. coli strain that has a hairpin loop upstream of a chloramphenicol resistance gene. In vitro dsDNA melting assays clearly demonstrated that WCSP1 melts dsDNA, an activity that was positively correlated to the ability to bind ssDNA. When mutations were introduced at critical residues within the consensus RNA binding motifs (RNP1 and RNP2) of WCSP1, it failed to melt dsDNA. Studies with WCSP1-GFP fusion proteins documented patterns that are consistent with ER and nuclear localization. In vivo and in vitro functional analyses, coupled with subcellular localization data, suggest that WCSP1 may function as a RNA chaperone to destabilize secondary structure and is involved in the regulation of translation under low temperature.
Nakaminami
Kentaro
K
Research Team for Crop Cold Tolerance, National Agricultural Research Center for Hokkaido Region, Hitsujigaoka 1, Toyohira-ku, Sapporo 062-8555, Japan.
Karlson
Dale T
DT
Imai
Ryozo
R
D002467:Cell Nucleus / Q000737:chemistry; D003080:Cold Temperature; D004277:DNA, Single-Stranded / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000737:chemistry; D004926:Escherichia coli / Q000235:genetics; D005786:Gene Expression Regulation; D005816:Genetic Complementation Test; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D009154:Mutation; D010940:Plant Proteins / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D017434:Protein Structure, Tertiary / Q000235:genetics; D014157:Transcription Factors / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic; D014908:Triticum / Q000737:chemistry / Q000378:metabolism*
D004277:DNA, Single-Stranded; D010940:Plant Proteins; D014157:Transcription Factors; D049452:Green Fluorescent Proteins