Mutagenesis of ribosomal protein S8 from Escherichia coli: expression, stability, and RNA-binding properties of S8 mutants.
Protein S8, a 129 amino acid component of the Escherichia coli ribosome, plays an essential role in the assembly of the 30S ribosomal subunit and in the translational regulation of the spc operon by virtue of its capacity to bind specifically to rRNA and mRNA. To study structure-function relationships within the protein, we have constructed a vector for its high-level expression in vivo and developed efficient methods for its purification. Under our conditions, S8 accumulates to a level of 35% of the cellular protein and can be prepared at a purity of over 98% using either HPLC or a combination of ion-exchange and gel-filtration chromatography. The unique cysteine residue at position 126 was replaced by alanine or serine by oligonucleotide-directed mutagenesis, and the two mutant proteins, CA126 and CS126, were expressed and isolated. The effects of the mutations on the RNA-binding ability, secondary structure, and stability of S8 were assessed. CD spectra indicated that wild-type S8 and the two mutant proteins have very similar secondary structures at 25 degrees C. In addition, both mutants are metabolically stable in vivo as inferred from pulse-chase labeling and immunoprecipitation experiments. However, while CA126 exhibits the same affinity for RNA and the same susceptibility to urea and thermal denaturation as wild-type S8, CS126 is severely impaired in its ability to interact with RNA and displays a dramatic reduction in conformational stability. Our results suggest that Cys126 is unlikely to play a specific role in RNA recognition but that it is an integral part of the RNA-binding domain of protein S8.
Wu
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Department of Biochemistry and Molecular Biology, University of Massachusetts, Amherst 01003.
Wower
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Zimmermann
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D000409:Alanine / Q000235:genetics; D017123:Bacteriophage T7 / Q000235:genetics; D002942:Circular Dichroism; D004926:Escherichia coli / Q000235:genetics*; D005822:Genetic Vectors; D007532:Isoleucine / Q000235:genetics; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic / Q000235:genetics; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000096:biosynthesis; D012269:Ribosomal Proteins / Q000096:biosynthesis / Q000235:genetics*; D012694:Serine / Q000235:genetics
D011994:Recombinant Proteins; D012269:Ribosomal Proteins; C030254:ribosomal protein S8; D007532:Isoleucine; D012694:Serine; D000409:Alanine