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 ribosom e, plays an essential role in the assembly of the 30S ribosomal subuni t and in the translational regulation of the spc operon by virtue of i ts capacity to bind specifically to rRNA and mRNA. To study structure- function relationships within the protein, we have constructed a vecto r for its high-level expression in vivo and developed efficient method s for its purification. Under our conditions, S8 accumulates to a leve l of 35% of the cellular protein and can be prepared at a purity of ov er 98% using either HPLC or a combination of ion-exchange and gel-filt ration 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 iso lated. The effects of the mutations on the RNA-binding ability, second ary structure, and stability of S8 were assessed. CD spectra indicated that wild-type S8 and the two mutant proteins have very similar secon dary structures at 25-degrees-C. In addition, both mutants are metabol ically stable in vivo as inferred from pulse-chase labeling and immuno precipitation experiments. However, while CA126 exhibits the same affi nity for RNA and the same susceptibility to urea and thermal denaturat ion as wild-type S8, CS126 is severely impaired in its ability to inte ract with RNA and displays a dramatic reduction in conformational stab ility. 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-bin ding domain of protein S8.