THE BINDING-SITE FOR RIBOSOMAL-PROTEIN S8 IN 16S RIBOSOMAL-RNA AND SPC MESSENGER-RNA FROM ESCHERICHIA-COLI - MINIMUM STRUCTURAL REQUIREMENTS AND THE EFFECTS OF SINGLE BULGED BASES ON S8-RNA INTERACTION

Through specific interactions with rRNA and mRNA, ribosomal protein S8 of Escherichia coil plays a central role in both assembly of the 30S ribosomal subunit and translational regulation of spc operon expressio n. To better understand S8 - RNA association, we have measured the aff inity of S8 for a number of variants of its rRNA and mRNA binding site s prepared by in vitro transcription or chemical synthesis. With the a id of site-directed deletions, we demonstrate that an imperfect, 33-nu cleotide helical stem encompassing nucleotides 588 - 603 and 635 - 651 possesses all of the structural information necessary for specific bi nding of S8 to the 16S rRNA. This segment consists of two short duplex es that enclose a conserved, assymetric internal loop which contains f eatures crucial for protein recognition. The S8 binding site in spe op eron mRNA is very similar in both primary and secondary structure to t hat in 16S rRNA except for the presence of two single bulged bases in one of the duplex segments. In addition, the apparent association cons tant for the S8 - mRNA interaction is approximately fivefold less than that for the S8 - rRNA interaction. We show that the difference in af finity can be attributed to the effects of the bulged bases. Deletion of the bulged bases from the mRNA site increases its affinity for S8 t o a level similar to that of the rRNA, whereas insertion of single-bas e bulges at equivalent positions within the rRNA site reduces its affi nity for S8 to a value typical of the mRNA. Single-base bulges in the proximity of essential recognition features are therefore capable of m odulating the strength of protein - RNA interactions.