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Nucleic Acids Research, 1994, Vol. 22, No. 9 1687-1695
© 1994

RNA

The binding site for ribosomal protein S8 in 16S rRNA and spc mRNA from Escherichia coli: minimum structural requirements and the effects of single bulged bases on S8 — RNA interaction

Herren Wu, Lihong Jiang and Robert A. Zimmermann*

Department of Biochemistry and Molecular Biology and Program in Molecular and Cellular Biology University of Massachusetts, Amherst, MA 01003, USA

*To whom correspondence should be addressed

Received December 22, 1993. Revised March 28, 1994. Accepted March 28, 1994.

Through specific interactions with rRNA and mRNA, ribosomal protein S8 of Escherichia coli plays a central role in both assembly of the 30S ribosomal subunit and translational regulation of spc operon expression. To better understand S8–RNA association, we have measured the affinity of S8 for a number of variants of its rRNA and mRNA binding sites prepared by in vitro transcription or chemical synthesis. With the aid of site-directed deletions, we demonstrate that an imperfect, 33-nucleotide helical stem encompassing nucleotides 588–603 and 635–651 possesses all of the structural information necessary for specific binding of S8 to the 16S rRNA. This segment consists of two short duplexes that enclose a conserved, assymetric internal loop which contains features crucial for protein recognition. The S8 binding site in spc operon mRNA is very similar in both primary and secondary structure to that in 16S rRNA except for the presence of two single bulged bases in one of the duplex segments. In addition, the apparent association constant for the S8–mRNA interaction is approximately fivefold less than that for the S8–rRNA interaction. We show that the difference in affinity can be attributed to the effects of the bulged bases. Deletion of the bulged bases from the mRNA site increases its affinity for S8 to a level similar to that of the rRNA, whereas insertion of single-base bulges at equivalent positions within the rRNA site reduces its affinity for S8 to a value typical of the mRNA. Single-base bulges in the proximity of essential recognition features are therefore capable of modulating the strength of protein–RNA interactions.


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