Minimal 16S rRNA binding site and role of conserved nucleotides in Escherichia coli ribosomal protein S8 recognition

Abstract

Escherichia coli ribosomal protein S8 was previously shown to bind a 16s rRNA fragment (nucleotides 584-756) with the same affinity as the complete 16s rRNA, and to shield an irregular helical region (region C) [Mougel, M., Eyermann, F., Westhof, E., Romby, P., Expert-Bezanqon, Ebel, J. P., Ehresmann, B. & Ehresmann, C. (1987). J. Mol. Biol. 198, 91-1071. Region C was postulated to display characteristic features : three bulged adenines (A595, A640 and A642), a noncanonical U598-U641 pair surrounded by two G . C pairs. In order to delineate the minimal RNA binding site, deletions were introduced by site-directed mutagenesis and short RNA fragments were synthesized. Their ability to bind S8 was assayed by filter binding. Our results show that the RNA binding site can be restricted to a short helical stem (588-609633-6.51) containing region C . The second part of the work focused on region C and on the role of conserved nucleotides as potential determinants of S8 recognition. Single and double mutations were introduced by site-directed mutagenesis in fragment 584-756, and their effect on S8 binding was measured. It was found that the three bulged positions are essential and that adenines are required at positions 640 and 642. U598 is also crucial and the highly conserved G597 . C643 pair cannot be inverted. These conserved nucleotides are either directly involved in the recognition process as direct contacts or required to maintain a specific conformation. The strong evolutionary pressure and the small number of positive mutants stress the high stringency of the recognition process.RNA-binding proteins recognize defined parts of RNA molecules in a highly specific manner, but in most cases the basic molecular mechanism of this recognition process remains not well known. Ribosomal components offer a valuable system for the possible dissection of the underlying principles of specific protein-RNA interactions. The Escherichia coli 30s subunit is composed of 21 proteins and some of them, named primary proteins, interact independently and specifically with selected regions of ribosomal 16s rRNA. Among them, protein S8 plays a central role both in 30s ribosomal subunit assembly (Held et al., 1974) and in the regulation of the Spc ribosomal protein operon (Yates et al., 1980: Dean et al., 1981. During 30s assembly, S8 binds to the central domain of 16s rRNA in the early stage of the process (Held et al., 1974) and may interact cooperatively with two other small subunit proteins: S6 and S38 Svensson et al., 1988). It was suggested that the region near S8, in the body of 30s particle, represents one of the two nucleation sites during assembly (Moore, 1987). A variety of methods, including partial nuclease digestion (Zimmermann et al., 1975;Ungewickell et al., 1975; Zim- (Mougel et al., 1987;Svensson et al., 1988) and sitedirected mutagenesis Gregory et al., 1988) was used to localize the region within 16s rRNA that mediates S8 recognition. In a previous work, we characterized the interactions between protein S8 and 16s rRN...

In vivo selection of functional ribosomes with variations in the rRNA-binding site of Escherichia coli ribosomal protein S8: Evolutionary implications

“…The RNA fragments encompassing nucleotides 576 to 764 were generated by PCR by using the pKKS8r vector from the isolated clones as template followed by in vitro transcription. Apparent K d s for EcS8 binding of the 16S rRNA fragments containing nucleotides 576-764 were estimated from saturation curves by using filter-binding assays (9). The results are summarized in Table 1.…”

“…The nucleotides essential for EcS8 binding ( Fig. 1) were mapped in the center of this stem (7)(8)(9)(10). Other protection sites were identified elsewhere in the 16S rRNA (11), but these additional protected sites are directly dependent on the binding of EcS8 to its anchoring site at 597-599/640-643 and are lost if this anchoring site is disrupted (H.M., unpublished work).…”

Ribosomal Protein S15 from Thermus Thermophilus

“…The same HindIII site was used for plasmid linearisation. Construction of the plasmid carrying an E. coli 16s rRNA gene fragment corresponding to nucleotides 584-756 was described previously (Mougel et al, 1993).…”

The ribosomal protein S8 from Thermus thermophilus VK1

“…The binding site on both types of RNAs has been identified. On the 16s rRNA, it has been located essentially by limited nuclease digestion (Ungewickell et al, 1975;Zimmermann et al, 1975;Zimmermann and SinghBergmann, 1979) and cross-linking experiments (Wower and Brimacombe, 1983), within the central domain of the 16s rRNA, in an imperfect helical structure as determined by structure probing, site-directed mutagenesis and phylogenetic comparisons (Mougel et al, 1987(Mougel et al, , 1993Gregory et al, 1988;Svenson et al 1988;Gutell et al, 1993). Nevertheless, there are some discrepancies in the conformations proposed by these authors.…”