NMR structure determination of the binding site for ribosomal protein S8 from Escherichia coli 16 S rRNA

The location of protein S8 and surrounding elements of 16S rRNA in the 70S ribosome from combined use of directed hydroxyl radical probing and X-ray crystallography

“…Distribution of probe-target distances for weak, medium, and strong directed hydroxyl radical cleavages measured from the S8-16S rRNA model+ Cleavage intensities were scored as defined in Materials and methods+ siderations (Moine et al+, 1997), in which S8 was proposed to interact with the major (deep) groove face of the RNA helix+ Phylogenetic sequence conservation of protein S8 can be attributed to its interactions with 16S rRNA (Table 1)+ Helix a1 runs nearly parallel to the 39 strand of the 820 helix of 16S rRNA (positions 874 to 878), within about 6-8 Å between their respective backbones+ The side-chains of the conserved Asp8, Thr11, Arg12, Arg14, and Asn15 on the RNA-proximal face of a1, as well as Arg79 in loop 7, are close enough to make contacts with the RNA backbone+ In contrast, the RNA sequence in this region is variable, except for conservation of a pyrimidine at position 875, whose 2-keto group could provide a minor groove recognition feature+ The helical axis of a1 points to the junction between helices 20 and 21, suggesting a possible electrostatic interaction between its permanent dipole and the phosphate backbone around position 587+ Similarly, the dipole of a2 is oriented toward the RNA backbone around positions 589-590+ Residues Ser29 and Lys30 are within contact distance of this same part of the RNA backbone+ Near the classical binding region in the 620 helix, a cluster of conserved amino acid sidechains is juxtaposed with the RNA+ The conserved Tyr85 faces the RNA minor groove at the G597-C643 base pair, one of the few strongly conserved features of the S8 RNA binding site, and could participate in a stacking interaction facilitated by irregularities in the RNA helix generated by the two bulges in the strands flanking the conserved G-C pair+ Several other conserved residues including Arg83, Ser104, Ser106, and Glu123 are also close enough to interact with the core of the bindingsite region+ The general orientation of S8 with respect to its classical binding site and the stacking of Tyr85 in the vicinity of base A642 is in agreement with a model for S8-RNA interaction proposed by Kalurachchi and Nikonowicz (1998)+ Genetic studies have identified mutants of S8 that disrupt its interaction with RNA (Wower et al+, 1992)+ Among these mutations, six map to conserved, exposed residues (Ser29, Lys30, Arg79, Tyr85, Ser106, and Glu123) that we predict will make direct contact with 16S rRNA (Table 1)+ Some understanding of the role of S8 in ribosome assembly can be inferred from the emerging structure of the S8 region of the 30S subunit+ During transcription of 16S rRNA, the first secondary structure element of the central domain that is able to fold into its mature conformation is the 620 hairpin loop, which could anchor the central domain to the 59 domain via its interactions with protein S16 (Stern et al+, 1988a;Powers et al+, 1993;Powers & Noller, 1995)+ Completion of the long 620 stem would then allow S8 to bind to its classical binding site around position 640+ Upon completion of transcription of the central domain, S8 would then be able to bind the 820 stem, fixing the locations of the two extremities of the domain+ Interaction between S8 and S5 would further help to orient the central domain relative to the 59 end of 16S rRNA near the point of convergence of its three major domains+ Thus, despite the limited size of its primary binding site in the 620 stem, S8 may play a central role in coordinatin...…”

“…The RNA wraps around two of the three edges of the delta-shaped structure of T. thermophilus S8 (Fig+ 7a,b,c)+ The 620 stem (helix 21) runs parallel to the lower edge of S8; its classical binding site at positions 595/640 contacts the C-terminal domain near its a3 corner, while the base of the stem and elements of the three-way junction interact with the N-terminal domain of S8 at its loop 5 corner+ The 820 and 840 stems (helices 25 and 26) flank the back side of the N-terminal domain where a1 interacts with the 820 stem+ Most, if not all, of the contacts are with the minor groove surfaces of the RNA+ The protein-RNA contacts are consistent with the regions of Bacillus stearothermophilus S8 predicted to bind RNA based on patches of basic and aromatic residues identified in the crystal structure (Davies et al+, 1996)+ S8 also appears to interact with other ribosomal proteins+ The right-hand edge of S8 faces S5, where an S5-S8 crosslink has been identified between positions 166 of S5 and 93 of S8 (Allen et al+, 1979)+ The resulting purified S5-S8 covalent dimer has been shown to restore activity to 30S subunits depleted for S5 and S8 in reconstitution experiments (Lutter & Kurland, 1973), providing strong evidence for interaction between S5 and S8+ A blob of density contacting the righthand side of b4-b5 at the top of the N-terminal domain of S8 (Fig+ 8a) is connected to the C-terminal end of S5 in the 7+8-Å electron-density map (not shown)+ This unassigned density probably represents the C-terminal segment that was disordered in the crystal structure of protein S5 (Ramakrishnan & White, 1992)+ In addition, the upper corner of S8 at loop 6 is continuous with a low-density feature coming from the head of the subunit that is likely to belong to one of the 39-domain proteins+ Thus, virtually all but the solvent face of S8 is involved in either protein-RNA or protein-protein interactions+ In contrast to the close detailed fit with the free hydroxyl radical footprinting data, the base-specific protections (Fig+ 1A) are only partially explained by the model+ The bases around positions 575, 812, and 860 are out of range of direct contact with S8, and are more likely to be protected as a result of S8-dependent RNA-RNA interactions, including the phylogenetically and genetically established tertiary interaction between G570 and C866 (Gutell et al+, 1985(Gutell et al+, , 1986)+ Even the protection of the three bases A595, A640, and A642 in the 595/640 region of the 620 stem appears to be the result of stabilization of RNA-RNA interactions by S8, rather than protein-RNA contact+ Protection of A640 can be explained by stabilization of its Watson-Crick pair with U598 by S8, whereas A595 is sterically inaccessible to the DMS probe because of tertiary folding of the RNA, according to an NMR structure of the S8-binding region (Kalurachchi et al+, 1997;Kalurachchi & Nikonowicz, 1998)+ S8 clearly contacts the minor groove surface of its classical RNA-binding region, in agreement with the NMR structure, but differing from a model derived from chemical probing and stereochemical con-FIGURE 9. Distribution of probe-target distances for weak, medium, and strong directed hydroxyl radical cleavages measured from the S8-16S rRNA model+ Cleavage intensities were scored as defined in Materials and methods+ siderations …”

Structural features of an influenza virus promoter and their implications for viral RNA synthesis

“…The consecutive adenosines (A10-A11) and a uracil (U22) on the opposite strand can form a (5Ј-A-A-3Ј)⅐U motif. This motif is a binding site for ribosomal protein S8 in Escherichia coli 16S rRNA (25) and constitutes the site of spliceosome binding to the branch-point helix and the RNA binding site for phage GA coat protein (26). We have not observed the U22 imino resonance even in low pH conditions (pH 5.5), probably because of the rapid solvent exchange.…”

Identity and geometry of a base triple in 16S rRNA determined by comparative sequence analysis and molecular modeling

“…The interaction that is predicted here involves an extra hydrogen bond between 16S rRNA nt 121 and 124 in the major groove of the helical region+ This is a different type of interaction than the recent examples of same-strand near-neighbor interactions in the group I ribozyme (Cate et al+, 1996) and in hepatitis delta virus ribozyme (Ferré-D'Amaré et al+, 1998) that occur in the minor groove of the helix+ However, the base triple interaction in the 16S rRNA at 595(596:644) has been shown to occur in the major groove (Kalurachchi & Nikonowicz, 1998) with the hydrogen bond occurring between nt 595 and 596+ There are also several examples in tRNA structures of base triple interactions that involve major groove interactions+ Furthermore, we are confident that the predictive modeling program MC-SYM has the capability of constructing minor groove interactions, because another base triple in 16S rRNA between nt 494(440:497) is predicted to contain its extra interaction in the minor groove+ Thus, in spite of the notoriety of the narrowness of the RNA major groove in regular helices, there is no clear rule about which groove is used in these types of interactions+ and C124 in hybrid 3+ The distance between the hydrogen of N4 (C121) and the O2 (C124) exceeds the maximum acceptable length of 2+17 Å+ B: Model for the base triple utilizing a hydrogen bond between C121 and U124 in hybrid 4+ The distance between the O4 (U124) and the hydrogen of N4 (C121) exceeds the maximum acceptable length of 2+17 Å+ Hydrogen-bond distances measured between heavy atoms also indicate unacceptably long distances in both cases (Saenger, 1984)+ By modeling "hybrid" sequences that utilized these nonoccurring base pairings, we demonstrated that a base triple in which nt 121 is hydrogen bonded to 124 is consistent with the coordinated set of base pairings at 124:237 and 125:236+ We conclude that specific base pairings at 124:237 (e+g+, CG and UA when 121 is U) and 125:236 (e+g+, CG and AU when 121 is U or C) were not allowed due to structural constraints at 121(124:237)+ The neighbor effect has been widely observed in tRNAs (Gautheret et al+ 1995), in Group I introns (Michel & Westhof, 1990), and in 16S and 23S rRNA (R+ Gutell, unpubl+ data); the work presented here is a demonstration that it is based at least partly on structural constraints+…”

“…The 122-128/233-239 16S rRNA helix first was proposed with comparative analysis based on the small number of sequences available in 1980 (Woese et al+, 1980)+ (We will refer to this interaction as helix 122 here)+ With a significant increase in the number of 16S rRNA sequences and the development of more powerful covariation algorithms (Gutell et al+, 1992;Maidak et al+, 1999; R+R+ Gutell, S+ Subrashchandran, M+ Schnare, Y+ Du, N+ Lin, L+ Madabusi, K+ Muller, N+ Pande, N+ Yu, Z+ Shang, S+ Date, D+ Konings, V+ Schweiker, B+ Weiser, & J+ Cannone, in prep+), all of the positions within the 122-128/233-239 helix have their strongest statistically significant covariation with their previously predicted base-pair partner, except for the 125:236 base pair, where position 125 is nearly always a U in approximately 5,000 prokaryotic sequences, and position 236 is G in 75% and A in 25% of the sequences, (forming UG and UA base pairs)+ With a smaller set of rRNA sequences, our earlier analysis revealed several strong base-triple candidates in 16S and 23S rRNA (Gutell, 1996), including 1072(1092:1099) in 23S rRNA, and 121(124:237)/ (125:236) in 16S rRNA+ Recently the putative 23S rRNA base triple [1072(1092:1099)] has been substantiated with experimental methods (Conn et al+, 1998(Conn et al+, , 1999)+ We have repeated this base-triple analysis utilizing the same methods, but applied them to a larger prokaryotic 16S rRNA alignment (Maidak et al+, 1999)+ Several strong candidates have been identified, including 595(596:644) and our previous 121(124:237)/(125:236) base triple+ There is a mutual best covariation between the unpaired position 595 and the base pair 596:644 with all three algorithms used to evaluate the significance of sequence covariations-chi square, pseudophylogenetic event counting (ec), and covary methods (see Materials and Methods)+ The phylogenetic distribution reveals that alternate versions of the triplets have evolved independently several times, thus increasing the likelihood that this comparatively inferred base triple is real+ Nuclear magnetic resonance analysis of this region of the 16S rRNA (Kalurachchi & Nikonowicz, 1998) The nucleotide at position 121 determines the base pairs and their arrangement at 124:237 and 125:236+ Note that position 121 is a pyrimidine in 98% of the prokaryotic 16S rRNA sequences, with C occurring in 76% of these sequences, U in 22%, and G and A each with 1% (Table 1A)+ When position 121 is a C, then 124:237 is a GC pair in 98% of the sequences, and 125:236 is a UG in 97% of the sequences (Table 1B)+ When position 121 is a U, then 124:237 is an AU (45%), CG (30%), UA (15%), or GC (9%), and 125:236 is a UA (94%), or UG (5%)+ Taken all together, four sequence motifs occur for these sequences (Table 2): when position 121 is a C, the 124:237 and 125:236 base pairs are predominantly GC/UG (76%, motif A), and when 121 is a U, then the 124:237 and 125:236 base pairs are predominantly AU/UA (10%, motif B), followed by CG/UA (7%, motif C) or UA/UA (3%, motif D)+ Comparative analysis should reveal, in addition to the nucleotide frequencies for the positions of interest, an approximate number of events or times that the nucleotides have changed coordinately during the evolution o...…”