A new model for the three-dimensional folding of Escherichia coli 16 S ribosomal RNA. I. fitting the RNA to a 3D electron microscopic map at 20 Å 1 1Edited by D. E. Drapper

Positions in the 30S ribosomal subunit proximal to the 790 loop as determined by phenanthroline cleavage

“…The results reported in this study provide definitive evidence that nt 582-584 and 693-694 are within 15 Å of the tethered site of phenanthroline at the 59 end of the DNA oligomer complementary to nt 787-795 of 16S rRNA in 30S ribosomal subunits that have not been reconstituted+ The cleavages in the 693 region do corroborate evidence from other studies+ Brimacombe's group (Atmadja et al+, 1986) found crosslinking between fragments 693-696 and 794 (or 799) using a nitrogen mustard+ This crosslink was placed into the Harvey model using a distance of 11 6 4 Å (Malhotra & Harvey, 1994)+ More recently Mundus & Wollenzien (1998) have reported a distance of ,25 Å between nt 788-789 and 693 using site-specific photocrosslinking+ Our results confirm the results reported by Brimacombe and refine the findings of Mundus and Wollenzien, limiting the 693 region to be within Mueller & Brimacombe (1997), wherein they showed helixes 24+3 (790 region) and 23+2 (690 region) to be both proximal and accessible to subunit association+…”

“…The 790 loop has been implicated as being on the surface of the 30S subunit and involved in subunit association (Tapprich & Hill, 1986;Tapprich et al+, 1989;Santer et al+, 1990)+ In addition, the 790 loop, although protected by P-site-bound tRNA (Moazed & Noller, 1990), along with 693, was found to be nonessential for tRNA binding (von Ahsen & Noller, 1995)+ These chemical protection studies also implied that A532 and G693, as well as numerous nucleotides in the decoding region, were protected by P-site-bound tRNA+ In addition, IF3 both footprints and crosslinks to the 790 loop (Ehresmann et al+, 1986;Muralikrishna & Wickstrom, 1989), and a mutation G791C inhibits IF3 binding (Tapprich et al+, 1989)+ These studies suggest the functional importance of the 790 region+ Numerous results have placed the 530 loop proximal to the 790 region (Dontsova et al+, 1992;RinkeAppel et al+, 1993;Alexander et al+, 1994;Bucklin et al+, 1997;Bullard et al+, 1998)+ The position of the 530 loop has been controversial, as immunoelectron microscopy places the 530 loop on the opposite side of the 30S subunit from the decoding site (Trempe et al+, 1982;Scheinman et al+, 1992;Malhotra & Harvey, 1994;Mueller & Brimacombe, 1997)+ We looked extensively for directed phenanthroline cleavages in this region; but none were found+ It is possible that the phenanthroline is near the 530 loop, but because of the short tether to the phenanthroline or its orientation, it cannot bookmark the loop, or perhaps the oP is on the distal side of the A-form RNA-DNA probe helix relative to the 530 loop (see Fig+ 7)+ Additional experiments that vary the length of the DNA oligomer, and thereby change the clockwise position around the 790 loop, may provide additional insight into the exact positions of the 790 and the 530 loops with respect to each other in the 30S ribosomal subunit+ The sensitivity and specificity provided by directed copper-phenanthroline cleavages should allow these and other delicate interactions within the dynamic 30S ribosomal subunits to be monitored+…”

Mutations in helix 27 of the yeast Saccharomyces cerevisiae 18S rRNA affect the function of the decoding center of the ribosome

“…The rDNA plasmid (see Materials and methods; Table 1) containing coding regions for all rRNAs as well as the TRP1 LEU2d genes (pRDN-wt-TL) was mutagenized with hydroxylamine in vitro, followed by transformation into L1524, bearing a stable deletion of the rDNA repeats on the chromosome and a wild-type rDNA locus on a multicopy URA3-plasmid, pRDNwt-U+ About 5,000 Trp ϩ transformants were patched on ϪLeu medium to select for cells in which the mutagenized pRDN-TL plasmid is amplified+ Following incubation on ϪLeu medium, cells were transferred to ϪAde ϪLeu and ϪHisϪLeu media to score for the efficiency of nonsense suppression+ One mutant, rdn8, was found to inhibit sup45-mediated readthrough of nonsense codons+ The level of inhibition of the ade1-14 UGA readthrough in the rdn8 mutant was also seen as a color difference on YPD medium: the darker the color, the stronger the inhibition of sup45-mediated readthrough (data not shown)+ The same phenotype was observed in this mutant after complete loss of host pRDN-wt-U plasmid on medium containing 5-fluoroorotic acid (5-FOA; see Materials and methods)+ Plasmid isolated from the rdn8 mutant cells was reintroduced into L1524, and resultant transformants were found to inhibit sup45-mediated nonsense suppression, thereby confirming plasmid-associated antisuppression+ Using a DNA fragment exchange technique, the rdn8 mutation was mapped to helix 27 of the 18S rRNA gene (Mueller & Brimacombe, 1997 The conformational switch model predicts that the rdn8 alteration should decrease translational fidelity, because the G886A change strengthens the 886-911 base pair of the 912-910/885-887 error-prone structure in helix 27 (Lodmell & Dahlberg, 1997; Fig+ 1B)+ Unexpectedly, we found that rdn8 decreases translational readthrough of stop codons+ To examine whether changes in the stability of the alternative 912-910/888-890 accurate helix would affect translational fidelity as predicted by the model, mutations that either stabilize or destabilize 912-888 base pairing were introduced into helix 27 by site-directed mutagenesis+ The effect of these changes on translational accuracy was characterized both in vivo and in vitro+…”

Organization of the 16S rRNA around its 5′ terminus determined by photochemical crosslinking in the 30S ribosomal subunit

“…The central part of the 30S ribosomal subunit that faces the 50S subunit in the working 70S ribosome contains the region essential for tRNA binding and movement+ Recent cryoelectron microscopy experiments have clearly shown electron density attributable to the tRNAs in the space between the 30S and 50S subunits (Frank et al+, 1995;Agrawal et al+, 1996;Malhotra et al+, 1998)+ The 30S subunit is responsible for mRNA association and the decoding process, and this requires a placement of the anticodon ends of the tRNAs in close proximity with parts of the 30S subunit (Dahlberg, 1989;Noller, 1991;Frank et al+, 1995)+ In terms of the molecular constituents in the 30S decoding region, ribosomal proteins S4, S5, and S12 have been implicated in influencing the fidelity of decoding (see Karimi & Ehrenberg, 1994)+ However, there are a number of 16S rRNA nucleotides that are footprinted by tRNA or at which mutations affect the decoding and translocation process (Noller, 1991)+ Photoaffinity labeling and site specific cleavage experiments by tRNA, tRNA derivatives, and mRNA analogs (see Green & Noller, 1997) also indicate important nucleotide positions+ Several proposals for the three-dimensional arrangement for the 16S rRNA already place many of these sites in positions in the central part of the subunit where they could participate in the expected interactions Noller et al+, 1995;Fink et al+, 1996;Mueller & Brimacombe, 1997;Wang et al+, 1999)+ However, the arrangements still are inconclusive about the proximity of some of the 16S rRNA sites with respect to the bound tRNA, and models are not available yet to describe the details of the binding pockets for the tRNAs+…”