Three-dimensional arrangement of the Escherichia coli 16 S ribosomal RNA

Alternative base pairing between 5′- and 3′-terminal sequences of small subunit RNA may provide the basis of a conformational switch of the small ribosomal subunit

“…3A). Expert-Bezancon and Wollenzien (15) in their earlier model place helix 48 and the 3'-terminal single stranded region at the base of the cleft more towards the centre of the particle, which would be in good accordance with our proposal. Neither of the models contradict our proposal of the open form (Fig.…”

“…This neighbourhood (though not immediate proximity) has gained support from several proposals of more detailed tertiary structure models of E. coli 16S rRNA (15,16,17). In these models neither the 5'-nor the 3'-terminal sequences are base paired and are, therefore, represented as flexible strings of 8 and 5 nucleotides, respectively, whereas the first two helices at the 5' terminus and the last helix at the 3' terminus are positioned in relatively close proximity to each other at the 30S/50S interface side near the cleft of the 30S particle.…”

“…Compatibility of the 5'/3'-terminal base pairing with the threedimensional models proposed for the E. col 30S subunit Three-dimensional models for the arrangement of the 16S rRNA secondary structure in relation to subunit shape and ribosomal proteins have been proposed independently by Brimacombe et al (16) and by Stern et al (17) more recently and an earlier version was proposed by Expert-Bezancon and Wollenzien (15). The former two models show large accordance with each other but as evident from various differences in details (also with the earlier model of Expert-Bezancon and Wollenzien 15) they must still be considered as approximations, open to further refinement and improvement.…”

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

“…Comparison of the conformation of the 59 terminal region in 30S versus 70S ribosomes+ A: APA-derivitized 16S rRNA (the ϩ5 construct) was irradiated as protein-free RNA (lane 16S) and after reconstitution and isolation of 30S subunits (lane 30S)+ To test for crosslinking in 30S versus 70S ribosomes, 30S and 50S subunits were incubated, irradiated, and then separated on sucrose gradients+ RNA was isolated from 30S, 50S, and 70S particles, labeled and examined by PAGE+ Tot is RNA from the 30S ϩ 50S mixture before sucrose gradient separation, 70S, 50S, and 30 are RNA samples from the corresponding parts of the sucrose gradient+ B: Comparison of crosslinking in 30S versus 70S for the ϩ8, ϩ5, and Ϫ5 constructs+ 30S subunits containing APA-modified 16S rRNA were prepared, mixed with 50S subunits, irradiated, and then separated on sucrose gradients+ Lanes marked 30S and 70S contain RNA from corresponding parts of sucrose gradients for each construct+ The lanes labeled 16S from 70S contain RNA that was isolated from 70S ribosomes, deproteinated, and then irradiated before PAGE analysis+ 1401,1413,[1492][1493]1330) have been determined as being tRNA footprinted sites (Moazed & Noller, 1986 and are expected therefore to be located in the front (interface) middle of the 30S subunit+ Three of the crosslinking sites in the first group, 298-300 [in helix H11 (helix numbering is according to ], 334-338 (in helix H13), and 343-347 (in helix H14) based on their crosslinking in the ϩ8 construct are within 16 Å of the G9 nucleotide+ The 313-314 crosslinking site (in helix H6) is 16-34 Å from G9+ A UV crosslink 244 ϫ 894 (Stiege et al+, 1986;Wilms et al+, 1997) and a chemical crosslink 31 ϫ 306 (Atmadja et al+, 1986) place strong constraints on the locations of H6 and H12, which must be close to the middle front of the subunit+ Chemical crosslinking done in the protein-free 16S rRNA included crosslinks that were between nucleotides at about 340 and nucleotides close to the 59 and 39 termini (Expert-Bezancon & Wollenzien, 1985;Wollenzien et al+, 1985)+ In addition, site-directed cleavage experiments in which the BABE reagent (Rana & Meares, 1991) was added to Cys C31 of protein S4 (Heilik et al+, 1995) or was added to Cys99 of protein S5 produced cleavages in the 16S rRNA in the interval 297-301+ S4 and S5 have been positioned in the middle left and middle of the subunit based on neutron scattering experiments (Capel et al+, 1987)+ Thus the APA crosslinking results confirm the locations of helices H6 and H11 and indicate new distances for helices H13 and H14 to G9 in the center of the subunit+ Sites in the intervals 556-563 and 912-920 are crosslinked in all of the constructs indicating a distance to G9 of less than 16 Å+ Crosslinking of 892 and 906 in helix H27 also indicates a distance of less than 16 Å to G9+ The crosslink involving 901 occurs only in the Ϫ5 construct; this suggests a much longer distance of 58-88 Å+ However, this would be physically unfeasible and there must be other reasons for the absence of crosslinking to 901 in the other constructs+ The sites 892 and 906 in helix H27 are expected to be ...…”

Quaternary structure of the small ribosomal subunit ofEscherichia colidetermined by neutron diffraction

“…A number of groups are in the process of solving atomic structures of ribosomal proteins via crystallography (Wilson, Krzysztof, Badger, Tanaka & White, 1986;Leijonmark, Erickson & Liljas, 1980) or NMR (van de Ven, de Bruin & Hilbers, 1984). The problems associated with the secondary and tertiary structures of ribosomal RNA are being attacked by chemical (Expert-Bezancon & Wollenzien, 1985;Brimacombe, Atmagja, Kyriatsoulis & Stiege, 1986) and NMR techniques (van de Ven, de Bruin & Hilbers, 1984). We can hope that in the not too distant future the structure and therefore a detailed mechanistic model of one of the most complex enzymatic systems will be in hand.…”