Neighborhood of 16S rRNA nucleotides U788/U789 in the 30S ribosomal subunit determined by site-directed crosslinking

Abstract

Site-specific photo crosslinking has been used to investigate the RNA neighborhood of 16S rRNA positions U788/ U789 in Escherichia coli 30S subunits. For these studies, site-specific psoralen (SSP) which contains a sulfhydryl group on a 17 Å side chain was first added to nucleotides U788/U789 using a complementary guide DNA by annealing and phototransfer. Modified RNA was purified from the DNA and unmodified RNA. For some experiments, the SSP, which normally crosslinks at an 8 Å distance, was derivitized with azidophenacylbromide (APAB) resulting in the photoreactive azido moiety at a maximum of 25 Å from the 49 position on psoralen (SSP25APA). 16S rRNA containing SSP, SSP25APA or control 16S rRNA were reconstituted and 30S particles were isolated. The reconstituted subunits containing SSP or SSP25APA had normal protein composition, were active in tRNA binding and had the usual pattern of chemical reactivity except for increased kethoxal reactivity at G791 and modest changes in four other regions. Irradiation of the derivatized 30S subunits in activation buffer produced several intramolecular RNA crosslinks that were visualized and separated by gel electrophoresis and characterized by primer extension. Four major crosslink sites made by the SSP reagent were identified at positions U561/U562, U920/U921, C866 and U723; a fifth major crosslink at G693 was identified when the SSP25APA reagent was used. A number of additional crosslinks of lower frequency were seen, particularly with the APA reagent. These data indicate a central location close to the decoding region and central pseudoknot for nucleotides U788/U789 in the activated 30S subunit.

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+…”

Nucleotides of 18S rRNA surrounding mRNA codons at the human ribosomal A, P, and E sites: A crosslinking study with mRNA analogs carrying an aryl azide group at either the uracil or the guanine residue

“…Because the structures of mammalian ribosomes are not available at a resolution that permits the location of specific helices of the rRNA, our data may be interpreted in terms of the partial atomic structures of the bacterial 30S ribosomal subunit+ Thus, the approximate positions of 18S rRNA nucleotides G961 (G693, the tip of helix 23), C1057 (U789, helix 24), G1207 (G926, helix 27) and G1702 (G1401), G1763-G1764 (;1450-1451), and A1823-A1824 (G1491-A1492) in helix 44 can all be discerned on the crystallographic maps of the Thermus thermophilus 30S subunit at a 5+5 Å resolution (Clemons et al+, 1999)+ Nucleotides G1207 (G926), G1702 (G1401), and A1823-A1824 (G1491-A1492), which are labeled from the ϩ1 to ϩ4 mRNA positions, are nicely clustered on the segment of 30S subunit known as the decoding site (see Clemons et al+, 1999); nucleotide C1057 (U789) is located in the vicinity of this segment+ Two other crosslinking sites, G961 (G693) and G1763-G1764 (1450-1451) are separated from this cluster and from each other+ Positions 1450-1451 lie at the tip of the long penultimate helix 44 very far from the decoding site (Cate et al+, 1999;Clemons et al+, 1999)+ Helix 44 is a very well conserved feature of small subunit rRNAs and it is quite unlikely that it differs substantially in eukaryotes and bacteria+ Thus, crosslinking of mRNA positions ϩ3/ϩ4 to G1763/G1764 should be considered as an artifact+ The source of the error might be the prolonged incubations at room temperature (required for the labeling of 80S ribosomes with the 39-alkylating mRNA analogs; see Malygin et al+, 1994) which could lead to a partial degradation of the ribosomes and, subsequently, to artifactual crosslinking in the fraction of the degraded ribosomes+ As for nucleotide G961 (G693), which crosslinks to the E site-bound codon, we do not see any reasons to doubt the validity of the result; moreover, the analogous crosslink has been detected for E. coli ribosome (see Table 2)+ One of the possible explanations for the separate position of this nucleotide from the decoding site on the structure of the free 30S (Mundus & Wollenzien, 1998) …”

Effects of Tetracycline and Spectinomycin on the Tertiary Structure of Ribosomal RNA in the Escherichia coli 30 S Ribosomal Subunit

“…6), 2 which shows the locations of the helices containing the tetracycline and spectinomycin binding sites and the RNA-RNA cross-links affected by their binding. This model is different from recent models (26 -28) in several regions because it incorporates new information including the UV cross-links C967ϫC1400 (9, 10), U793ϫG1517, and G976ϫG1361 3 and site-specific psoralen cross-links (25). 4 Approximate distances from G1300, G1338, and G693 to C1400 (the P-site) are 45, 35, and 25 Å, respectively, and distances from G1300, G1338, and G693 to A1408 (associated with the A-site) are 57, 50, and 36 Å, respectively.…”