Covalent crosslinking of tRNA1Val to 16S RNA at the ribosomal P site: identification of crosslinked residues.

Abstract

N-Acetylvalyl-tRNAYl (AcVal-tRNA 1) was bound to the P site of uniformly 32P-labeled 70S ribosomes from Escherichia coli and crosslinked to 16S RNA in the 30S ribosomal subunit by irradiation with light of300-400 nm. To identify the crosslinked nucleotide in 16S RNA, AcVal-tRNAY. l-16S [32P]RNA was digested completely with RNase TI and the band containing the covalently attached oligonucleotides from tRNA and rRNA was isolated by polyacrylamide gel electrophoresis. The crosslinked oligonucleotide, and the 32P-labeled rRNA moiety released from it by photoreversal of the crosslink at 254 nm, were then analyzed by secondary hydrolysis with pancreatic RNase A and RNase U2. The oligonucleotide derived from 16S RNA was found to be the evolutionarily conserved sequence, U-A-C-A-C-A-C-C-G1401, and the nucleotide crosslinked to tRNAI , C14W. The identity of the covalently attached residue in the tRNA was established by using AcVal-tRNAlv-16S RNA prepared from unlabeled ribosomes.This complex was digested to completion with RNase T1 and the resulting RNA fragments were labeled at the 3' end with [5'-32P]pCp. The crosslinked Ti oligonucleotide isolated from the mixture yielded one major end-labeled component upon photoreversal. Chemical sequence analysis demonstrated that this product was derived from the anticodon-containing pentadecanucleo-

Orientation of the tRNA anticodon in the ribosomal P-site: Quantitative footprinting with U33-modified, anticodon stem and loop domains

“…In addition to showing differential protection of the individual P-site nucleosides in 16S rRNA, quantitative footprinting also highlighted subtle differences between the ribosomal binding of native tRNA Phe and the ASL-U 33 + The ASL-U 33 protected the 16S P-site nucleosides very similarly to yeast tRNA Phe , except for two notable differences, G530 and C1400+ Residue G530 was protected to a higher degree by tRNA Phe than by ASL-U 33 (tRNA Phe : K p ϭ 25 6 18 nM; ASL-U 33 : K p ϭ 96 6 60 nM), whereas C1400, whose chemical reactivity was enhanced upon yeast tRNA Phe binding, did not exhibit any enhancement with the binding of ASL-U 33 + A fully modified yeast tRNA Phe ASL (with all the nucleoside modifications) binds to 30S ribosomal subunits with the same binding affinity (K d ) as yeast tRNA Phe (Rose et al+, 1983) and produces the same footprints (including the C1400 enhancement) on 16S rRNA as yeast tRNA Phe (Moazed & Noller, 1986)+ The anticodon stem and loop is the only region of the tRNA molecule that is protected from hydroxyl radical probing by the 30S ribosomal subunit (Huttenhofer & Noller, 1992)+ Therefore, we believe the differences in the K p s observed for the interaction of the 16S rRNA nucleosides with ASL-U 33 versus yeast tRNA Phe (Table 1) are due to the lack of modified nucleosides in the ASL, and not due to its smaller size or sequence length+ The enhancement of C1400 reactivity with the binding of yeast tRNA Phe was thought to result from the 29-O-methyl modification of G 34 (Gm 34 ) in that tRNA's anticodon, as this enhancement was not observed upon binding of E. coli tRNA Phe that has an unmethylated G at position 34 (Moazed & Noller, 1986)+ In fact, we have recently observed that Gm 34 was the only modification in the yeast tRNA Phe ASL able to produce the C1400 enhancement+ This effect was observed only with Gm 34 -containing ASLs (Ashraf et al+, in prep+) and is consistent with the fact that C1400 has been directly cross-linked to the anticodon position 34 of tRNA (Prince et al+, 1982)+ Thus, C1400 lies very close to the tRNA anticodon in the P-site, and probably adjacent to position 34+…”

“…Transfer and ribosomal RNAs play critical roles in both the decoding (Moazed & Noller, 1986, 1990) and peptidyl-transferase (Barta et al+, 1984;Noller et al+, 1992;Noller, 1993;Green et al+, 1997) activities of the ribosome+ Binding of tRNA to the mRNA-programmed ribosome involves tRNA-rRNA, as well as tRNA-mRNA contacts+ Some tRNA-rRNA contacts have been identified with chemical footprinting techniques (Moazed & Noller, 1990;von Ahsen & Noller, 1995)+ Numerous cross-linking studies have provided insights into tRNA binding sites on the ribosome (Prince et al+, 1982;Doring et al+, 1994;Osswald et al+, 1995)+ However, two of the more difficult challenges have been the identification of specific nucleoside interactions of tRNA's anticodon stem and loop with 16S rRNA and the orientation of the tRNA anticodon relative to the 16S nucleosides at the decoding site+…”

Structure and function of ribosomal RNA

“…The mutations in 23S RNA are implicated in peptidyl transfer, and as already mentioned above the same region of the 23S RNA structure has been identified in a cross-link to a peptidyl-tRNA affinity analogue (Barta et al, 1984). This latter RNA region and the corresponding region in 16S RNA that is cross-linked to the anticodon of tRNA (Prince et al, 1982;cf. Figs. 1 and 2) are both part of the highly conserved secondary structural core (see the section on secondary structure), and preliminary modelbuilding studies in our laboratory indicate that the conserved core is concentrated in the interface regions of both subunits.…”

“…The early studies Glotz et al, 1981) only led to the identification of secondary structural cross-links, but, by using different partial digestion conditions, a number of tertiary as well as secondary intra-RNA cross-links have now been localized in both 50S (Stiege et al, 1982(Stiege et al, , 1983 tRNA has also been used as a target for crosslinking studies. In one series of experiments, Prince et al (1982) showed that the hypermodified uridine at the 5'-anticodon position of tRNAval or tRNASer is cross-linked to C-1400 in the E. coli 16S RNA sequence. Furthermore a precisely analogous cross-link has been identified in yeast 18S RNA , and the same authors have described an elegant method for the localization of such cross-link sites by a combined endlabelling and cleavage procedure (Ehresmann & Ofengand, 1984).…”

Alterations in the peptidyltransferase and decoding domains of ribosomal RNA suppress mutations in the elongation factor G gene

“…Percentage of mutant rRNA in the total cellular rRNA and the rRNA fractions of PEM100 pSTL102, JM83 pSTL102, PEM100 pKK1400U/2058G, and JM83 pKK1400U/2058G when grown at 30 8C (light gray) and 42 8C (dark gray) as determined by primer extension analysis+ Values are given for 16S rRNA (A and C) and 23S rRNA (B and D) in the total cellular rRNA pool (Tot), polysome fraction (Pol), free-subunit fraction (Sub) and 70S monosome fraction (70S)+ major determinant of growth rate under optimal conditions is cellular protein-synthetic capacity (Nomura et al+, 1984;Lindahl & Zengel, 1986)+ The increases in doubling time observed in a wild-type EF-G background (i+e+, in PEM100 at 30 8C or in JM83 at either 30 8C or 42 8C) reflect a reduced translational efficiency due either to overloading of ribosomes per cell by plasmidencoded rrn operons (pSTL102) (Gourse et al+, 1982) or to a defect in the decoding region of 16S rRNA (C1400U mutation)+ However, despite the detrimental effect on growth rate, both plasmids effectively suppress the ts EF-G mutation at 42 8C (Table 2)+ The sites of both suppressor mutants in 16S rRNA are located in the decoding region of the 30S subunit (Dahlberg, 1989)+ The site of the EF-G mutation, domain IV, binds to the ribosome in close proximity to C1400 (Agrawal et al+, 1998;Wilson & Noller, 1998a), and UV crosslinking experiments have revealed that position 1400 associates with the anticodon loop of the aa-tRNA in the ternary complex with EF-Tu-GTP (Prince et al+, 1982), of which EF-G domain IV is a structural mimic (Nissen et al+, 1995)+ Position 1192 in 16S rRNA is located in helix 34, which has also been placed in the decoding region of the ribosome (Dontsova et al+, 1992;Moine & Dahlberg, 1994)+ The resistance to spectinomycin that is conferred by the C1192U substitution suggests that this nucleotide is involved in the translocation step of elongation, as spectinomycin is believed to inhibit EF-G-ribosome interactions (O'Connor et al+, 1995)+ Furthermore, the spectinomycin-resistance phenotype conferred by pKK1192U can be suppressed by a mutation in EF-G (Johanson & Hughes, 1994)+ Interestingly, mutants in 16S rRNA alone are not sufficient to suppress the ts EF-G mutant; there is a need for the 23S rRNA mutation A2058G, which resides in the peptidyltransferase loop of domain V (Douthwaite, 1992)+ Both of these functional centers on the ribosome are directly involved in the elongation cycle (Wilson & Noller, 1998b), but mutations at the two known EF-G binding sites in 23S rRNA, 1067 and 2661, did not give suppression and, in fact, were incompatible with the EF-G mutation at any temperature (Table 1)+ The preferential incorporation of mutant 16S and 23S rRNA into the polysomes of PEM100 pSTL102 and PEM100 pKK1400U/2058G at 42 8C (Fig+ 1) suggests the suppressor mutations are more functional than wildtype rRNAs at the restrictive temperature+ It is clear, however, that the polysome fractions are not composed exclusively of mutant rRNA, but do contain some wild-type rRNA+ Suppression of the ts EF-G defect must be achieved by ribosomes containing either a 16S or a 23S rRNA mutation+ Mutat...…”