Specific recognition of the 3′-terminal adenosine of tRNAPhe in the exit site of Escherichia coli ribosomes

Interactions between 23S rRNA and tRNA in the ribosomal E site

“…The importance of interaction of the tRNA 39 end with the 2390 region is further strengthened by the results of modification-interference selection+ Among E site-specific nucleotides in 23S rRNA, only modification of C2394 interfered with binding of uncharged tRNA to the 50S subunit E site+ Therefore, C2394 is apparently the primary 23S rRNA residue responsible for establishing contacts with A76 of tRNA in the E site+ Besides C2394, modification of three other residues, G2252, U2506, and U2585, also destabilized the complex between the 50S subunit and deacylated tRNA+ These three residues were among those that were found to be essential for binding of peptidyl-tRNA in the P site in the previous modificationinterference experiments (Bocchetta et al+, 1998) and thus, in the current experiments their modification apparently interfered with binding of deacylated tRNA to the large subunit P site+ Though modification of A2451 with DMS was found previously to interfere with binding of peptidyl-tRNA to the P site of the 50S subunit, in experiments with deacylated tRNA, modification of A2451 did not preclude tRNA binding+ Thus, modification-interference results confirm the conclusion of Noller et al+ (1990), made on the bases of tRNA footprinting, that A2451 may interact with the aminoacyl moiety of the P site-bound tRNA+ Crosslinking experiments utilizing tRNA azidoderivatives placed the 39 end of the E site-bound tRNA near ribosomal protein L33 (Wower et al+, 1993(Wower et al+, , 2000+ At the same time, L33 was mapped close to C2394 (Osswald et al+, 1990)+ Therefore, the interaction between tRNA A76 and 23S rRNA C2394 could be mediated by L33+ This model seems unlikely, however, because deacylated tRNA binds efficiently to the socalled KSP particles and protects E site-specific C2394 (our unpubl+ results)+ KSP particles, which are prepared from Thermus aquaticus 50S subunits by treatment with proteinase K, SDS, and phenol, retain only a subset of large ribosomal subunit proteins and apparently lack L33 (Noller et al+, 1992;Khaitovich et al+, 1999)+ Thus, protein L33 appears to be dispensable for the interaction of the tRNA 39 end with C2394+ Footprinting and modification-interference results are compatible with a direct interaction of A76 of E sitebound tRNA and C2394 of 23S rRNA+ Because tRNA protects C2394 from DMS modification at N3 of the cytidine base and, furthermore, because binding of tRNA to the E site is abolished when C2394 is modified by DMS, it is reasonable to assume that N3 of C2394 participates directly in bond formation with A76 of tRNA+ Reduced binding of tRNA containing etheno A or formycin as a 39 terminal base suggests that N6 and N7 of A76 may be involved in bond formation+ These considerations can be accommodated by a model in which A76 of tRNA forms a reverse Hoogsteen pair with C2394 of 23S rRNA (Fig+ 5B), though other types of interaction between tRNA 39-terminal residue and C2394 are also possible+ Hoogsteen-type base pairing between A76 of tRNA and U2506 or U2585 in ribosomal A-and P-sites, respectively, was proposed previously (Porse et al+, 1996;Wower et al+, 1999)+ It was suggested previously that A76 may form a Watson-Crick base pair with U2111 (Lill et al+, 1988(Lill et al+, , 1989+ This hypothesis appears ...…”

“…The most straightforward explanation of this result is that A2392 and C2394 are in immediate proximity to the tRNA 39 terminal nucleotide+ This conclusion is in good agreement with the observation that the Fe(II)-EDTA complex, tethered to the end of the E site-bound tRNA, cleaves preferentially the 2390-2440 region of 23S rRNA (Joseph & Noller, 1996)+ It is further supported by a recently reported crosslink between the E site-bound tRNA containing 2-azidoadenosine at the 39 terminus and C2422 of 23S rRNA (Wower et al+, 2000); in the ribosome tertiary structure the 23S rRNA position 2422 is located in a close proximity from C2394 (Ban et al+, 2000)+ Although removal of the tRNA 39 terminal nucleotide completely eliminates protections at A2392 and C2394, tRNA ox-red with the disrupted 39 terminal ribose still strongly protects these positions, showing that the ribose moiety is not essential for the interaction+ This conclusion is supported by the binding studies, in which disruption of the 39 terminal ribose only marginally affected binding of tRNA to the E site (Lill et al+, 1988)+ In contrast to the ribose modification, the nature of the nitrogen base of the tRNA 39 terminal nucleotide is critically important for tRNA binding and its interaction with 23S rRNA+ Replacement of adenine 76 with C, G, or etheno-A reduced tRNA binding more than 100-fold (Lill et al+, 1988)+ Replacement of adenine 76 with formycin also notably lessens tRNA binding to the E site of 50S subunits, even in the presence of methanol, as can be judged by reduced protection of all E sitespecific positions (Fig+ 3)+ At the same time, quantification of the footprints produced by tRNA F76 at G2112 and C2394 showed a more significant loss of protection at C2394 and A2392 than at G2112, compatible with the direct participation of the 39 terminal base in interaction with the 2390 region+…”

“…Binding studies and functional tests showed that, in the presence of 33% methanol, peptidyl-tRNA analogs bind preferentially to the P site of the isolated 50S subunit (Monro & Marcker, 1967;Parfenov & Saminskii, 1985;Moazed & Noller, 1991;Bocchetta et al+, 1998)+ Under these conditions, deacylated tRNA binds to two sites whose biochemical characteristics are reminiscent of those of P and E sites (Parfenov & Saminskii, 1985)+ In agreement with these findings, our footprinting experiments showed that while peptidyl-tRNA bound to 50S subunit in the presence of 33% methanol protects exclusively P site-specific positions in 23S rRNA, deacylated tRNA protects three additional positions, G2112, A2392, and C2394+ The same three positions become protected when deacylated tRNA is bound to a 50S subunit-peptidyl-tRNA complex, indicating that these positions belong to the E site+ Two of these positions, G2112 and C2394, were assigned previously to the E site based on footprinting of tRNA on the 70S ribosome (Moazed & Noller, 1989)+ Protection of A2392 has not been described previously+ It appears, however, that the A2392 footprint is not a result of subunit dissociation or the presence of methanol because, in our hands, A2392 was also protected if deacylated tRNA (either tRNA Tyr or tRNA Phe ) was bound to 70S ribosomes in the absence of methanol (not shown)+ Footprinting of tRNA derivatives in the E site of the 50S subunit revealed intimate details of tRNA-rRNA contacts+ Interaction of deacylated tRNA with the E site depends critically on the presence of 39-terminal A76+ In the absence of methanol, removal of A76 decreases tRNA binding to the ribosomal E site at least 100-fold (Grajevskaja et al+, 1982;Lill et al+, 1988)+ In the presence of methanol, both tRNA C75 and tRNA A73 can still bind to the E site of isolated 50S subunits, as follows from protection of E site-specific G2112 (Figs+ 2 and 3)+ However, in spite of the apparent binding to the E site, tRNA C75 and tRNA A73 do not protect A2392 and C2394+…”

“…The footprinting experiments presented have another implication+ In contrast with the 50S subunit P site, in which the only tRNA component required for proper interaction is the 39-terminal CCA (Monro & Marcker, 1967;Moazed & Noller, 1991;Samaha et al+, 1995), a more extended portion of tRNA is required for its placement in the E site+ This was evidenced by the inability of microhelix, RNA16-CCA, or pCCA to protect any E site-specific residues+ Because A76 accounts for a significant part of tRNA binding free energy in the E site (Lill et al+, 1988), we suggest that the requirement for a complete T⌿C-acceptor stem-loop is related to proper steric presentation of A76 to the 2394 region+…”

“…In the course of translation, tRNA interacts with the ribosome at three different sites+ Prior to formation of a new peptide bond, aminoacyl-and peptidyl-tRNA are positioned in the acceptor (A) and donor (P) sites, respectively+ After peptidyl transfer and translocation, newly deacylated tRNA is transferred to the exit (E) site+ Existence of the E site was realized considerably after the A and P sites were described (Rheinberger et al+, 1981;Grajevskaja et al+, 1982;Kirillov et al+, 1983)+ Significantly less is known about E site location, composition, interaction with tRNA, and function compared to the other two tRNA-binding sites+ Nevertheless, the E site is thought to play an important role in translation+ Binding of tRNA in the E site prior to its release from the ribosome may promote translocation (Lill et al+, 1986(Lill et al+, , 1989 and/or improve accuracy of translation (Geigenmuller & Nierhaus, 1990)+ The critical characteristic of the E site is its strong preference for a deacylated tRNA 39 end (Rheinberger et al+, 1981;Grajevskaja et al+, 1982;Kirillov et al+, 1983;Lill et al+, 1984;Parfenov & Saminsky, 1985)+ Modifications of the 39 terminal tRNA residue or its removal dramatically affect E site binding of tRNA, indicating the importance of the tRNA 39 end for binding in the E site (Lill et al+, 1988)+ The recently deduced X-ray crystallographic structure of the ribosome-tRNA functional complexes showed that in the E site, tRNA contacts the ribosome in three locations: the 39 end and T⌿C loop of tRNA make contacts with the large ribosomal subunit in the cleft between the central protuberance and the L1 stalk, and the anticodon loop interacts with the small subunit between the platform and the head (Cate et al+, 1999)+ Uncharged tRNA bound to the E site of Escherichia coli 70S ribosomes protects from chemical modification a specific set of bases in 23S rRNA, namely, U2111, G2112, G2116, A2169, and C2394 (Moazed & Noller, 1989)+ This evidence suggested a close proximity of tRNA to rRNA in the E site+ From this, and from the observation that modifications of the 39 terminal adenine of tRNA decreased tRNA binding to the E site, a base pairing between the 39 terminal A of tRNA and U2111 of 23S rRNA was proposed (Lill et al+, 1988)+ However, tRNA-protein interactions can contribute to tRNA binding in the E site as well+ tRNA probes carrying 2-azidoadenosine near their 39 ends could be crosslinked to the protein L33 in the ribosomal E site, suggesting that L33 may facilitate tRNA binding (Wower et al+, 1993)+ At the same time, the anticodon loop of the E site-bound tRNA was crosslinked to protein S11 and 16S rRNA, revealing the contacts between the tRNA anticodon arm and the small ribosomal subunit+ Consensus has not yet been reached on the extent of contribution of codon-anticodon interaction to the tRNA binding in the E site …”