Non-bridging Phosphate Oxygen Atoms within the tRNA Anticodon Stem-loop are Essential for Ribosomal A Site Binding and Translocation

Phelps, Steven S.; Joseph, Simpson

doi:10.1016/j.jmb.2005.03.079

Cited by 8 publications

(9 citation statements)

References 44 publications

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“…The U-turn has been suggested to be essential for translocation of normal tRNAs (Phelps et al 2002;Phelps and Joseph 2005), but the present structures show that it is not essential for translocation in these particular tRNAs. In these two structures neither U33 nor the base at position 33.5 is in a position where it can interact with the other side of the anticodon loop (Fig.…”

Section: Resultscontrasting

confidence: 76%

Structures of tRNAs with an expanded anticodon loop in the decoding center of the 30S ribosomal subunit

Dunham

Selmer²,

Phelps³

et al. 2007

RNA

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During translation, some +1 frameshift mRNA sites are decoded by frameshift suppressor tRNAs that contain an extra base in their anticodon loops. Similarly engineered tRNAs have been used to insert nonnatural amino acids into proteins. Here, we report crystal structures of two anticodon stem-loops (ASLs) from tRNAs known to facilitate +1 frameshifting bound to the 30S ribosomal subunit with their cognate mRNAs. ASL CCCG and ASL ACCC (59-39 nomenclature) form unpredicted anticodon-codon interactions where the anticodon base 34 at the wobble position contacts either the fourth codon base or the third and fourth codon bases. In addition, we report the structure of ASL ACGA bound to the 30S ribosomal subunit with its cognate mRNA. The tRNA containing this ASL was previously shown to be unable to facilitate +1 frameshifting in competition with normal tRNAs (Hohsaka et al. 2001), and interestingly, it displays a normal anticodon-codon interaction. These structures show that the expanded anticodon loop of +1 frameshift promoting tRNAs are flexible enough to adopt conformations that allow three bases of the anticodon to span four bases of the mRNA. Therefore it appears that normal triplet pairing is not an absolute constraint of the decoding center.

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Section: Resultscontrasting

confidence: 76%

Structures of tRNAs with an expanded anticodon loop in the decoding center of the 30S ribosomal subunit

Dunham

Selmer²,

Phelps³

et al. 2007

RNA

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“…Since the ASL is composed of only 15 nt, it can be easily synthesized with various substitutions. This has facilitated the identification of functional groups within the ASL that are essential for Asite binding and translocation (Phelps et al 2002;Phelps and Joseph 2005). It is important to note that even though an ASL is translocated from the A-site, the rate of translocation is at least 350-fold slower than a peptidyl-tRNA (Studer et al 2003), suggesting that specific contacts between the acceptor arm of the tRNA and the ribosome or the three-dimensional shape of the A-site tRNA may accelerate the translocation of the mRNA-tRNA complex.…”

Section: Discussionmentioning

confidence: 99%

“…Previous studies have demonstrated that the ribosome is capable of translocating an anticodon stem-loop analog (ASL) of tRNA containing a 7-nt anticodon loop and a 4-bp stem from the ribosomal Asite to the ribosomal P-site (Joseph and Noller 1998). More recent studies have identified 2¢-hydroxyl groups and nonbridging phosphate oxygen atoms within the A-site ASL that are important for translocation into the P-site (Phelps et al 2002;Phelps and Joseph 2005). This shows that specific contacts between the backbone of the ASL and the 30S subunit are essential for translocation.…”

Section: Introductionmentioning

confidence: 99%

Ribose 2′-hydroxyl groups in the 5′ strand of the acceptor arm of P-site tRNA are not essential for EF-G catalyzed translocation

Feinberg

Joseph²

2006

RNA

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The coupled movement of tRNA-mRNA complex through the ribosome is a fundamental step during the protein elongation process. We demonstrate that the ribosome will translocate a P-site-bound tRNA Met with a break in the phosphodiester backbone between positions 17 and 18 in the D-loop. Crystallographic data showed that the acceptor arms of P-and E-site tRNA interact extensively with the ribosomal large subunit. Therefore, we used this fragmented P-site-bound tRNA Met to investigate the contributions of single 2¢ -hydroxyl groups in the 5¢ strand of the acceptor arm for translocation into the ribosomal E-site. EF-G-dependent translocation of the tRNAs was monitored using a toeprinting assay and a fluorescence-based rapid kinetic method. Surprisingly, our results show that none of the 2¢ -hydroxyl groups in the 5¢ strand of the acceptor arm of P-site-bound tRNA Met between positions 1-17 play a critical role during translocation. This suggests that either these 2¢ -hydroxyl groups are not important for translocation or they are redundant and the three-dimensional shape of the P-site tRNA is more important for translocation.

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“…In fact, RNA modifications have been shown to stabilize the pyrimidine-rich ASL Lys UUU by pre-ordering the loop into a structure that is more suitable for presentation to the codon in the decoding center. 33 Phosphorothioate substitutions in the canonical ASL 34 have shown that disruption of the contact between the N3 of uridine 33 and the pro-R P nonbridging phosphate oxygen atom at position 36 has little effect on A site binding but severely inhibits translocation to the P site. Destabilization of the Uturn motif does not affect codon-dependent A site binding and this correlates with the finding that a lack of a stabilized U-turn motif in the ASL CCCG also has little effect on A site binding.…”

Section: The Four Nucleotide Asl In Solution Is Structurally Differenmentioning

confidence: 99%

“…12,34 Ribosomes (0.40 μM), tRNA CCG and tRNA CCCG (or ASL5) (6.0 μM), and charged (2.0 μM) or uncharged (6.0 μM) tRNA Phe and tRNA Val were prepared individually in 80 mM potassium cacodylate (pH 7.2), 20 mM MgCl 2 , 150 mM NH 4 Cl, 3 mM 2-mercaptoethanol. A 32 P-labeled AL2 primer was annealed to the mRNA (0.8 μM) and added to pre-programmed and activated ribosomes.…”

Section: Translocation Assaymentioning

confidence: 99%

Translocation of a tRNA with an Extended Anticodon Through the Ribosome

Phelps¹,

Gaudin²,

Yoshizawa³

et al. 2006

Journal of Molecular Biology

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Non-bridging Phosphate Oxygen Atoms within the tRNA Anticodon Stem-loop are Essential for Ribosomal A Site Binding and Translocation

Cited by 8 publications

References 44 publications

Structures of tRNAs with an expanded anticodon loop in the decoding center of the 30S ribosomal subunit

Structures of tRNAs with an expanded anticodon loop in the decoding center of the 30S ribosomal subunit

Ribose 2′-hydroxyl groups in the 5′ strand of the acceptor arm of P-site tRNA are not essential for EF-G catalyzed translocation

Translocation of a tRNA with an Extended Anticodon Through the Ribosome

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