Photolabile anticodon stem–loop analogs of tRNA^Phe as probes of ribosomal structure and structural fluctuation at the decoding center

Abstract

With the recent availability of high-resolution structures of bacterial ribosomes, studies of ribosome-catalyzed protein biosynthesis are now focusing on the nature of conformational changes that occur as the ribosome exerts its complex catalytic function. Photocrosslinking can be relevant for this purpose by providing clues to ribosomal structural fluctuations and dynamics. Here we describe crosslinking experiments on 70S ribosomes using two photolabile anticodon stem-loop derivatives of Escherichia coli tRNA Phe carrying a 4-thiouridine in either position 33 or 37 and denoted Ph-ASLs. One or both of these Ph-ASLs bind to the tRNA A-, P-, and E-sites on the ribosome, with both binding to and photocrosslinking from the E-site showing strong dependence on the presence of a tRNA in the P-site. Both Ph-ASLs crosslink to the extreme 3-end of 16S rRNA from both the P-and E-sites, providing direct confirmatory evidence in solution for the folding back of the 3-end toward the decoding region. This suggests that the 3-end of 16S rRNA may act as a switch in controlling mRNA access to the decoding center, a phenomenon of potential relevance for the translation of leaderless mRNA. E-site bound Ph-ASLs also form photocrosslinks to nucleotides 1395-1398, 1399-1400, and 1491-1494 at the top of helix 44 of 16S rRNA, indicating movement of the decoding center from a position between the A-and P-sites seen in the crystal structure to one neighboring the E-site.

A functional relationship between helix 1 and the 900 tetraloop of 16S ribosomal RNA within the bacterial ribosome

“…Site-directed cross-linking studies with 4-thiouridine identified a cross-link between residue U14 of helix 1 and the 903-904 region of helix 27, very close to the 900 tetraloop (Juzumiene and Wollenzien 2001), but such a cross-link is incompatible with the crystal structure of the 30S subunit. It is known that the top of helix 44 and neighboring helices, such as helices 1 and 27, have some conformational flexibility (VanLoock et al 2000;Gabashvili et al 2003;Druzina and Cooperman 2004), but a direct contact between nucleotides U14 and 902-903 would require the disruption of helix 2 and a drastic displacement of helices 1 and 27. Rather, we propose that mutations in helix 1 affect the 900 tetraloop indirectly.…”

EF-G-Dependent GTPase on the Ribosome. Conformational Change and Fusidic Acid Inhibition

“…Except as specified below, materials were obtained and methods were performed as described elsewhere ( − ). 5-Iodoacetamido-fluorescein (IAF), 7-diethylamino-3-((((2-maleimidyl)ethyl)amino)carbonyl)coumarin (MDCC), and 7-diethylamino-3-(4‘-maleimidylphenyl)-4-methylcoumarin (CPM) were obtained from Molecular Probes.…”

RNA Crosslinking Methods

“…Specific reaction conditions will be based on our studies with RNase P RNA; however, such conditions are similar to that in other in vitro studies of RNA biochemistry. Indeed, some or all of the methods described in this chapter have been successfully applied in analysis of structural and catalytic RNAs as well as the major cellular RNPs, including the ribosome, and the spliceosome (Christian et al , 1998; Druzina and Cooperman, 2004; Harris et al , 1994, 1997; Huggins et al , 2005; Juzumiene and Wollenzien, 2001; Juzumiene et al , 2001; Kim and Abelson, 1996; Leung and Koslowsky, 1999; Maroney et al , 1996; Montpetit et al , 1998; Newman et al , 1995; Pinard et al , 1999; Pisarev et al , 2008; Podar et al , 1998; Rinke-Appel et al , 2002; Ryan et al , 2004; Sergiev et al , 1997; Wassarman and Steitz, 1992; Yu and Steitz, 1997). Crosslinking is thus likely to remain a very useful tool in deconstructing the structure and function of RNAs and RNPs for many years to come.…”