tRNA dynamics on the ribosome during translation

Abstract

Using single-molecule fluorescence spectroscopy, time-resolved conformational changes between fluorescently labeled tRNA have been characterized within surface-immobilized ribosomes proceeding through a complete cycle of translation elongation. Fluorescence resonance energy transfer was used to observe aminoacyl-tRNA (aa-tRNA) stably accommodating into the aminoacyl site (A site) of the ribosome via a multistep, elongation factor-Tu dependent process. Subsequently, tRNA molecules, bound at the peptidyl site and A site, fluctuate between two configurations assigned as classical and hybrid states. The lifetime of classical and hybrid states, measured for complexes carrying aa-tRNA and peptidyl-tRNA at the A site, shows that peptide bond formation decreases the lifetime of the classical-state tRNA configuration by Ϸ6-fold. These data suggest that the growing peptide chain plays a role in modulating fluctuations between hybrid and classical states. Single-molecule fluorescence resonance energy transfer was also used to observe aa-tRNA accommodation coupled with elongation factor G-mediated translocation. Dynamic rearrangements in tRNA configuration are also observed subsequent to the translocation reaction. This work underscores the importance of dynamics in ribosome function and demonstrates single-particle enzymology in a system of more than two components. P rotein synthesis, catalyzed by the ribosome, is rapid, processive, and highly regulated. In bacteria, two RNA-protein subunits consisting of a small 30S subunit and a large 50S subunit assemble around a mRNA template into a roughly spherical 70S particle that translates the nucleotide sequence into a polypeptide chain through repetitive, codon-dependent binding of aminoacylated tRNA.The landmark structures of the 30S (1, 2), 50S (3, 4), and 70S (5) ribosomal particles provide a molecular basis for understanding ribosome function. tRNA molecules bind to the ribosome in a solvent-accessible channel at the subunit interface. Three binding sites for tRNA, called the aminoacyl site (A site), peptidyl site (P site), and exit site (E site), have been identified on both the large and small subunit (Fig. 1). Each tRNA is separated from its neighbor at the elbow region (the site of Ϸ90°b ending) by 25-45 Å (5, 6). The anticodon stem loops of A-and P-site tRNA form Watson-Crick base pairs with adjacent mRNA codons on the 30S subunit (5, 7), whereas the 3Ј CCA terminal residues of A-and P-site tRNAs base-pair with conserved ribosomal RNA (rRNA) loops (8-10) within the 50S subunit peptidyltransferase center, the site of peptide bond formation (11). Additional interactions with rRNA and ribosomal proteins position tRNA molecules on the ribosome (5).The essential components and principal steps of translation have been delineated through genetic, biochemical, and kinetic methods (12). The process of polypeptide elongation has been most extensively characterized (13). Binding of aminoacyl-tRNA (aa-tRNA) to the A site occurs as a ''ternary complex'' with the GTPase elongation ...

Elongation Factor Ts Directly Facilitates the Formation and Disassembly of the Escherichia coli Elongation Factor Tu·GTP·Aminoacyl-tRNA Ternary Complex

“…Akin to earlier steady state measurements of ternary complex formation (16), the fluorescence-based assays described here are based on changes in relative fluorescence intensity. However, unlike earlier work, the approach we describe is based on the environmentally sensitive Cy3 fluorophore (43,58) at a distinct site of tRNA attachment, the naturally occurring modified nucleotide acp 3 U present at position 47 in E. coli tRNA Phe . Although this modification is not as ubiquitous as the s 4 U residue utilized previously, tRNA molecules modified at this site have been shown to be fully functional in all aspects of the translation process, including aminoacylation, tRNA selection, and translocation (43,46,60).…”

“…Native tRNA Phe molecules were site-specifically labeled with the small molecule organic fluorophore Cy3 via the naturally occurring modified nucleotide acp 3 U present at position 47 (43,45). tRNA molecules labeled in this fashion have been shown previously to retain wild-type activities in both aminoacylation and translation reactions (43,46).…”

“…Native tRNA Phe molecules were site-specifically labeled with the small molecule organic fluorophore Cy3 via the naturally occurring modified nucleotide acp 3 U present at position 47 (43,45). tRNA molecules labeled in this fashion have been shown previously to retain wild-type activities in both aminoacylation and translation reactions (43,46). Purified tRNA Phe was aminoacylated by mixing 7.5 pmol of labeled tRNA Phe with 6 pmol of phenylalanyl-tRNA synthetase and 10 nmol of phenylalanine amino acid in a volume of 10 l in buffer C (50 mM Tris-HCl, pH 8, 20 mM KCl, 100 mM NH 4 Cl, 1 mM DTT, 2.5 mM ATP, 0.5 mM EDTA, 10 mM MgCl 2 ).…”

“…Purification of Elongation Factors-His 6 -EF-Tu (tufA) and -EF-Ts (tsf) were expressed recombinantly in E. coli and purified by nickel-nitrilotriacetic acid affinity chromatography in the absence of magnesium as described previously (43). After cleavage, protein factors were further purified using Superdex 75 gel filtration chromatography equilibrated in buffer A (50 mM Tris-HCl, pH 7.5, 100 mM NH 4 Cl, 10 mM MgCl 2 , 0.5 mM EDTA, 50 mM KCl, 1 mM DTT).…”

The importance of surfaces in single-molecule bioscience

“…For example, the folding of an RNA molecule, [81][82][83] structural transitions in DNA molecules, 84,85 assembly and behavior of protein-DNA complexes [86][87][88] and ribosomal structural transitions during translation have all been studied using spFRET. [89][90][91] TIRFM studies have also been conducted with λ DNA (48 502 bp) since these long DNA substrates are particularly useful for studying protein-DNA interactions that occur at multiple, well-resolved locations or those that involve movement of the proteins along the DNA. The contour length of λ DNA (~16 μm) is considerably larger than the effective depth of the evanescent field and therefore the DNA must be confined parallel to the surface to allow visualization along its entire length.…”