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The ribosome translates the genetic information encoded in messenger RNA into protein. Folded structures in the coding region of an mRNA represent a kinetic barrier that lowers the peptide elongation rate, as the ribosome must disrupt structures it encounters in the mRNA at its entry site to allow translocation to the next codon. Such structures are(More)
We have followed individual ribosomes as they translate single messenger RNA hairpins tethered by the ends to optical tweezers. Here we reveal that translation occurs through successive translocation--and-pause cycles. The distribution of pause lengths, with a median of 2.8 s, indicates that at least two rate-determining processes control each pause. Each(More)
We report the crystal structure of a translation termination complex formed by the Thermus thermophilus 70S ribosome bound with release factor RF2, in response to a UAA stop codon, solved at 3 A resolution. The backbone of helix alpha5 and the side chain of serine of the conserved SPF motif of RF2 recognize U1 and A2 of the stop codon, respectively. A3 is(More)
Coupled translocation of messenger RNA and transfer RNA (tRNA) through the ribosome, a process catalyzed by elongation factor EF-G, is a crucial step in protein synthesis. The crystal structure of a bacterial translocation complex describes the binding states of two tRNAs trapped in mid-translocation. The deacylated P-site tRNA has moved into a partly(More)
Translocation of messenger and transfer RNA (mRNA and tRNA) through the ribosome is a crucial step in protein synthesis, whose mechanism is not yet understood. The crystal structures of three Thermus ribosome-tRNA-mRNA-EF-G complexes trapped with β,γ-imidoguanosine 5'-triphosphate (GDPNP) or fusidic acid reveal conformational changes occurring during(More)
Ribosome recycling factor (RRF) disassembles posttermination complexes in conjunction with elongation factor EF-G, liberating ribosomes for further rounds of translation. The striking resemblance of its L-shaped structure to that of tRNA has suggested that the mode of action of RRF may be based on mimicry of tRNA. Directed hydroxyl radical probing of 16S(More)
A detailed understanding of tRNA/mRNA translocation requires measurement of the forces generated by the ribosome during this movement. Such measurements have so far remained elusive and, thus, little is known about the relation between force and translocation and how this reflects on its mechanism and regulation. Here, we address these questions using(More)
The sequence and secondary structure of the 5'-end of mRNAs regulate translation by controlling ribosome initiation on the mRNA. Ribosomal protein S1 is crucial for ribosome initiation on many natural mRNAs, particularly for those with structured 5'-ends, or with no or weak Shine-Dalgarno sequences. Besides a critical role in translation, S1 has been(More)
Measuring the binding of antibiotics and other small-molecular-weight ligands to the 2.5 MDa ribosome often presents formidable challenges. Here, we describe a general method for studying binding of ligands to ribosomes that carry a site-specific fluorescent label covalently attached to one of the ribosomal proteins. As a proof of principle, an(More)