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Reverse translocation of tRNA in the ribosome.

ABSTRACT: A widely held view is that directional movement of tRNA in the ribosome is determined by an intrinsic mechanism and driven thermodynamically by transpeptidation. Here, we show that, in certain ribosomal complexes, the pretranslocation (PRE) state is thermodynamically favored over the posttranslocation (POST) state. Spontaneous and efficient conversion from the POST to PRE state is observed when EF-G is depleted from ribosomes in the POST state or when tRNA is added to the E site of ribosomes containing P-site tRNA. In the latter assay, the rate of tRNA movement is increased by streptomycin and neomycin, decreased by tetracycline, and not affected by the acylation state of the tRNA. In one case, we provide evidence that complex conversion occurs by reverse translocation (i.e., direct movement of the tRNAs from the E and P sites to the P and A sites, respectively). These findings have important implications for the energetics of translocation.

SUBMITTER: Shoji S

PROVIDER: S-EPMC2661759 | biostudies-literature | 2006 Dec

REPOSITORIES: biostudies-literature

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Reverse translocation of tRNA in the ribosome.

Shoji Shinichiro S Walker Sarah E SE Fredrick Kurt K

Molecular cell 20061201 6

A widely held view is that directional movement of tRNA in the ribosome is determined by an intrinsic mechanism and driven thermodynamically by transpeptidation. Here, we show that, in certain ribosomal complexes, the pretranslocation (PRE) state is thermodynamically favored over the posttranslocation (POST) state. Spontaneous and efficient conversion from the POST to PRE state is observed when EF-G is depleted from ribosomes in the POST state or when tRNA is added to the E site of ribosomes con ...[more]

PMID: 17189194

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Project description:In addition to the conserved translation elongation factors eEF1A and eEF2, fungi require a third essential elongation factor, eEF3. While eEF3 has been implicated in tRNA binding and release at the A and E sites, its exact mechanism of action is unclear. Here we show that eEF3 acts at the mRNA–tRNA translocation step by promoting the dissociation of the tRNA from the E site, but independent of aminoacyl-tRNA recruitment to the A site. Depletion of eEF3 in vivo leads to a general slow-down in translation elongation due to accumulation of ribosomes with an occupied A site. Cryo-EM analysis of native eEF3-ribosome complexes shows that eEF3 facilitates late steps of translocation by favoring non-rotated ribosomal states as well as by opening the L1 stalk to release the E-site tRNA. Additionally, our analysis provides structural insights into novel translation elongation states, enabling presentation of a revised yeast translation elongation cycle.

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Eukaryotic translation elongation factor 2 (eEF2) catalyzes reverse translocation of the eukaryotic ribosome.

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