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Model of ribosomal translocation coupled with intra- and inter-subunit rotations.

ABSTRACT: The ribosomal translocation involves both intersubunit rotations between the small 30S and large 50S subunits and the intrasubunit rotations of the 30S head relative to the 30S body. However, the detailed molecular mechanism on how the intersubunit and intrasubunit rotations are related to the translocation remains unclear. Here, based on available structural data a model is proposed for the ribosomal translocation, into which both the intersubunit and intrasubunit rotations are incorporated. With the model, we provide quantitative explanations of in vitro experimental data showing the biphasic character in the fluorescence change associated with the mRNA translocation and the character of a rapid increase that is followed by a slow single-exponential decrease in the fluorescence change associated with the 30S head rotation. The calculated translation rate is also consistent with the in vitro single-molecule experimental data.

SUBMITTER: Xie P

PROVIDER: S-EPMC5668647 | biostudies-literature | 2015 Jul

REPOSITORIES: biostudies-literature

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Model of ribosomal translocation coupled with intra- and inter-subunit rotations.

Xie Ping P

Biochemistry and biophysics reports 20150609

The ribosomal translocation involves both intersubunit rotations between the small 30S and large 50S subunits and the intrasubunit rotations of the 30S head relative to the 30S body. However, the detailed molecular mechanism on how the intersubunit and intrasubunit rotations are related to the translocation remains unclear. Here, based on available structural data a model is proposed for the ribosomal translocation, into which both the intersubunit and intrasubunit rotations are incorporated. Wi ...[more]

PMID: 29124148

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Project description:Glycine receptors (GlyRs) mediate inhibitory neurotransmission and are targets for alcohols and anesthetics in brain. GlyR transmembrane (TM) domains contain critical residues for alcohol/anesthetic action: amino acid A288 in TM3 forms crosslinks with TM1 (I229) in the adjacent subunit as well as TM2 (S267) and TM4 (Y406, W407, I409, Y410) in the same subunit. We hypothesized that these residues may participate in intra-subunit and inter-subunit sites of alcohol/anesthetic action. The following double and triple mutants of GLRA1 cDNA (encoding human glycine receptor alpha 1 subunit) were injected into Xenopus laevis oocytes: I229C/A288C, I229C/A288C/C290S, A288C/Y406C, A288C/W407C, A288C/I409C, and A288C/Y410C along with the corresponding single mutants and wild-type GLRA1. Butanol (22 mM) or isoflurane (0.6 mM) potentiation of GlyR-mediated currents before and after application of the cysteine crosslinking agent HgCl2 (10 μM) was measured using two-electrode voltage clamp electrophysiology. Crosslinking nearly abolished butanol and isoflurane potentiation in the I229C/A288C and I229C/A288C/C290S mutants but had no effect in single mutants or wild-type. Crosslinking also inhibited butanol and isoflurane potentiation in the TM3-4 mutants (A288C/Y406C, A288C/W407C, A288C/I409C, A288C/Y410C) with no effect in single mutants or wild-type. We extracted proteins from oocytes expressing I229C/288C, A288C/Y410C, or wild-type GlyRs, used mass spectrometry to verify their expression and possible inter-subunit dimerization, plus immunoblotting to investigate the biochemical features of proposed crosslinks. Wild-type GlyR subunits measured about 50 kDa; after crosslinking, the dimeric/monomeric 100:50 kDa band ratio was significantly increased in I229C/288C but not A288C/Y410C mutants or wild-type, providing support for TM1-3 inter-subunit and TM3-4 intra-subunit crosslinking. A GlyR homology model based on the GluCl template provides further evidence for a multi-site model for alcohol/anesthetic interaction with human GLRA1.

Dataset Information

Model of ribosomal translocation coupled with intra- and inter-subunit rotations.

Publications

Model of ribosomal translocation coupled with intra- and inter-subunit rotations.

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