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Kinetically competent intermediates in the translocation step of protein synthesis.

ABSTRACT: Translocation requires large-scale movements of ribosome-bound tRNAs. Using tRNAs that are proflavin labeled and single-turnover rapid kinetics assays, we identify one or possibly two kinetically competent intermediates in translocation. EF-G.GTP binding to the pretranslocation (PRE) complex and GTP hydrolysis are rapidly followed by formation of the securely identified intermediate complex (INT), which is more slowly converted to the posttranslocation (POST) complex. Peptidyl tRNA within the INT complex occupies a hybrid site, which has a puromycin reactivity intermediate between those of the PRE and POST complexes. Thiostrepton and viomycin inhibit INT formation, whereas spectinomycin selectively inhibits INT disappearance. The effects of other translocation modulators suggest that EF-G-dependent GTP hydrolysis is more important for INT complex formation than for INT complex conversion to POST complex and that subtle changes in tRNA structure influence coupling of tRNA movement to EF-G.GTP-induced conformational changes.

SUBMITTER: Pan D

PROVIDER: S-EPMC1995019 | biostudies-literature | 2007 Feb

REPOSITORIES: biostudies-literature

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Kinetically competent intermediates in the translocation step of protein synthesis.

Pan Dongli D Kirillov Stanislav V SV Cooperman Barry S BS

Molecular cell 20070201 4

Translocation requires large-scale movements of ribosome-bound tRNAs. Using tRNAs that are proflavin labeled and single-turnover rapid kinetics assays, we identify one or possibly two kinetically competent intermediates in translocation. EF-G.GTP binding to the pretranslocation (PRE) complex and GTP hydrolysis are rapidly followed by formation of the securely identified intermediate complex (INT), which is more slowly converted to the posttranslocation (POST) complex. Peptidyl tRNA within the IN ...[more]

PMID: 17317625

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Project description:In the present work, the conformational dynamics and folding pathways of i-motif DNA were studied in solution and in the gas-phase as a function of the solution pH conditions using circular dichroism (CD), photoacoustic calorimetry analysis (PAC), trapped ion mobility spectrometry-mass spectrometry (TIMS-MS), and molecular dynamics (MD). Solution studies showed at thermodynamic equilibrium the existence of a two-state folding mechanism, whereas during the pH = 7.0 → 4.5 transition a fast and slow phase (ΔHfast + ΔHslow = 43 ± 7 kcal mol-1) with a volume change associated with the formation of hemiprotonated cytosine base pairs and concomitant collapse of the i-motif oligonucleotide into a compact conformation were observed. TIMS-MS experiments showed that gas-phase, kinetically trapped i-motif DNA intermediates produced by nanoESI are preserved, with relative abundances depending on the solution pH conditions. In particular, a folded i-motif DNA structure was observed in nanoESI-TIMS-MS for low charge states in both positive and negative ion mode (e.g., z = ±3 to ±5) at low pH conditions. As solution pH increases, the cytosine neutralization leads to the loss of cytosine-cytosine+ (C·CH+) base pairing in the CCC strands and in those conditions we observe partially unfolded i-motif DNA conformations in nanoESI-TIMS-MS for higher charge states (e.g., z = -6 to -9). Collisional induced activation prior to TIMS-MS showed the existence of multiple local free energy minima, associated with the i-motif DNA unfolding at z = -6 charge state. For the first time, candidate gas-phase structures are proposed based on mobility measurements of the i-motif DNA unfolding pathway. Moreover, the inspection of partially unfolded i-motif DNA structures (z = -7 and z = -8 charge states) showed that the presence of inner cations may or may not induce conformational changes in the gas-phase. For example, incorporation of ammonium adducts does not lead to major conformational changes while sodium adducts may lead to the formation of sodium mediated bonds between two negatively charged sides inducing the stabilization towards more compact structures in new local, free energy minima in the gas-phase.

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Kinetically competent intermediates in the translocation step of protein synthesis.

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Kinetically competent intermediates in the translocation step of protein synthesis.

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