A frameshifting stimulatory stem loop destabilizes the hybrid state and impedes ribosomal translocation

Hee-Kyung Kim; Fei Liu; Jingyi FeiCarlos BustamanteRuben L. Gonzalez Jr.Ignacio Tinoco Jr.

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A frameshifting stimulatory stem loop destabilizes the hybrid state and impedes ribosomal translocation

机译：A frameshifting stimulatory stem loop destabilizes the hybrid state and impedes ribosomal translocation

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Ribosomal frameshifting occurs when a ribosome slips a few nucleotides on an mRNA and generates a new sequence of amino acids. Programmed ?1 ribosomal frameshifting (?1PRF) is used in various systems to express two or more proteins from a single mRNA at precisely regulated levels. We used single-molecule fluorescence resonance energy transfer (smFRET) to study the dynamics of ?1PRF in the Escherichia coli dnaX gene. The frameshifting mRNA (FSmRNA) contained the frameshifting signals: a Shine– Dalgarno sequence, a slippery sequence, and a downstream stem loop. The dynamics of ribosomal complexes translating through the slippery sequence were characterized using smFRET between the Cy3-labeled L1 stalk of the large ribosomal subunit and a Cy5- labeled tRNA~(Lys) in the ribosomal peptidyl-tRNA–binding (P) site. We observed significantly slower elongation factor G (EF-G)–catalyzed translocation through the slippery sequence of FSmRNA in comparison with an mRNA lacking the stem loop, ΔSL. Furthermore, the P-site tRNA/L1 stalk of FSmRNA-programmed pretranslocation (PRE) ribosomal complexes exhibited multiple fluctuations between the classical/open and hybrid/closed states, respectively, in the presence of EF-G before translocation, in contrast with ΔSL-programmed PRE complexes, which sampled the hybrid/closed state approximately once before undergoing translocation. Quantitative analysis showed that the stimulatory stem loop destabilizes the hybrid state and elevates the energy barriers corresponding to subsequent substeps of translocation. The shift of the FSmRNA-programmed PRE complex equilibrium toward the classical/open state and toward states that favor EF-G dissociation apparently allows the PRE complex to explore alternative translocation pathways such as ?1PRF.

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《Proceedings of the National Academy of Sciences of the United States of America》 |2014年第15期|5538-5543|共6页
作者
Hee-Kyung Kim; Fei Liu; Jingyi FeiCarlos BustamanteRuben L. Gonzalez Jr.Ignacio Tinoco Jr.;
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作者单位

Department of Chemistry, Columbia University, New York, NY 10027;

Department of Chemistry, Department of Physics and Department of Molecular and Cellular Biology, and Howard Hughes Medical Institute, University of California, Berkeley, CA 94720;

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收录信息美国《科学引文索引》(SCI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
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正文语种英语
中图分类自然科学总论;
关键词
programmed ribosomal frameshifting; ribosomal dynamics; single-molecule FRET; mRNA secondary structure;

A frameshifting stimulatory stem loop destabilizes the hybrid state and impedes ribosomal translocation

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