A frameshifting stimulatory stem loop destabilizes the hybrid state and impedes ribosomal translocation
A frameshifting stimulatory stem loop destabilizes the hybrid state and impedes ribosomal translocation
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DOI：
10.1073/pnas.1403457111
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发表时间：
2014-04-15
影响因子：
11.1
通讯作者：
Tinoco, Ignacio, Jr.
中科院分区：
文献类型：
--
作者：
Kim, Hee-Kyung;Liu, Fei;Tinoco, Ignacio, Jr.
当核糖体在mRNA上滑动几个核苷酸并产生一个新的氨基酸序列时，就会发生核糖体移码。程序性-1核糖体移码（-1PRF）在各种系统中用于以精确调节的水平从单个mRNA表达两种或多种蛋白质。我们使用单分子荧光共振能量转移（smFRET）的大肠杆菌dnaX基因的动力学研究-1PRF。移码mRNA（FSmRNA）包含移码信号：Shine-Dalgarno序列，滑动序列和下游茎环。核糖体复合物通过滑动序列翻译的动力学的特点是使用之间的Cy 3-标记的L1柄的核糖体大亚基和Cy 5-标记的tRNA（Lys）的核糖体肽基-tRNA结合（P）网站的smFRET。我们观察到显着较慢的延伸因子G（EF-G）催化易位通过FSmRNA的光滑序列相比，缺乏茎环，三角洲SL的mRNA。此外，P-位点tRNA/L1柄的FSmRNA程序化的前易位（PRE）核糖体复合物表现出多个波动之间的经典/开放和混合/封闭状态，分别在EF-G的存在下易位前，在与三角洲SL程序化的PRE复合物，采样的混合/封闭状态大约一次之前进行易位。定量分析表明，刺激茎环不稳定的混合状态，并提高相应的后续子步骤的易位的能量障碍。FSmRNA编程的PRE复合物平衡向经典/开放状态和有利于EF-G解离的状态的转变显然允许PRE复合物探索替代易位途径，如-1PRF。
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, Delta SL. 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 Delta SL-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 sub-steps 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.