期刊
JOURNAL OF MOLECULAR BIOLOGY
卷 318, 期 2, 页码 287-303出版社
ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD
DOI: 10.1016/S0022-2836(02)00054-2
关键词
retrotransposon; ribozyme; RNA-protein interaction; RNA splicing; translational control
资金
- NIGMS NIH HHS [GM37949] Funding Source: Medline
Mobile group II introns encode reverse transcriptases that bind specifically to the intron RNAs to promote both intron mobility and RNA splicing (maturase activity). Previous studies with the Lactococcus lactis L1.LtrB intron suggested a model in which the intron-encoded protein (LtrA) binds first to a primary high-affinity binding site in intron subdomain DIVa, an idiosyncratic structure at the beginning of the LtrA coding sequence, and then makes additional contacts with conserved regions of the intron to fold the RNA into the catalytically active structure. Here, we analyzed the DIVa binding site by iterative in vitro selection and in vitro mutagenesis. Our results show that LtrA binds to a small region at the distal end of DIVa that contains the ribosome-binding site and initiation codon of the LtrA open reading frame. The critical elements are in a small stem-loop structure emanating from a purine-rich internal loop, with both sequence and structure playing a role in LtrA recognition. The ribosome-binding site falls squarely within the LtrA-binding region and is sequestered directly by the binding of LtrA or by stabilization of the small stem-loop or both. Finally, by using LacZ fusions in Escherichia coli, we show that the binding of LtrA to DIVa down-regulates translation. This mode of regulation limits accumulation of the potentially deleterious intron-encoded protein and may facilitate splicing by halting ribosome entry into the intron. The recognition of the DIVa loop-stem-loop structure accounts, in part, for the intron specificity of group II intron maturases and has parallels in template-recognition mechanisms used by other reverse transcriptases. (C) 2002 Elsevier Science Ltd. All rights reserved.
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