Ooms Marcel, Cupac Daniel, Abbink Truus E M, Huthoff Hendrik, Berkhout Ben
Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center of the University of Amsterdam, The Netherlands.
Nucleic Acids Res. 2007;35(5):1649-59. doi: 10.1093/nar/gkm046. Epub 2007 Feb 18.
Initiation of reverse transcription of a retroviral RNA genome is strictly regulated. The tRNA primer binds to the primer binding site (PBS), and subsequent priming is triggered by the primer activation signal (PAS) that also pairs with the tRNA. We observed that in vitro reverse transcription initiation of the HIV-1 leader RNA varies in efficiency among 3'-end truncated transcripts, despite the presence of both PBS and PAS motifs. As the HIV-1 leader RNA can adopt two different foldings, we investigated if the conformational state of the transcripts did influence the efficiency of reverse transcription initiation. However, mutant transcripts that exclusively fold one or the other structure were similarly active, thereby excluding the possibility of regulation of reverse transcription initiation by the structure riboswitch. We next set out to determine the availability of the PAS element. This sequence motif enhances the efficiency of reverse transcription initiation, but its activity is regulated because the PAS motif is initially base paired within the wild-type template. We measured that the initiation efficiency on different templates correlates directly with accessibility of the PAS motif. Furthermore, changes in PAS are critical to facilitate a primer-switch to a new tRNA species, demonstrating the importance of this enhancer element.
逆转录病毒RNA基因组的逆转录起始受到严格调控。tRNA引物与引物结合位点(PBS)结合,随后的引发由同样与tRNA配对的引物激活信号(PAS)触发。我们观察到,尽管存在PBS和PAS基序,但HIV-1前导RNA的体外逆转录起始在3'端截短的转录本中效率各不相同。由于HIV-1前导RNA可以呈现两种不同的折叠形式,我们研究了转录本的构象状态是否确实影响逆转录起始的效率。然而,仅折叠一种或另一种结构的突变转录本具有相似的活性,从而排除了通过结构核糖开关调控逆转录起始的可能性。接下来,我们着手确定PAS元件的可及性。这个序列基序提高了逆转录起始的效率,但其活性受到调控,因为PAS基序最初在野生型模板中碱基配对。我们测量发现,不同模板上的起始效率与PAS基序的可及性直接相关。此外,PAS的变化对于促进引物转换到新的tRNA种类至关重要,这证明了这个增强元件的重要性。