HIV-1转录物的功能部分受孪生转录起始位点的使用控制,其中以单个鸟苷(1G)开始的5个加帽的RNA优先作为基因组RNA(gRNA)包装到子代病毒粒子中,而以三个连续鸟苷(3G)开始的RNA作为mRNA保留在细胞中。在3G成绩单中,一个额外的鸟嘌呤碱基对与位于保守的5'polyA元件内的胞嘧啶,导致形成延伸的5'polyA结构,而不是1GRNA中形成的发夹结构。要了解这种重塑如何影响整体转录本功能,我们将细胞内基因组包装和竞争性翻译测定的体外生物物理研究应用于天然和5'polyA突变体转录本,该转录本由差异产生1G或3GRNA的启动子产生。我们确定了稳定3GRNA中5'polyA发夹结构的突变,这促进RNA二聚化和Gag结合而不螯合5'帽。这些3G成绩单都不是竞争性包装的,确认帽暴露是病毒基因组包装的主要负决定因素。对于所有检查的RNA,与那些有利于5'帽隔离的构象相比,有利于5'帽暴露的构象包装不良且翻译效率更高。我们建议5'polyA和其他保守RNA元件的结构可塑性将5'前导序列置于热力学临界点上,用于低能量(〜3kcal/mol)控制整体转录本结构和功能。
HIV-1 transcript function is controlled in part by twinned transcriptional start site usage, where 5\' capped RNAs beginning with a single guanosine (1G) are preferentially packaged into progeny virions as genomic RNA (gRNA) whereas those beginning with three sequential guanosines (3G) are retained in cells as mRNAs. In 3G transcripts, one of the additional guanosines base pairs with a cytosine located within a conserved 5\'
polyA element, resulting in formation of an extended 5\'
polyA structure as opposed to the hairpin structure formed in 1G RNAs. To understand how this remodeling influences overall transcript function, we applied in vitro biophysical studies with in-cell genome packaging and competitive translation assays to native and 5\'
polyA mutant transcripts generated with promoters that differentially produce 1G or 3G RNAs. We identified mutations that stabilize the 5\'
polyA hairpin structure in 3G RNAs, which promote RNA dimerization and Gag binding without sequestering the 5\' cap. None of these 3G transcripts were competitively packaged, confirming that cap exposure is a dominant negative determinant of viral genome packaging. For all RNAs examined, conformations that favored 5\' cap exposure were both poorly packaged and more efficiently translated than those that favored 5\' cap sequestration. We propose that structural plasticity of 5\'
polyA and other conserved RNA elements place the 5\' leader on a thermodynamic tipping point for low-energetic (~3 kcal/mol) control of global transcript structure and function.