Cannon R E, Wu G J, Railey J F
Proc Natl Acad Sci U S A. 1986 Mar;83(5):1285-9. doi: 10.1073/pnas.83.5.1285.
The internal transcriptional control region (ITCR) of VARNA1 gene consists of a 33-base-pair (bp) interblock sequence and two 12-bp sequence blocks that are highly conserved in most of the genes transcribed by RNA polymerase III. To define the functions of and study the interactions between the two blocks, we have constructed mutants with altered interblock sequence or spacing for transcription. The results of transcription efficiencies and competing strengths indicated that the interblock sequence was dispensable and the A and B blocks were essential for transcription control. One of the major functions of the interblock sequence was to maintain an optimal spacing for an intimate interaction between the two essential blocks. Shortening or elongating the interblock spacing in the mutants beyond this range drastically decreased the transcription efficiencies and competing strengths of these mutated genes. To further study how the interaction between the two blocks leads to initiation, the start sites and sizes of RNA products of the mutants were determined. When the interblock spacing was less than 105 bp, the wild-type start site was dictated by the A block after an interaction with the B block through proteins. However, when the interblock spacing was longer than 105 bp, several new start sites located closer to the B block were preferentially used. This suggests that new start sites may be dictated by the B block when its interaction with the A block is weakened by longer spacing. The mechanisms of interaction between the bipartite domain in this gene leading to initiation are different from those in tRNAs and Alu-family RNA genes.
VARNA1基因的内部转录控制区域（ITCR）由一个33个碱基对（bp）的间隔序列和两个12 bp的序列块组成，这两个序列块在大多数由RNA聚合酶III转录的基因中高度保守。为了确定这两个序列块的功能并研究它们之间的相互作用，我们构建了间隔序列或间隔发生改变的突变体用于转录研究。转录效率和竞争强度的结果表明，间隔序列是可有可无的，而A块和B块对于转录控制至关重要。间隔序列的一个主要功能是为两个必需序列块之间的紧密相互作用维持最佳间隔。在突变体中缩短或延长间隔序列超出这个范围会大幅降低这些突变基因的转录效率和竞争强度。为了进一步研究这两个序列块之间的相互作用如何导致起始，我们确定了突变体RNA产物的起始位点和大小。当间隔序列小于105 bp时，野生型起始位点由A块决定，A块通过蛋白质与B块相互作用之后确定起始位点。然而，当间隔序列长于105 bp时，几个更靠近B块的新起始位点被优先使用。这表明当B块与A块的相互作用因间隔延长而减弱时，新起始位点可能由B块决定。该基因中二分结构域之间导致起始的相互作用机制与tRNA和Alu家族RNA基因中的不同。
