Barman Anik, Shaw Rahul, Bhawsinghka Niketa, Das Gupta Sujoy K
Department of Microbiology, Bose Institute, P-1/12 C.I.T. Scheme VIIM, Kolkata-700054, India.
Present address: Genome Integrity and Structural Biology Laboratory, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, USA.
Microbiology (Reading). 2022 Nov;168(11). doi: 10.1099/mic.0.001276.
A unique feature found in the genomes of mycobacteriophages such as L5 belonging to the A cluster is the presence of multiple dispersed repeated elements known as stoperators. The phage repressor binds these repeat elements, shutting off transcription globally and thereby promoting lysogeny. Interestingly, the sequence of these stoperators closely matches that of the consensus -35 region of prokaryotic promoters, leading us to propose that they may have a role to play in the initiation of transcription by serving as RNA polymerase binding sites. Mycobacteriophage D29 is closely related to phage L5, and their genome organizations are very similar. As in L5, there are multiple stoperators in the genome of D29. The positions occupied by the stoperators in the two genomes are almost identical. The significant difference between the two phages is that D29 lacks the gene encoding the equivalent of the L5 repressor. Since phage D29 does not produce a repressor, we considered it to be a suitable model for testing our hypothesis that the stoperators function as promoters in the absence of the repressor. To prove our point, we targeted CRISPR guide RNAs against six stoperators. In the case of five out of the six, we found a significant reduction in downstream gene expression and phage growth. Based on this observation and primer extension assays, we conclude that promoting gene expression is likely to be the primary function of stoperators.
在属于A簇的分枝杆菌噬菌体（如L5）的基因组中发现的一个独特特征是存在多个分散的重复元件，即所谓的终止子。噬菌体阻遏物与这些重复元件结合，全局关闭转录，从而促进溶原性。有趣的是，这些终止子的序列与原核启动子的共有-35区域的序列紧密匹配，这使我们提出它们可能通过作为RNA聚合酶结合位点在转录起始中发挥作用。分枝杆菌噬菌体D29与噬菌体L5密切相关，它们的基因组组织非常相似。与L5一样，D29的基因组中也有多个终止子。这两个基因组中终止子占据的位置几乎相同。这两种噬菌体之间的显著差异在于D29缺乏编码与L5阻遏物等效物的基因。由于噬菌体D29不产生阻遏物，我们认为它是检验我们的假设（即终止子在没有阻遏物的情况下作为启动子发挥作用）的合适模型。为了证明我们的观点，我们针对六个终止子设计了CRISPR引导RNA。在六个中的五个案例中，我们发现下游基因表达和噬菌体生长显著降低。基于这一观察结果和引物延伸分析，我们得出结论，促进基因表达可能是终止子的主要功能。
