Sharma S, Gopinathan K P
Department of Microbiology and Cell Biology and Center for Genetic Engineering, Indian Institute of Science, Bangalore 560 012, India.
J Biol Chem. 1996 Nov 8;271(45):28146-53. doi: 10.1074/jbc.271.45.28146.
Individual copies of tRNA1Gly from within the multigene family in Bombyx mori could be classified based on in vitro transcription in homologous nuclear extracts into three categories of highly, moderately, or weakly transcribed genes. Segregation of the poorly transcribed gene copies 6 and 7, which are clustered in tandem within 425 base pairs, resulted in enhancement of their individual transcription levels, but the linkage itself had little influence on the transcriptional status. For these gene copies, when fused together generating a single coding region, transcription was barely detectable, which suggested the presence of negatively regulating elements located in the far flanking sequences. They exerted the silencing effect on transcription overriding the activity of positive regulatory elements. Systematic analysis of deletion, chimeric, and mutant constructs revealed the presence of a sequence element TATATAA located beyond 800 nucleotides upstream to the coding region acting as negative modulator, which when mutated resulted in high level transcription. Conversely, a TATATAA motif reintroduced at either far upstream or far downstream flanking regions exerted a negative effect on transcription. The location of cis-regulatory sequences at such farther distances from the coding region and the behavior of TATATAA element as negative regulator reported here are novel. These element(s) could play significant roles in activation or silencing of genes from within a multigene family, by recruitment or sequestration of transcription factors.
家蚕多基因家族中的tRNA1Gly单拷贝，根据其在同源核提取物中的体外转录情况，可分为高转录、中转录或低转录三类基因。串联聚集在425个碱基对内的低转录基因拷贝6和7分离后，其各自的转录水平得到提高，但基因间的连锁本身对转录状态影响不大。对于这些基因拷贝，当融合在一起形成单个编码区域时，几乎检测不到转录，这表明在侧翼较远的序列中存在负调控元件。它们对转录产生沉默作用，超过了正调控元件的活性。对缺失、嵌合和突变构建体的系统分析表明，在编码区域上游800多个核苷酸处存在一个序列元件TATATAA，它作为负调节剂，突变后会导致高水平转录。相反，在上下游侧翼区域重新引入的TATATAA基序对转录产生负面影响。本文报道的顺式调控序列在距编码区域如此远的位置以及TATATAA元件作为负调节剂的行为都是新颖的。这些元件可能通过招募或隔离转录因子，在多基因家族内基因的激活或沉默中发挥重要作用。
