Downing W L, Dennis P P
Department of Biochemistry, University of British Columbia, Vancouver, Canada.
J Mol Biol. 1987 Apr 20;194(4):609-20. doi: 10.1016/0022-2836(87)90238-5.
Transcripts from the rplKAJL-rpoBC ribosomal protein-RNA polymerase gene cluster have been quantified and their ends mapped using RNA-DNA hybridization, sucrose density-gradient sedimentation, Northern hybridization and S1 nuclease protection. The results indicate that the most abundant transcript is the 2600 nucleotide tetracistronic L11-L1-L10-L12 mRNA initiated at the upstream major PL11 promoter and terminated at the transcription attenuator in the L12-beta intergenic space. Somewhat less abundant 1300 nucleotide L11-L1 and L10-L12 bicistronic transcripts were observed. The 3' ends of the L11-L1 transcripts were heterogeneous; most of the ends were localized to three sites within a 110 base-pair region in the L1-L10 intergenic space. This intergenic space encodes also the major PL10 promoter and the mRNA binding site for the L10 translational control protein. Two 5' ends were observed for L10-L12 bicistronic mRNA, one at the PL10 promoter and the other 150 nucleotides further downstream in a region in which promoter activity has not been detected. It is suggested that this second downstream 5' end is generated by processing of the transcripts initiated at the major PL10 promoter. No transcript initiation in the L10-L12 intergenic space was detected. About 80% of the transcripts reading through the L12 gene were terminated in the vicinity of the transcription attenuator that is responsible for the reduction in the expression of the downstream RNA polymerase genes. Transcripts reading through the attenuator were partially processed by RNase III within a potential hairpin structure in the RNA transcript. Processing appears to produce 3' and 5' transcript end sites separated by about ten nucleotides. No other major 5' ends were observed in the L12-beta intergenic space. These results indicate that the two major promoters, PL11 and PL10, are both utilized to drive the interrelated transcriptional expression of this ribosomal protein-RNA polymerase gene cluster.
利用RNA-DNA杂交、蔗糖密度梯度沉降、Northern杂交和S1核酸酶保护技术，对核糖体蛋白-RNA聚合酶基因簇rplKAJL-rpoBC的转录本进行了定量分析，并确定了其末端位置。结果表明，最丰富的转录本是2600个核苷酸的四顺反子L11-L1-L10-L12 mRNA，它从上游主要的PL11启动子起始，在L12-β基因间隔区的转录衰减子处终止。还观察到丰度稍低的1300个核苷酸的L11-L1和L10-L12双顺反子转录本。L11-L1转录本的3'末端具有异质性；大多数末端定位于L1-L10基因间隔区内110个碱基对区域的三个位点。这个基因间隔区还编码主要的PL10启动子和L10翻译控制蛋白的mRNA结合位点。L10-L12双顺反子mRNA观察到两个5'末端，一个在PL10启动子处，另一个在下游150个核苷酸处，该区域未检测到启动子活性。推测这个第二个下游5'末端是由在主要PL10启动子起始的转录本加工产生的。在L10-L12基因间隔区未检测到转录起始。约80%通读L12基因的转录本在负责下游RNA聚合酶基因表达降低的转录衰减子附近终止。通读衰减子的转录本在RNA转录本中的潜在发夹结构内被RNase III部分加工。加工似乎产生了相隔约十个核苷酸的3'和5'转录本末端位点。在L12-β基因间隔区未观察到其他主要的5'末端。这些结果表明，两个主要启动子PL11和PL10都被用于驱动这个核糖体蛋白-RNA聚合酶基因簇的相关转录表达。
