Departamento de Medicina Preventiva, Facultad de Medicina, Universidad Autónoma, Madrid, Spain.
PLoS One. 2010 Jul 14;5(7):e11575. doi: 10.1371/journal.pone.0011575.
Except for the ribosomal protein L12 (rplL), ribosomal proteins are present as one copy per ribosome; L12 (rplL) is unusual because it is present as four copies per ribosome. Thus, the strategies used by Mycobacterium fortuitum to regulate ribosomal protein synthesis were investigated, including evaluations of the rates of chain elongations of 16S rRNA, rplL and ribosomal protein S12 (rpsL).
RNA was isolated from cell cultures and cDNA was prepared. The numbers of cDNA copies of 16S rRNA, precursor-16S rRNA and transcripts of rpsL and rplL were quantified by qRT-PCR and then related to the rates of 16S rRNA, rpsL and rplL chain elongations by means of a mathematical framework for coupled transcription/translation.
The rates of synthesis of 16S rRNA, rpsL and rplL respectively were found to be approximately 50 x 10(3) nucleotides h(-1), 1.6 x 10(3) amino acid residues h(-1) and 3.4 x 10(3) amino acid residues h(-1). The number of transcripts of rplL was approximately twice that of rpsL. These data account for the presence of one copy of rpsL and four copies of rplL per ribosome, and reveal that the rate of M. fortuitum ribosome synthesis was closer to that of M. tuberculosis than to E. coli. Except for rplJ, the elongation rate obtained for rpsL was inferred to be appropriate for all other proteins present as one copy per ribosome.
The results obtained provide the basis for a comprehensive view of the kinetics of ribosome synthesis, and of the ways that bacterial cells utilize genes encoding ribosomal proteins. The methodology also applies to proteins involved in transcription, energy generation and to bacterial proteins in general. The method proposed for measuring the fidelity of cDNA preparations is intrinsically much more sensitive than procedures that measure the integrity of 16S rRNA.
除了核糖体蛋白 L12（rplL）外，核糖体蛋白每个核糖体中存在一个拷贝；L12（rplL）是不寻常的，因为它每个核糖体中有四个拷贝。因此，研究了分枝杆菌 Fortunatum 调节核糖体蛋白合成的策略，包括评估 16S rRNA、rplL 和核糖体蛋白 S12（rpsL）的链延伸率。
从细胞培养物中分离 RNA，并制备 cDNA。通过 qRT-PCR 定量测定 16S rRNA、前体-16S rRNA 和 rpsL 和 rplL 转录本的 cDNA 拷贝数，并通过转录/翻译偶联的数学框架将其与 16S rRNA、rpsL 和 rplL 链延伸率相关联。
分别发现 16S rRNA、rpsL 和 rplL 的合成率约为 50 x 10(3)核苷酸 h(-1)、1.6 x 10(3) 个氨基酸残基 h(-1)和 3.4 x 10(3)个氨基酸残基 h(-1)。rplL 的转录本数量约为 rpsL 的两倍。这些数据解释了每个核糖体中存在一个 rpsL 和四个 rplL 拷贝的情况，并表明分枝杆菌 Fortunatum 的核糖体合成率更接近分枝杆菌结核，而不是大肠杆菌。除了 rplJ 之外，推断 rpsL 的延伸率适合每个核糖体存在一个拷贝的所有其他蛋白质。
获得的结果为核糖体合成动力学和细菌细胞利用编码核糖体蛋白的基因的方式提供了基础。该方法也适用于参与转录、能量产生的蛋白质以及一般的细菌蛋白质。用于测量 cDNA 制剂保真度的方法在本质上比测量 16S rRNA 完整性的方法灵敏得多。
