Withey J, Friedman D
Graduate Program in Cellular and Molecular Biology, University of Michigan, Ann Arbor, Michigan 48109-0620, USA.
J Bacteriol. 1999 Apr;181(7):2148-57. doi: 10.1128/JB.181.7.2148-2157.1999.
The small, stable RNA molecule encoded by ssrA, known as tmRNA or 10Sa RNA, is required for the growth of certain hybrid lambdaimmP22 phages in Escherichia coli. tmRNA has been shown to tag partially synthesized proteins for degradation in vivo by attaching a short peptide sequence, encoded by tmRNA, to the carboxyl termini of these proteins. This tag sequence contains, at its C terminus, an amino acid sequence that is recognized by cellular proteases and leads to degradation of tagged proteins. A model describing this function of tmRNA, the trans-translation model (K. C. Keiler, P. R. Waller, and R. T. Sauer, Science 271:990-993, 1996), proposes that tmRNA acts first as a tRNA and then as a mRNA, resulting in release of the original mRNA template from the ribosome and translocation of the nascent peptide to tmRNA. Previous work from this laboratory suggested that tmRNA may also interact specifically with DNA-binding proteins, modulating their activity. However, more recent results indicate that interactions between tmRNA and DNA-binding proteins are likely nonspecific. In light of this new information, we examine the effects on lambdaimmP22 growth of mutations eliminating activities postulated to be important for two different steps in the trans-translation model, alanine charging of tmRNA and degradation of tagged proteins. This mutational analysis suggests that, while charging of tmRNA with alanine is essential for lambdaimmP22 growth in E. coli, degradation of proteins tagged by tmRNA is required only to achieve optimal levels of phage growth. Based on these results, we propose that trans-translation may have two roles, the primary role being the release of stalled ribosomes from their mRNA template and the secondary role being the tagging of truncated proteins for degradation.
由ssrA编码的小的、稳定的RNA分子，即tmRNA或10Sa RNA，是某些杂交λimmP22噬菌体在大肠杆菌中生长所必需的。已表明tmRNA通过将由tmRNA编码的短肽序列连接到这些蛋白质的羧基末端，在体内标记部分合成的蛋白质以便降解。该标签序列在其C末端包含一个被细胞蛋白酶识别的氨基酸序列，导致被标记蛋白质的降解。描述tmRNA此功能的一个模型，即反式翻译模型（K.C.基勒、P.R.沃勒和R.T.索尔，《科学》271:990 - 993，1996）提出，tmRNA首先作为tRNA起作用，然后作为mRNA起作用，导致原始mRNA模板从核糖体释放以及新生肽转移到tmRNA。本实验室先前的工作表明，tmRNA也可能与DNA结合蛋白特异性相互作用，调节它们的活性。然而，最近的结果表明，tmRNA与DNA结合蛋白之间的相互作用可能是非特异性的。鉴于这一新信息，我们研究了消除假定对反式翻译模型中两个不同步骤重要的活性的突变对λimmP22生长的影响，这两个步骤是tmRNA的丙氨酸装载和被标记蛋白质的降解。这种突变分析表明，虽然tmRNA的丙氨酸装载对于λimmP22在大肠杆菌中的生长至关重要，但tmRNA标记的蛋白质的降解仅在达到噬菌体生长的最佳水平时才是必需的。基于这些结果，我们提出反式翻译可能有两个作用，主要作用是使停滞的核糖体从其mRNA模板上释放，次要作用是标记截短的蛋白质以便降解。
