Santer M, Bennett-Guerrero E, Byahatti S, Czarnecki S, O'Connell D, Meyer M, Khoury J, Cheng X, Schwartz I, McLaughlin J
Department of Biology, Haverford College, PA 19041.
Proc Natl Acad Sci U S A. 1990 May;87(10):3700-4. doi: 10.1073/pnas.87.10.3700.
To investigate the function of base 792 of 16S rRNA in 30S ribosomes of Escherichia coli, the wild-type (adenine) residue was changed to guanine, cytosine, or uracil by oligonucleotide-directed mutagenesis. Each base change conferred a unique phenotype on the cells. Cells containing plasmid pKK3535 with G792 or T792 showed no difference in generation time in LB broth containing ampicillin, whereas cells with C792 exhibited a 20% increase in generation time in this medium. To study the effect on cell growth of a homogeneous population of mutant ribosomes, the mutations were cloned into the 16S rRNA gene on pKK3535 carrying a spectinomycin-resistance marker (thymine at position 1192), and the cells were grown with spectinomycin. Cells containing G792 or C792 showed 16% and 56% increases in generation time, respectively, and a concomitant decrease in 35S assimilation into proteins. Cells with T792 did not grow in spectinomycin-containing medium. Maxicell analyses indicated decreasing ability to form 70S ribosomes from 30S subunits containing guanine, cytosine, or uracil at position 792 in 16S rRNA. It appeared that C792-containing 30S ribosomes had lost the ability to bind initiation factor 3.
为了研究大肠杆菌30S核糖体中16S rRNA第792位碱基的功能，通过寡核苷酸定向诱变将野生型（腺嘌呤）残基分别替换为鸟嘌呤、胞嘧啶或尿嘧啶。每种碱基替换都赋予细胞独特的表型。含有携带G792或T792的质粒pKK3535的细胞，在含有氨苄青霉素的LB肉汤中培养时，其代时没有差异，而含有C792的细胞在此培养基中的代时增加了20%。为了研究突变核糖体的同质群体对细胞生长的影响，将突变克隆到携带壮观霉素抗性标记（第1192位为胸腺嘧啶）的pKK3535上的16S rRNA基因中，然后让细胞在壮观霉素存在的情况下生长。含有G792或C792的细胞，其代时分别增加了16%和56%，同时35S掺入蛋白质的量减少。含有T792的细胞在含有壮观霉素的培养基中无法生长。最大细胞分析表明，由16S rRNA第792位含有鸟嘌呤、胞嘧啶或尿嘧啶的30S亚基形成70S核糖体的能力下降。似乎含有C792的30S核糖体失去了结合起始因子3的能力。
