Goldman E, Korus M, Mandecki W
Department of Microbiology, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103, USA.
FASEB J. 2000 Mar;14(3):603-11. doi: 10.1096/fasebj.14.3.603.
An unusual nucleotide sequence, called H10, was previously isolated by biopanning with a random peptide library on filamentous phage. The sequence encoded a peptide that bound to the growth hormone binding protein. Despite the fact that the H10 sequence can be expressed in Escherichia coli as a fusion to the gene III minor coat protein of the M13 phage, the sequence contained two TGA stop codons in the zero frame. Several mutant derivatives of the H10 sequence carried not only a stop codon, but also showed frameshifts, either +1 or -1 in individual isolates, between the H10 start and the gene III sequences. In this work, we have subcloned the H10 sequence and three of its derivatives (one requiring a +1 reading frameshift for expression, one requiring a -1 reading frameshift, and one open reading frame) in gene fusions to a reporter beta-galactosidase gene. These sequences have been cloned in all three reading frames relative to the reporter. The non-open reading frame constructs gave (surprisingly) high expression of the reporter (10-40% of control vector expression levels) in two out of the three frames. A site-directed mutant of the TGA stop codon (to TTA) in the +1 shifter greatly reduced the frameshift and gave expression primarily in the zero frame. By contrast, a site-directed mutant of the TGA in the -1 shifter had little effect on the pattern of expression, and alteration of the first TGA (of two) in H10 itself paradoxically reduced expression by half. We believe these phenomena to reflect a translational recoding mechanism in which ribosomes switch reading frames or read past stop codons upon encountering a signal encoded in the nucleotide sequence of the mRNA, because both the open reading frame derivative (which has six nucleotide changes from parental H10) and the site-directed mutant of the +1 shifter, primarily expressed the reporter only in the zero frame.
一种名为H10的异常核苷酸序列，先前通过用丝状噬菌体上的随机肽库进行生物淘选而分离得到。该序列编码一种与生长激素结合蛋白结合的肽。尽管H10序列可以在大肠杆菌中作为与M13噬菌体基因III小衣壳蛋白的融合体进行表达，但该序列在零框架中包含两个TGA终止密码子。H10序列的几个突变衍生物不仅携带一个终止密码子，而且在H10起始序列和基因III序列之间的个别分离物中还显示出移码，要么是+1，要么是-1。在这项工作中，我们将H10序列及其三个衍生物（一个需要+1读码框移码才能表达，一个需要-1读码框移码，一个开放阅读框）亚克隆到与报告β-半乳糖苷酶基因的基因融合体中。这些序列已相对于报告基因在所有三个阅读框中进行克隆。非开放阅读框构建体在三个阅读框中的两个中（令人惊讶地）给出了报告基因的高表达（对照载体表达水平的10-40%）。+1移码器中TGA终止密码子的定点突变（变为TTA）大大减少了移码，并主要在零框架中表达。相比之下，-1移码器中TGA的定点突变对表达模式影响很小，而H10本身中两个TGA中的第一个TGA的改变反而使表达减半。我们认为这些现象反映了一种翻译重编码机制，其中核糖体在遇到mRNA核苷酸序列中编码的信号时切换阅读框或越过终止密码子进行阅读，因为开放阅读框衍生物（与亲本H10有六个核苷酸变化）和+1移码器的定点突变体主要仅在零框架中表达报告基因。
