Sera T, Schultz P G
Howard Hughes Medical Institute, Department of Chemistry, University of California, Berkeley 94720, USA.
Proc Natl Acad Sci U S A. 1996 Apr 2;93(7):2920-5. doi: 10.1073/pnas.93.7.2920.
A transcription interference assay was used to generate mutant basic region-leucine zipper proteins with altered DNA-binding specificities. A library of mutants of a CCAAT/enhancer binding protein was constructed by randomizing five DNA-contacting amino acids in the basic region Asn-18, Ala-15, Val-14, Ser-11, and Arg-10. These mutants were then selected for their ability to bind mutant recognition sequences containing substitutions at the 2 and 3 positions of the wild-type sequence 5'-A5T4T3G2C1G1'C2'A3A4'T5'-3'. Mutants containing the sequence Leu-18Tyr-15Xaa-14Tyr-11Arg-10, in which four of the five contact residues are altered, were identified that recognize the palindromic sequence 5'-ATCYCGY'GAT-3' (Xaa = asparagine when Y = G; Xaa = methionine when Y = A). Moreover, in a selection against the sequence 5'-ATTACGTAAT-3', mutants were obtained containing substitutions not only in the basic region but also in the hinge region between the basic and leucine zipper regions. The mutant proteins showed high specificity in a functional transcription interference assay. A model for the interaction of these mutants with the target DNA sequences is discussed.
采用转录干扰试验来生成具有改变的DNA结合特异性的突变型碱性区域-亮氨酸拉链蛋白。通过随机改变碱性区域中五个与DNA接触的氨基酸（Asn-18、Ala-15、Val-14、Ser-11和Arg-10）构建了CCAAT/增强子结合蛋白的突变体文库。然后选择这些突变体，以检测它们结合在野生型序列5'-A5T4T3G2C1G1'C2'A3A4'T5'-3'的2和3位置含有取代的突变识别序列的能力。鉴定出含有Leu-18Tyr-15Xaa-14Tyr-11Arg-10序列的突变体，其中五个接触残基中的四个发生了改变，这些突变体识别回文序列5'-ATCYCGY'GAT-3'（当Y = G时，Xaa = 天冬酰胺；当Y = A时，Xaa = 甲硫氨酸）。此外，在针对序列5'-ATTACGTAAT-3'的筛选中，获得的突变体不仅在碱性区域，而且在碱性区域和亮氨酸拉链区域之间的铰链区也有取代。在功能转录干扰试验中，突变蛋白表现出高特异性。讨论了这些突变体与靶DNA序列相互作用的模型。
